Revealing ETC-1922159 affected unknown 3 rd order WNT 10 B-XX combinations , in silico †

WNT10B belongs to the family of WNT proteins that are implicated in a range of phenomena that are affected by the Wnt signaling pathway. Recent studies have shown that WNT10B plays a role in colorectal cancer. WNTs have been found to directly affect the stemness of the tumor cells via regulation of ASCL2. Switching off the ASCL2 literally blocks the stemness process of the tumor cells and vice versa. Furthermore, recent findings suggest BVES to be highly suppressed in malignancies and in vitro deletions of BVES show higher Wnt signaling activity to induce stemness. WNT10B was found to be highly expressed in such cases. Often, in biology, we are faced with the problem of exploring relevant unknown biological hypotheses in the form of myriads of combination of factors that might be affecting the pathway under certain conditions. For example, WNT10B-ASCL2 is one such 2nd order combination whose relation needs to be tested under the influence of recently developed porcupine-WNT inhibitor ETC-1922159. The inhibitor is known to suppress PORCN (porcupine) and thus inhibit a range of oncogenes known to be directly or indirectly affected by the Wnts. In a recent unpublished work in bioRxiv, Sinha1, we had the opportunity to rank these unknown biological hypotheses for down regulated genes at 2nd order level after the drug was administered. The in silico observations showed that the combination of WNT10B-ASCL2 was assigned a relatively lower rank, thus validating the pipeline’s efficacy with the confirmed wet lab experiment that indicate that both WNT10B and ASCL2 were down regulated after treatment in cancer cells. Here, we take one step further by in silico analysis of the 3rd order combinations of WNT10B-X-X (X can be known or unknown factor), from a range of 100 randomly picked down regulated genes after ETC-1922159 treatment. The pipeline uses the density based HSIC (Hilbert Schmidt Information Criterion) sensitivity index with an rbf (radial basis function) kernel, which is known to be highly effective in sensitivity analysis. Various unknown/unexplored/untested 3rd order biological hypotheses emerge some of which are confirmed in wet lab, while others need to be tested. The potential of such ranking is indispensable in the current era of search in a vast combinatorial forest.


Introduction
Wnt signaling and secretion 2 's accidental discovery of the Wingless played a pioneering role in the emergence of a widely expanding research field of the Wnt signaling pathway.A majority of the work has focused on issues related to • the discovery of genetic and epigenetic factors affecting the pathway 3 & 4 , • implications of mutations in the pathway and its dominant role on cancer and other diseases 5 , • investigation into the pathway's contribution towards embryo development 6 , homeostasis 7 & 8 and apoptosis 9 and • safety and feasibility of drug design for the Wnt pathway 10 , 11 , 12 , 13 & 14 .
The Wnt phenomena can be roughly segregated into signaling and secretion part.The Wnt signaling pathway works when the WNT ligand gets attached to the Frizzled(FZD)/LRP coreceptor complex.FZD may interact with the Dishevelled (DVL) causing phosphorylation.It is also thought that Wnts cause phosphorylation of the LRP via casein kinase 1 (CK1) and kinase GSK3.These developments further lead to attraction of Axin which causes inhibition of the formation of the degradation complex.The degradation complex constitutes of AXIN, the β-catenin transportation complex APC, CK1 and GSK3.When the pathway is active the dissolution of the degradation complex leads to stabilization in the concentration of β-catenin in the cytoplasm.As β-catenin enters into the nucleus it displaces the GROUCHO and binds with transcription cell factor TCF thus instigating transcription of Wnt target genes.GROUCHO acts as lock on TCF and prevents the transcription of target genes which may induce cancer.In cases when the Wnt ligands are not captured by the coreceptor at the cell membrane, AXIN helps in formation of the degradation complex.The degradation complex phosphorylates β-catenin which is then recognised by F BOX/WD repeat protein β-TRCP.β-TRCP is a component of ubiquitin ligase complex that helps in ubiquitination of β-catenin thus marking it for degradation via the proteasome.A cartoon of the signaling transduction snapshot is shown in figure 1.
Contrary to the signaling phenomena, the secretion phenomena is about the release and transportation of the WNT protein/ligand in and out of the cell, respectively.Briefly, the WNT proteins that are synthesized with the endoplasmic reticulum (ER), are known to be palmitoyleated via the Porcupine (PORCN) to form the WNT ligand, which is then ready for transportation 15 .It is believed that these ligands are then transported via the EVI/WNTLESS transmembrane complex out of the cell 16 & 17 .The EVI/WNTLESS themselves are known to reside in the Golgi bodies and interaction with the WNT ligands for the later's glycosylation 18 & 19 .Once outside the cell, the WNTs then interact with the cell receptors, as explained in the foregoing paragraph, to induce the Wnt signaling.Of importance is the fact that the EVI/WNTLESS also need a transporter in the from of a complex termed as Retromer.A cartoon of the signaling transduction snapshot is shown in figure 2.

WNT10B
WNT10B has been found to be implicated in a range of cancers.In gastric cancer, the knockdown of WNT10B showed reduced expression of cell proliferation and migration as well as inhibition of epithelial-mesenchymal transition 20 .On the other hand, WNT10B is also involved in the formation of bone mass and progenitor maintenance of various kinds of tissue, while deletion of the same leads to loss of bone mass and mesenchymal progenitor cells 21 .Their contribution is also reported in axonal regeneration in injured CNS 22 .Furthermore, like WNT10B, WNT10A and WNT6 have shown to play a major role in inhibiting adipogenesis and stimulates osteoblastogenesis while regulating the mesenchymal stem cells 23 & 24 .Involvement in heptocellular carcinoma of WNT10B has been found wherein it is shown that stable silencing of WNT10B leads to significant reduction in proliferation, colony formation, migration and invasion in HepG2 HCC cell line 25 .Its implication in breast cancer 26 & 27 as well as endometrial cancer 28 has also been reported.
In colorectal cancer, WNT10B has shown to play a dual function of both oncogenesis promotion via β-catenin/TCF pathway and the inhibition of cell growth, possibly via FGF family of proteins 29 .Methylation of WNT10B has been found in the some of the cancer cell lines while its reversal has lead to over-expression of the WNT10B.However, the over-expression of WNT10B has lead to reduced cell growth in cancer, indicating a β-catenin independent component to be behind such a phenomena.Methylation of over-expressed WNT10B and synergistic work with FGF family of proteins later indicate the promotion of oncogenesis, as has been demonstrated in 29 .
In a more recent work, ASCL2 has been found to play a major role in stemness in colon crypts and is implicated in colon cancer 30 .Switching off the ASCL2 leads to a literal blockage of the stemness process and vice versa.At the downstream level, ASCL2 is regulated by TCF4/β-catenin via non-coding RNA target named WiNTRLINC1 31 .Activation of ASCL2 leads to feedforward transcription of the non-coding RNA and thus a loop is formed which helps in the stemness and is highly effective in colon cancer.At the upstream level, ASCL2 is known act as a WNT/RSPONDIN switch that controls the stemness 32 .It has been shown that removal of RSPO1 lead to decrease in the Wnt signaling due to removal of the FZD receptors that led to reduced expression of ASCL2.Also, low levels of LGR5 were observed due to this phenomena.The opposite happened by increasing the RSPO1 levels.After the drug treatment, it was found that ASCL2 was highly suppressed pointing to the inhibition of stemness in the colorectal cancer cells.Also, 32 show that by genetically disrupting PORCN or inducing a PORCN inhibitor (like IWP-2), there is loss of stem cell markers like LGR5 and RNF43, which lead to disappearance of stem cells and moribund state of mice.A similar affect can be found with ETC-1922159, where there is suppression of RNF43 and LGR5 that lead to inhibition of the Wnt pathway and thus the ASCL2 regulation.These wet lab evidences are confirmed in the relatively low ranking of the combination ASCL2-RNF43 via the inhibition of PORCN-WNT that leads to blocking of the stemness that is induced by ASCL2.Since ASCL2 is directly mediated by the WNT proteins, the recorded ASCL2-WNT10B combination showed low priority ranking of 488, 497 and 321 for rbf, laplace and linear kernels, respectively, thus indicating a possible connection between WNT10B and ASCL2 activation.WNT10B might be playing a crucial role in stemness.This is further confirmed by wet lab experiments in 33 , which show BVES deletion results in amplified stem cell activity and Wnt signaling after radiation.WNT10B has been implicated in colorectal cancer 29 .

PORCN-WNT inhibitors
The regulation of the Wnt pathway is dependent on the production and secretion of the WNT proteins.Thus, the inhibition of a causal factor like PORCN which contributes to the WNT secretion has been proposed to be a way to interfere with the Wnt cascade, which might result in the growth of tumor.Several groups have been engaged in such studies and known PORCN-WNT inhibitors that have been made available till now are IWP-L6 34 & 35 , C59 36 , LGK974 37 and ETC-1922159 38 .In this study, the focus of the attention is on the implications of the ETC-1922159, after the drug has been administered.The drug is a enantiomer with a nanomolar activity and excellent bioavailability as claimed in 38 .

Combinatorial search problem and a possible solution
We have already addressed the issue of combinatorial search problem and a possible solution in 39 and 1 .The details of the methodology of this manuscript have been explained in great detail in 39 & its application in 1 and readers are requested to go through the same for gaining deeper insight into the working of the pipeline and its use for published data set generated from ETC-1922159.In order to understand the significance of the solution proposed to the problem of combinatorial search that the biologists face in revealing unknown biological search problem, these works are of importance.Using the same code with minor modifications in 39 and 1 , it was possible to generate the rankings for 3 rd order combinations.100 genes were randomly selected from the list of down regulated genes, by the pipeline and a 3 rd order combination was generated from those 100 genes.The total number of gene combination with C 100 3 = 161700.Out of these the WNT10B associated 3 rd order combinations were selected, which account to a total of 4851 combinations.The goal of this manuscript is to analyse these 3 rd order ranked WNT10B associations.

Results & discussion
We present here the 3 rd order combinations associated with the WNT10B and represent them as WNT10B-X-X were X can be known or unknown factor from a list of genes that were affected after the administration of the ETC-1922159 drug.There are a total of 4851 combinations of randomly selected 100 genes from a list of 2500± genes.Out of these 100, WNT10B was one of them.Here we analyse some of the ranked combinations out of 4851 3 rd order interactions.Note that the rankings were generated using only the HSIC density index using the radial basis function kernel.Also, the rankings for a particular gene might change over different combinations and the biologists/oncologists are advised to cross check across the different tables presented.However, where possible, we report confirmatory results by the pipeline that fall in line with the published and known mechanism of a particular gene under consideration.Also, many of the combinations are yet to be tested and we make opennings for the deeper analysis and exploration of the combinations as future work.

SCO1-WNT10B-X combinations
The most important functionality of mitochondria is the production of ATP through respiration process and the regulation of the cellular metabolism.Illingworth 40 in a tutorial indicate -" The study of mitochondrial functionality is usually done via toxic compounds.Inhibitors help in distinguishing the electron transport system from the phosphorylation system and help in defining the redox carriers along the respiratory chain.If the chain is blocked then all the intermediates on the substrate side of the block become more reduced, while all those on the oxygen side become more oxidised.It is easy to see what has happened because the oxidised and reduced carriers often differ in their spectral properties.There are six kinds of poisons which might affect the mitochondrial functioning -• respiratory chain inhibitors • phosphorylation inhibitors • uncoupling agents • transport inhibitors • ionophores and • Krebs cycle inhibitors."The distribution of copper and its homeostasis plays a major role in many biological processes 41 and this is facilitated by the work of metal transporters and chaperones 42 , 43 & 44 .Disruption in pathways that transport copper can cause major damages in the form of metallostasis found in tumors 45 .The pathway is regulated by copper homeostasis genes and recent transcriptome analysis of copper homeostasis genes have shown the upregulation of SLC31A1, SCO and COX11 in colorectal cancer cases 46 .
It has been shown that human SCO1 and SCO2 have independent cooperative functions in copper delivery to cytochrome c oxidase (CcO) 47 , 48 & 49 .CcO is a multimeric protein complex with 13 subunits working in tandem to transfer electrons from reduced cytochrome c to molecular oxygen in the terminal step of the respiratory chain.It is embedded in the inner mitochondrial membrane (IMM) and composed of three mitochondrially encoded subunits (COX I -COX III) and ten nuclear encoded subunits.Additional factors are required for the proper functioning of the complex.Mutations in SCO1 and SCO2 that code for metallochaperone proteins with roles of copper delivery to COX, lead to severe deficiencies in the respiratory mechanism.Leary et al. 47 show that overexpression of SCO1 in a SCO2 patient background, and vice versa, has a dominant-negative effect on COX activity, indicating the non-overlapping functions of SCO1 and SCO2.Finally, Leary et al. 47 propose a model were COX17 a metallochaperone, transfers copper to SCO2, which in turn delivers it to COX II.SCO1 facilitates the latter interaction, thereby promoting the biogenesis of the copper site.The metallation of COX II occurs at an early stage of COX assembly and is required for the incorporation of this structural subunit into the assembling holoenzyme.To confirm the matter, different configurations of SCO1 have been found to be existant while interacting with COX II, for copper transfer 50 .
Armed with this information we begin with the analysis of the combinations of SCO1-WNT10B-X (X a known or unknown combination) that is presented in table 1.Since it is known that both WNT10B and SCO1 are implicated and upregulated in colorectal cancer cases, low rankings of these match with the fact that after the administration of the drug ETC-1922159 WNT10B and SCO1 were suppressed.In a majority of the rankings that we observe in table 1, most of them are assigned a very low rank (nearing to 1) in the randomly chosen list of down regulated genes.We analyse the functions of the 3 rd component X which might be either a known factor or an unknown factor.

X -known/unknown/untested factor with SCO1-WNT10B
Madan et al. 51 report that both WNT10B and SCO1 were down regulated by the administration of ETC-1922159.Out of the 100 randomly selected genes, XX172, an unknown factor was found to have a very low ranking of 1 by the pipeline.This points to the fact that this unknown factor is suppressed by the drug and can be a major factor in the propagation of colorectal cancer.The pipeline reveals its strength by pointing towards this unknown and unexplored factor and gives the oncologists/biologist an insight into the newly observed factor that might be contributing heavily in the colorectal cancer and is found to be highly suppressed after the drug treatment.Further wet-lab test might reveal major implications regarding XX172.Similarly, the unknown factors XX91 (ranked 21), XX81 (ranked 31), XX134 (ranked 51), XX16 (ranked 77), XX148 (ranked 171) and XX228 (ranked 199) out of the 4850± combinations of WNT10B-SCO1 combinations in randomly selected 100 genes show a similar behaviour and require further wet lab investigations based on the pipeline's in-RANKING USING HSIC -RBF KERNEL W.R.T WNT10B-SCO1  1 3 rd order interaction ranking using HSIC for radial basis function kernel.Total number of 3 rd order interactions in a set of 100 genes -161700.4851 3 rd order combinations for WNT10B associated work.Rankings for SCO1-WNT10B-X have been tabulated.

dication.
Mechanism of amplification of oncogenes is a property of many of the tumors.DM or double minute chromosomes are DNA segments containing amplified oncogenes and their frequency is high in tumor cases.Ji et al. 52 observe the amplification of ZNF572 in colorectal cancer cases via these DMs.After the administration of ETC-1922159 in different colorectal cancer cases, ZNF572 is found to be down regulated and its rankings along with SCO1-WNT10B is found to be extremely low (48) in the randomly selected set of 100 genes.Chemotherapeutic treatment of metastatic CRC is usually based on combination of 5FU antimetabolite drug and DNA binding agent Oxaliplatin.Jensen et al. 53 observed that ZNF502 was found to be down regulated in Oxaliplatin resistant cell lines indicating that associated gene that is resistant the drug in cancer cell line.However, Bash-Imam et al. 54 observed the upregulation of ZNF502 after treatment with 10μM of 5-FU in HCT-116 cells.After the treatment of ETC-1922159, the ZNF502 was found to be downregulated.Its combination with SCO1-WNT10B was confirmed to have lower rank (141) indicating possible direct or indirect combinatorial play in the Wnt pathway during the cancer stages.Similar interpretations could be found for ZNF594 and ZNF740.
RET is a member of GDNF family receptor complex and is a receptor for the different kinds of ligands.It binds the ligands to form a multi-receptor complex that includes GFRα (GFRA) proteins to activate receptor tyrosine kinases in a variety of signaling.However, it has been found to be a tumor suppressor in colorectal cancer case and is methylated 55 .Moreover, the functional RET receptor complex includes RET and one of four glycosylphosphatidylinositol-anchored co-receptors, designated GDNF-α receptors, GFRα1, GFRα2, GFRα3 (GFRA3), and GFRα4 and Luo et al. 55 observe that GFRA3 is expressed in the colorectal cancer cases.GFRA3 was found to be downregulated on treatment with the ETC-1922159 and might have been highly expressed in the colorectal cancer case.Its consequent downregulation along with SCO1-WNT10B gets one of the lowest priority of 2 in a randomly selected set of 100 genes.
DHX9 is a RNA helicase that belongs to the family of DExD/Hbox family and is found to be involved in the transcriptional regulation in cancer 56 & 57 .DHX9 has been found to be upregulated in colorectal cancer cases and after the treatment of the ETC-1922159, it was downregulated.The combination of SCO1-WNT10B-DHX9 acquires lower priority of 4, indicating a possible potential synergistic affect (within the selected 100 genes).
ABCA2 belongs to the category of ABC transporters that play an essential role in the development of resistance by the efflux of anticancer agents outside of cancer cells 58 .Hlavata et al. 58 observed that ABCA2 had no significant change/affect in colorectal cancer cases.Kobayashi et al. 59 found ABCA2 to be downregulated in colorectal cancer case.Contrary to this, ETC-1922159 affected cancer cells showed down regulation of ABCA2 and the pipeline points towards the low rank (of 5) associated with the combination of WNT10-SCO1.This entails further investigation in wet lab regarding the functionality of ABCA2.
PABPC1L (as recorded by 51 ) or the cytoplasmic 1-like poly(A)binding protein 60 , belong to a family of multifunctional proteins PABPs which regulate and stabilize the mRNA translation.They are observed to be helpful in the transportation of the mRNA also from the nucleus and there exists a nucleic version of PABP also 61 .PABPC1 contains four non identical RRMs that are joined with the main PABC domain and separated by a linker.Though their mechanism of import and export of mRNA has not been understood deeply, a few models/observations have been made.Association with translation complexes or mRNA at cytoplasmic level inhibits the transportation of PABPC1 to the nucleus.Contrary to this, its release leads to bindation with Importin-α/ β complex that facilitates in nuclear import.There are various mechanisms by which PABPC1L might be exported out of the nucleus.Modes of export involve association with (1) cytoplasmic eEF1α (2) mRNA as well as TAP that mediates the mRNA export and (3) Paxillin via CRM1 pathway.In gastric cancer cases, PABPC1 has been found to be an oncoprotein 62 and observed to exert carcinogenesis.In colon cancer mutations in PABPC1 have been found in minor tumor clones 63 .Genomic correlates of immune-cell infiltrates in CRC have found the existence of significantly mutated CRC genes 64 .PABPC1L was found to be downregulated after the treatment of ETC-1922159 and a low ranking (of 6) is associated with SCO1-WNT10B.In CRC, PABPC1L might be mutated and work as oncoprotein and facilitate the transmission of other oncogenic factors.The administration of the drug down regulated the gene in drug treated CRC cells and the low ranking confirms at in silico level the suppression at cytoplasmic level.Additionally, since RRM1 is linked to the PABPs, the pipeline found a similar low ranking (of 71) with the combination SCO1-WNT10B (See table 1).Discussion on RRM1 combination will be done a little later.
LEFTY1 belongs to the family of LEFTY genes involved in the left-right determination 65 .Yashiro et al. 65 study the LEFTY pair for human cases in colon.They show that the LEFTY1 is highly expressed in colorectal cancer as well as normal colon and point that it is not easy to correlate the two phenomena.Naba et al. 66 also identify LEFTY1 in primary colon tumor signatures.After the ETC-1922159 drug, LEFTY1 was found to be down regulated and the pipeline shows this confirmatory result by assigning a low rank (of 8) to LEFTY1 along with SCO1-WNT10B.Again, association of SCO1-WNT10B-LEFTY1 needs to be explored at wet lab level.
Tetratricopeptide repeat protein 26 (TTC26, also known as Intraflagellar transport protein 56), has found to be involved in cilia formation in zebrafish 67 .It has been found to impair hedgehog signaling on mutation 68 .Not much is known about TTC26 in case of colorectal cancer and after the ETC-1922159 treatment, it was found to be down regulated.The pipeline shows a very low ranking (of 9) in a set of 100 genes which confirms the wet lab results on drug treatment and further investigation of the role of TTC26 in colorectal cancer is required.
CMR1 is found to be involved in response to DNA replication stress which contributes to genomic instability.Gallina et al. 69 also observe that, its human orthologue, WDR76 responds to DNA damage via association with CCT to recover from genomic instability via regulation of turnover of sumoylated and phosphorylated proteins.Human WDR76 is known to interact with XRCC-5/6 which are known to mediate or stabilize RAD51 during the homologous recombination (HR) process.WDR76 was found to be down regulated by the ETC-1922159 treatment and the pipeline assigned a low ranking of 10.This suppression of the WDR76 indicates the drug's affect in destabilizing the colorectal cancer cells whose stability might be sustained by the WDR76 or its mutated version which might have lead to propagation of CRC, has been suppressed.Further investigation is necessary.
Recently, UHRF1 is a newly found gene that translates to (Ring)-finger domain containing associated protein which is re-quired for the survival and tumorigenicity 70 .Taniue et al. 70 show that UPAT a long noncoding RNA UPAT interacts with UHRF1 to interfere with the β-TRCP that is involved in the ubiquitination process.This interaction is basically the inhibition of the degradation of UHRF1.Wang et al. 71 also show that UHRF1 is upregulated in colorectal cancer case and facilitates in cell proliferation and metastasis via suppression of p16 nk4 .Similar findings have been made in 72 , 73 & 74 .Consistent with these, the UHRF1 was down regulated in cancer cells, on the treatment with ETC-1922159.The pipeline confirms this with a low rank of 12 along with WNT10B-SCO1 combination.
Mitochondrian DNA transcription happens via the phenomena of the formation of the D-Loop structure 75 .The transcription requires mitochondrial RNA polymerase and Tfam, a DNA binding stimulatory factor.In presence of this Tfam and the mitochondrial RNA polymerase, the mitochondrial transcription specificity factors TFB1M and TFB2M enhance the transcription.Gleyzer et al. 76 show that these are further controlled by NRF-1/2 and PGC-1 coactivators.Furthermore, Cyclophilin-D is found to interact with TFB2M for mitochondrial transcription 77 .TFB1M and TFB2M is found to be significantly reduced on FOXO3a activation 78 .While in colorectal cancer cases FOXO3a is found to be highly inhibited 79 & 80 .In colorectal cancer cases TFB2M is found to be upregulated and after the ETC-1922159 treatment, it was found to be down regulated.The low ranking of 15 of TFB2M by the pipeline with WNT10B-SCO1 suggests the effectiveness of the framework to assign a low priority to the suppression of TFB2M by the drug.
It has been found that Nodal promotes the self-renewal of human colon cancer stem cells 81 and it signals through activation of receptor complex, including ALK-4/7.ALK-7 is the protein that is encoded by ACVR1C.ACVR1C plays a major role for embryogenesis and left-right patterning in the mouse 82 .However, Nagaoka et al. 83 show that the modulation of the β-catenin pathway by Cripto-1 in response to Wnt3a stimulation is independent of the Nodal/Alk4/Alk7 signaling pathway.Nevertheless, ACVR1C is known to be activated in colorectal cancer case as has been observed in 81 .After the administration of ETC-1922159, ACVR1C was found to be down regulated and the pipeline assigns this down regulation with a low priority of 18.
Importin-β family proteins are transport receptors that help in the transportation of proteins and RNAs in and out of the nucleus via the nucleus pores.Kimura and Imamoto 84 give a detailed review of the importins.It was observed that Importin-α/ β was responsible in the transportation of PABPC1L (the cytoplasmic 1-like poly(A)-binding protein 60 that belong to a family of multifunctional proteins PABPs which regulate and stabilize the mRNA translation.The ranking of the PABC1L was found to be low after its suppression by ETC-1922159.Since Importin-α/ β facilitate the transportation of PABPC1L, it is likely that it is also suppressed after the drug treatment.Furthermore, Importin-9 is implicated in transport of ARID3A into the nucleus where it forms a complex with ARID3B which is responsible for stemness in cancer.Liao et al. 85 show that deletion of LET7 which suppresses ARID3A and Importin-9 and is thus a tumor suppressor, leads to initiation of cancer via ARID3B.In their experiment 51 , show that the drug treatment suppresses Importin-9 that is IPO9 and thus the stemness property of the colorectal cancer cases.This is indicated by the pipeline with a low rank of 19.Similarly, IPO11 gets a low rank of 103.
Lysophosphatidic acid receptors (LPAR6) are commonly overexpressed in HCC and supports tumorigenicity in the same 86 .LPAR are known to be implicated in tumor metastasis and p2y5 has been found to be a LPAR and was designated as LPAR6 87 .Not much is known about the LPAR6 in colorectal cancers and it was found to be down regulated after the administration of the drug.The pipeline correlates with the down regulation of the LPAR6 and shows a ranking of 20 after the drug treatment.
Uchida et al. 88 show on knockdown, that CDCA2 or Cell division cycle associated 2 inhibit the cellular proliferation by arresting cell-cycle progression at G1 phase and upregulating the cyclin-dependent kinase inhibitors (p21 cp1 , p27 kp1 , p15 nk4b , and p16 nk4 ), in human squamous cell carcinoma.Earlier, we saw that also Wang et al. 71 show that UHRF1 is upregulated in colorectal cancer case and facilitates in cell proliferation and metastasis via suppression of p16 nk4 .It is evident that overexpression of CDCA2 leads to repression of p16 nk4 and cell proliferation.Consistent with these, Kwon et al. 89 show that CDCA2 is upregulated in the colorectal cancer case.Wang et al. 90 also show the upregulation of CDCA2 in recurrent and non-recurrent types of CRC.After the treatment of ETC-1922159 in the subtype of colorectal cancer cells, CDCA2 was down regulated and facilitated in inhibition of growth of cancer cells 51 .This confirmatory result has been indicated by the pipeline with a rank of 23.
Matsumura et al. 91 identified that the inactivation of the Klotho (KI) gene in mice showed disorders that resemble human aging.Knockdown of Klothoγ (KLG) or LCLT has been implicated in immortalization of normal human colonic epithelial cells 92 .Reversibly, this means that overexpression/up-regulation of LCLT would not immortalize the colonic cells.Kim et al. 92 show that the KLG/LCTL knockdown lead to disappearance of KLA (Klothoα) which is a tumor suppressor and canonical Wnt antagonist.In colorectal cancer, LCLT might be highly regulated and after the ETC-1922159 administration, the LCLT was found to be downregulated.The pipeline points to the suppression of LCLT by assigning a low ranking of 27.
Liao et al. 93 show via machine learning methods that DEP (Dishevelled/EGL-10/Pleckstrin) domain containing (DC) proteins are expressed in HCC.It has been found to be highly expressed in colorectal cancer 94 and 95 .After the administration of the ETC-1922159, DEPDC1B and DEPDC7 were down regulated and the pipeline indicates a low rank of 28 and 119 for the same down regulation, respectively.Exosome is a highly conserved complex that mediates the degradation and processing of multiple classes of RNA Liu et al. 96 .It is composed of 9 subunits marked EXOSC-1/2/.../9.In cancer cells it might be that the exosome is disrupted from its execution of the degradation of the multiple tumor causing RNAs.EXOSC3 (RRP40) and EX-OSC5 (RRP46) show low rank of 195 and 30 and were found to be suppressed after the treatment of ECT-1922159.
ZIC1 (Zinc finger of the cerebellum) is found to be highly suppressed via hypermethylation in colorectal cancer and is known to be a tumor suppressor Gan et al. 97 .However, in breast cancer cases as Nakakido et al. 98 show, the expression of ZIC1 is found to be regulated by the knockdown of PIGX as well as RCN1 (reticulocalbin 1) and (reticulocalbin 2) RCN2.Thus upregulation or overexpression of RCN2 negatively regulates ZIC1 for cancer proliferation.RCN2 was found to be overexpressed in gastrointestinal cancer cells lines 99 .After treatment with ETC-1922159 in colorectal cancer samples, RCN2 was found to be down regulated and the pipeline assigns this down regulation to a rank of 32 along with WNT10B-SCO1.
COLEC11, has been found to be differentially expressed in colorectal tumor cases 100 .Not much is known about COLEC11 in colorectal cancer and it was found to be down regulated after the treatment of ETC-1922159 drug and indicated with a low ranking of 35 along with WNT10B-SCO1 combination.
Binding of SRL (Sclerotium rolfsii lectin) to human colon has been found to induce cell apoptosis and suppression of tumor growth 101 .SRL treatment also downregulated POLA2 101 .POLA2 encodes DNA polymerase α subunit 2 and pairs with PARP to do a DNA damage survey.Consistent with these POLA2 should be upregulated in colorectal cancer and reversibly after treatment with ETC-1922159 it was found to be down regulated.The pipeline assigns a low rank of 36 for this down regulation.
CDK5RAP2 (CDK5 regulatory subunit-associated protein 2) has been found to function in centrosome to spindle pole attachment and DNA damage response Barr et al. 102 .Mutations in CDK5RAP2 have caused premature depletion in neural stem cells and thus microcephaly.Its role in colorectal cancer is not much known.However, CDK5RAP2 was found to be down regulated after the drug treatment and assigned a low rank of 38 for down regulation.
Yu et al. 103 have elucidated the combination of various WNTs and (Frizzled) FZD in ventricular septal defects.In one of the observations, they find the combination of WNT10B and FZD7 to be very high.However, 104 did not include the WNT10B for the combinatorial study.FZD7 has been found to be highly expressed in colorectal cancer cells 105 & 106 .Ater the ETC-1922159 treatment, FZD7 was found to be highly suppressed.Further ranked confir-mation in the 3 rd order combination of SCO1-WNT10B-FZD7 is depicted by a very low priority of 39.
SGOL1 or Shugoshin-like 1 is a protein encoded by SGOL1 gene and is a key protein that protects sister chromatids from premature separation during mitosis 107 .Kahyo et al. 107 found SGOL1 to be down regulated in colorectal cancer cases.SGOL1 are known to be tumor suppressor gene and found to be mutated in colorectal cancer cases also 108 .Mutations in SGOL1 or its down regulation means there is no prevention of premature separation which leads to different kinds of instability as can be found by presence of microsatellite instability in colorectal cancer cases.After treatment of ETC-1922159, SGOL1 has been found to be down regulated in treated colorectal cancer cells.This points to the fact that mutated versions of SGOL1 might have been suppressed as wild type SGOL1 would have been upregulated in cured cells.The pipeline points to this down regulation with a rank of 40.
TP73 or p73 is a tumor suppressor belonging to the family of p53 transcription factors 109 .It is known to be upregulated in colorectal cancer case 110 .Dysfunction in p73 leads to mitotic abnormalities causing polyploidy and aneuploidy which contributes to tumorigenesis 111 .Consistent with these, after the ETC-1922159 treatment, TP73/p73 was found to be down regulated.Probably the dysfunction of TP73 was suppressed after the drug administration.A low rank of 41 confirms the pipeline's indication of down regulation.
PAK4 has been found to be expressed in breast cancer cells and depletion of the same modifies cell adhesion dynamics 112 .Dart et al. 112 show that reduced expression of PAK4 leads to loss of RHOU and RHOU is ubiquitinated via RAB40A-CULLIN5 complex.Expression of PAK4 rescues RHOU ubiquitination.Also RHOU expression assists PAK4 expression.Thus RAB40A is often found to be underexpressed in breast cancer.Furthermore, knockdown of PAK4 inhibits proliferation of mutant KRAS colorectal cancer cases 113 .Its expression would lead to proliferation.Analogous to the breast cancer case, RAB40A might be underexpressed in colorectal cancer case as PAK4 helps in proliferation.However, in colorectal cancer cases mutations in RAB40A could be present which do not help in targeting RHOU degradation and thus via PARK4 and RHOU are expressed in colorectal cancer case.Following this line of thought, ETC-1922159 administration lead to down regulation of RAB40A.It might be that the mutated versions of RAB40A have been suppressed after the drug treatment.The pipeline indicates the low rank of 42 apropos this down regulation.
TGIF2 is found to be expressed in colorectal cancer case 114 and is actually a transcriptional repressor that works by recruitment of HDAC3 (histone deacetylase 3).After administration of ETC-1922159, TGIF2 was found to be down regulated and this down regulation was assigned a value of 43.
PDRG1 is a novel p53 and DNA damage-regulated gene that has been found to be up regulated in colon cancer cases and knock down of the same has shown marked slowdown in tumor growth 115 .Jiang et al. 115 showed that PDCD7 (programmed cell death 7) has been found to be interacting with PDRG1 and implicates PDRG1 in cell growth regulation via involvement in apoptosis and cell cycle regulation.After the treatment of ETC-1922159 drug PDCD7 was found to be down regulated.This down regulation indicates the inactivation of PDRG1 which thereby slows down tumor growth.The pipeline points to this down regulation via a low rank of 45.
Glutathione peroxidase 1 or GPX1 is an antioxidant enzyme that helps in protecting cells from oxidative stress via reduction of hydrogen peroxide to H 2 0. Polymorphisms of the gene have been related to increased risk in cancer 116 and Goldberg et al. 117 show that GPX1 has a loss of heterozygosity at later stages of colon carcinogenesis.Consistent with these GPX1 was found to be down regulated after the ETC-1922159 drug, indicating that the drug might be restricting the process of oxidative stress by suppressing the polymorphed GPX1.The pipeline assigns this down regulation with a value of 47.
Kinesin superfamily (KIF) members share a highly conserved protein family and are known to be involved in motor binding as well as transportation of vesicles and organelles 118 .Liu et al. 118 show that KIF20B is known to be overexpressed in colorectal cancer case.After the ETC-1922159 administration KIF20B is found to be down regulated and the pipeline points to this observation by assigning a low rank of 49.
LAT2 (Large neutral amino acids transporter small subunit 2) is a family of LAT proteins that is encoded by SLC7A8 gene.These are Na + -independent transporters that deliver neutral amino acids into cells and have been responsible for cellular leucine uptake, protein translation and cell growth 119 .The LAT2 is found to be expressed in colorectal cancer and is regulated by the expression of MYC 120 .Satoh et al. 120 show that the knockdown of the MYC that is involved in metabolic reprogramming, lead to decrease in the levels LAT2 (SLC7A8).SLC7A8 was found to be down regulated in colorectal cancer cells treated with ETC-1922159 and the pipeline points to this down regulation with a low rank of 52.
The role of IL-17 (Interleukin-17) family is known to be controversial in CRC, however there are cases were it has been reported to be a prognostic marker for colorectal cancer Lin et al. 121 & 122 .A homologue of the family, IL-17D a novel cytokine has been discovered 123 and found to play a role in many of the cancers.In cells treated with ETC-1922159, IL-17D was found to be down regulated and reversibly it must have been regulated in the colorectal cancer cases.A low ranking of 55 along with WNT10B-SCO1 relates to the down regulation after the drug treatment.
ING5 (Inhibitor growth protein 5) has a controversial role and sometime it is found to work as a tumor suppressor by binding to p53 and enhancing p53 activity 124 while at other times it has been reported to play a role of oncogene at cytoplasmic level but not at nuclear level 125 .Tallen and Riabowol 126 claim overexpression of ING5 in colorectal cancer cases.After treatment with ETC-1922159, ING5 was found to be down regulated and in context of the WNT10B-SCO1 combination, the pipeline indicates the suppression with a rank of 56.Probably, as Zheng et al. 125 suggest, ING5 is up regulated at cytoplasmic level in colorectal cancer cases.MNS1 (meiosis specific nuclear structural 1) was found to be down regulated via knockdown of KLK6 127 .KLK6 is observed to be highly regulated in colorectal cancer cases and facilitates in the invasion-metastasis formation via specific downstream network of miRNA-mRNA effectors.Furthermore, oxaliplatin treatment lead to down regulation of MNS1 in colorectal cancer cell lines 128 .Not much is known about MNS1 in colorectal cancer case and after treatment with ETC-1922159 in colorectal cancer cells, it was found to be down regulated.This down regulation is assigned a rank of 57.Further investigations are needed for MNS1 with respect to WNT10B and SCO1.
DDN or dendrin is a neural protein that is usually found to be functional in brain and kidney.The authors are unclear how and why DDN was chosen for study after the administration of ETC-1922159 in 51 .However, DDN was found to be down regulated after the administration of the drug and the pipeline assigned a ranking of 61 for the downregulation.
CBX5(Chromobox Protein Homolog 5) also known as heterochromatin protein 1α or HP1α in humans is known to act as a gene silencer 129 .Unphosphorylated STAT5 is a tumor suppressor that inhibits multiple oncogenes by binding to CBX5/HP1α to stabilize heterochromatin 130 .This formation of heterochromatin and involvement of epigenetics leads to tight packing of the genes and consecutive folding of DNA such that transcription of oncogenes is inhibited.Reduction in HP1α/CBX5 levels have been found to instigate cancer progression 131 .CBX5 was found to the down regulated in colorectal cancer cells after the treatment of ETC-1922159 51 .Mechanistically, over expression of CBX5 should suppress the tumor progression.This points to the fact that mutations/defects in CBX5 might have been present in colorectal cancer cases which could not help in stabilization of heterochromatin and the administration of the drug lead to its down regulation.This down regulation is indicated by a low rank of 64 by the pipeline.
C4orf46 (Chromosome 4 open reading frame 46) was found to be down regulated after the administration of the drug ETC-1922159.Not much is known about the role of C4orf46 in colorectal cancer.The pipeline indicates a down regulation with a low rank assignement of 69.
FAM131B (Family with sequence similarity 31 member B) has been found make fusions with BRAF and is involved in some of the cancers like pilocytic astrocytoma 132 .Oncogenic fusions like FAM131B-BRAF are found mostly in brain tumors.Not much is known about FAM131B in colorectal cancer and administration of drug in experiments 51 showed that FAM131B is down regulated in treated colorectal cancer cells.Consistent with these, the pipeline points to this down regulation with a rank of 70.Family with sequence similarity 168 member A (FAM168A) was found to under go somatic mutations in colorectal cancer case 133 .A low rank of 191 was allocated by our pipeline and indicates to the observed down regulation after administration of ETC-1922159.RRM1 (Ribonucleoside-diphosphate reductase large subunit) 134 helps in the formation of deoxyribonucleotides prior to DNA synthesis.The role of RRM1 is also known from its association with PABC1L (see earlier discussion in the section of SCO1-WNT10B-X combinations).PABPC1L (as recorded by 51 ) or the cytoplasmic 1-like poly(A)-binding protein 60 , belong to a family of multifunctional proteins PABPs which regulate and stabilize the mRNA translation.They are observed to be helpful in the transportation of the mRNA also from the nucleus and there exists a nucleic version of PABP also 61 .PABPC1 contains four non identical RRMs that are joined with the main PABC domain and separated by a linker.We know that PABC family is down regulated after the treatment of ETC-1922159 and expect that RRM1 should also be down regulated after the drug administration.This is confirmed by experiments in 51 .Additionally, wild type RRM1 is known to be highly expressed in colorectal cancer 135 & 136 .Consistent with these, after the treatment of ETC-1922159, RRM1 was found to be down regulated and the pipeline points to this via a low rank of 71 along with SCO1-WNT10B.
Long non-coding RNA facilitate in protein coding and non coding and recently, aberrations in the same have been found to promote various cancers.FOXD2-AS1 has been implicated in gastric cancer 137 as well as the non-small lung cancer 138 .FOXD2-AS1 has recently been found to be expressed in colorectal cancer and promotes the same via regulating EMT and Notch signaling pathway 139 .Consistent with the recent finding and the experiments of ETC-1922159 drug administration 51 , FOXD2-AS1 was found to be down regulated after the treatment in colorectal cancer cells.A low rank of 72 was assigned to FOXD2-AS1 along with WNT10B and SCO1.Further investigation for FOXD2-AS1 is needed at in vitro/in vivo level.
MARVELD1 (MARVEL domain containing protein 1 and unfortunately not the MARVEL comics) has been found to bind to the importin-β1 (IPOβ1, a kind of nucleocytoplasmic protein that helps in transportation of proteins between the nucleus and the cytoplasm).It has been found to be MARVELD1 shows decreased expression in tumor cases and binds to IPOβ1 140 .However, the functionality of MARVELD1 in colorectal cancer is not known and Madan et al. 51 report the down regulation of the same after the administration of ETC-1922159 drug.The pipeline assigns a rank of 75 for this reported down regulation.
NUBPL (or the nucleotide-binding protein-like) encodes the iron-sulfur (Fe/S) protein (IND1) and has a role in the assembly of mitochondrial complex 1 141 .Mitochondrial complex 1 is a member of the mitochondrial respiratory chain.Recently, NUPBL has been found to be highly expressed in colorectal cancer cases and Wang et al. 142 show that this is due to the induced affect of NUPBL expression in EMT.EMT is a major process which helps in metastasis in cancers.Consistent with these, NUBPL was found to be down regulated after the treatment of ETC-1922159.This down regulation is assigned a low rank of 78 which is in line with the findings in 51 .
TAMM41 or mitochondrial translocator assembly and maintenance protein was found to be down regulated after treatment of ETC-1922159.Currently, the authors are not much aware of the affects of TAMM41 in colorectal cancer cases, however, the reversible picture is that TAMM41 is highly regulated in colorectal cancer cell and after the administration of the drug the down regulation was assigned a value of 80.
ARHGAP11B has been found to be highly expressed in the development of mouse and human neocortex 143 .However, its role in colorectal cancer is not known explicitly.Madan et al. 51 report the down regulation of this gene after the administration of ETC-1922159.We do not know why this is so, however the pipeline indicates this down regulation with a low rank of 85.
RRS1 (Ribosome biogenesis regulatory protein homolog) is been found to be a highly conserved gene, deletions/mutations of which lead to transcription repression of ribosomal protein 144 .It has also been found to up regulated in colorectal cancer 145 , 146 & 147 , indicating the expression of ribosomal proteins which might be effective in tumor.Consistent with these, RRS1 was down regulated after the treatment of ETC-1922159 and the pipeline pointed to the down regulation with a low rank of 89 along with SCO1-WNT10B combination.
PPT1 (Palmitoyl-protein thioesterase 1) is a small glycoprotein involved in the catabolism of lipid modified proteins during lysosomal degradation.Defects in these genes have been implicated in infantile neuronal ceroid lipofuscinosis 148 & 149 .Tsukamoto et al. 150 observe overexpression of CLN1 in colorectal cancer which encodes PPT for removal of fatty acids from fattyacylated cysteineresidues in proteins 148 .Consistent with these, ETC-1922159 treatment of colorectal cancer cells lead to the the down regulation of PPT1 and the pipeline indicates this with a low rank of 91.
Cortactin-binding protein 2 is encoded by CTTNBP2 and has been found to be up regulated in APC driven tumorigenesis Gaspar et al. 151 .Tuupanen et al. 152 observe mutations in CTTNBP2 in colorectal cancer cases.Nehrt et al. 153 also somatic mutations in colon cancer.After the treatment of ETC-1922159, CT-TNBP2 was found to be down regulated and this is indicated by the pipeline with a low rank of 93.
MEGF8 encoded Multiple Epidermal Growth Factor-like Domains 8, is a single pass membrane protein that facilitates cell communication and developmental regulation 154 .MEGF8 has been found to be significantly associated with colorectal cancer cases in 155 .Mechanistic role of MEGF8 has not been explored much in colorectal cancer and after the administration of the drug, it was found to be down regulated.The pipeline indicates this down regulation with a low rank of 97.
Hydroxyacyl-Coenzyme A dehydrogenase is encoded by gene HADH and mutations in the same have been found to cause hyperinsulinemic hypoglycemia 156 .Deficiency in HADH can lead to a rare condition where body stops converting fat into energy.The authors are not aware of HADH in colorectal cancer and the pipeline indicated a low rank of 107 along with SCO1-WNT10B.After the administration of the drug, HADH was found to be down regulated in the treated colorectal cancer cells with ETC-1922159.
ATAD3A (ATPase family AAA domain containing 3A) was found to interact with WASF3 which is a metastasis promoting gene 157 .ATAD3A is a mitochondrial membrane protein and Teng et al. 157 show that knockdown of ATAD3A lead to decreased levels of WASF3.Furthermore, silencing of ATAD3A causes loss of invasion and suppression of tumor growth.Consistent with these, administration of ETC-1922159 lead of down regulation of ATAD3A and the pipeline assigns a low rank of 110.
Mitochondrial Intermembrane Chaperone TIMM9 has been found to be over expressed in gastric cancer cases 158 .Encoded protein are involved in the transportation of the membrane proteins into the mitochondrial inner membrane.TIMM9 was found to be up regulated in colorectal cancer case 145 .Consistent with these, our pipeline indicated the down regulation of TIMM9 by ETC-1922159 51 , with an assignment of low rank of 124.
TARS2 has been found to be implicated in epilipsey 159 .Its role in colorectal cancer is not much known and after ETC-1922159 treatment TARS2 was found to be down regulated.This down regulation is pointed to with a rank of 133 by the pipeline.
MTHFD2L (NAD-dependent methylenetetrahydrofolate dehydrogenase 2-like protein) 160 functions within the inner mitochondrial membrane.MTHFD2L is a part of mitochondrial pathway and facilitates in the conversion of folate to formate.Mitochondrial folate-coupled metabolism plays role in cell proliferation and MTHFD2L has been found to be highly expressed in many tumors 161 & 162 .Consistent with these MTHFD2L was found to be expressed in colorectal cancer cells and after the administration of ETC-1922159 it was down regulated 51 .Our pipeline allocates a low rank of 136 for this down regulation.Similarly, another variant MTHFD1L was allotted a low rank of 157.
We earlier saw the role of PDCD7 were 115 showed that PDCD7 (programmed cell death 7) has been found to be interacting with PDRG1 and implicates PDRG1 in cell growth regulation via involvement in apoptosis and cell cycle regulation.PDRG1 has been found to be up regulated in colon cancer cases.A variant of PDCD7, i.e.PDCD4, also inhibits migration and invasion in colorectal cancer.Silencing of HNRNPC lead to the inhibition of migration and invasion in T98G cells, thus supporting the fact that HNRNPC regulates invasion and metastasis via regulation of PDCD4 163 .HNRNPC (Heterogeneous nuclear ribonucleoproteins C1/C2) 164 is usually found to be expressed in fetuses and lead to birth defects Zhang et al. 165 .Nevertheless, after the ETC-1922159 treatment, HNRNPC was found to be down regulated and the pipeline assigned a low rank of 137.
Overexpression of GINS has been found in colorectal cancer 166 .Furthermore, PSF3 which is a component of the tetrameric complex GINS, has a major role in colon cacner cell proliferation 167 .Consistent with these, ETC-1922159 administration lead to GINS3 suppression 51 and our pipeline allocated a low rank of 149.
METTL16 (methyltransferase-like protein 16) is known to bind with metastasis associated lung adenocarcinoma transcript 1 MALAT1 which is a cancer promoting long noncoding RNA.This binding happens via a triple RNA binding helix element as Brown et al. 168 have observed.MALAT1 is a long non coding RNA whose 3' functional motiff plays a role in the cell proliferation, invasion and metastasis in CRC 169 .Yeon et al. 170 show frame shift mutations in METTL16 in cases of colon cancer.Consistent with these, ETC-1922159 induced inhibition in cancer growth and METTL16 was found to be down regulated.Probably, MALAT1 must also have been down regulated as it works in combination with METTL16.This needs to be verified in vitro/in vivo.Our pipeline also indicated a low rank for this down regulation with a rank of 151.A variant METTL12 171 was also found to be down regulated and the pipeline assigned a low rank of 174.However, how METTL12 plays a role in colorectal cancer needs to be investigated and research is ongoing.
SEC31 is a protein in yeasts essential for endoplasmic recticulum-golgi body transport 172 .Its homologue SEC31A and SEC31B are prevalent in humans.In human intestinal epithelial cells, SEC31 depletion was shown to causes defective epithelial polarity and organization on permeable supports 173 .Sec13-Sec31 heterotetramer 174 is thought to link with a pre-budding complex and drive the membrane deformation to form COPII vesicles 175 .SEC24C is an essential component of COPII and a potential marker for colorectal cancer 176 .Consistent with these, SEC31B was found to be down regulated after ETC-1922159 and our pipeline points to this with a low rank of 159.
Similar to role of SCO1 in the respiratory chain reactions in mitochondria, BOLA3 has been found to be play a role in mitochondria.Iron-sulfur (Fe-S) clusters in the mitochondria have been found to play crucial role in many of the cellular processes 177 .Genes NFU1 and BOLA3 (and encoded proteins) facilitate in the formation of complexes along with other factors that help in the biogenesis and stabilization of the Fe-S centers for assembly of respiratory chain complexes in mitochondria and normal maturation of lipoate-containing 2-oxoacid dehydrogenases 178 , among the various other processes.Mutations in NFU1 and BOLA3 have been found to cause genetic diseases with defects in mitochondrial Fe-S centers 179 .Expression of BOLA3 was found to be significantly altered in colorectal cancer cases 180 and down regulated after the ETC-1922159 treatment in colorectal cancer cells 51 and our pipeline indicates this down regulation with a low rank of 188.
Tubulin alpha-1B chain (TUBA1B) was found to be significantly expressed in colorectal cancer cases 181 .MKI67 and TUBA1B were found to be expressed in cycling LGR5 + intestinal stem cells 182 .LGR-4/5/6 is known to work RNF families to inhibit the FZD families and thus inhibit the Wnt signaling.Along with RSPO, the signaling is up regulated as LGR-RNF go through degradation process.TUBA1B was found to be down regulated after the administration of ETC-1922159 and our pipeline assigned the down regulation with a low rank of 202.
ACTL6A (Actin-like protein 6A) was found to be up regulated in HCC and play major role in metastasis and EMT of HCC 183 .It has been found to be co-amplified with p63 in squamous cell carcinoma and is a poor proginator 184 .Also, ARID1A normally targets SWI/SNF complexes and acts a tumor suppressor in colon cancer 185 .Finally, ACTL6A prevents SWI/SNF chromatin-remodelling complexes to regulate many of the differentiation genes to maintain epidermal progenitor state 186 .It might be that in colorectal cancer case ACTL6A is highly active and prevents the prevents SWI/SNF for regulation of oncogenes.This needs verification, however, ACTL6A was found to be down regulated after the treatment of ETC-1922159 and this down regulation was allotted a low rank of 216.
GC-rich sequence DNA-binding factor (GCFC2) factor were found to be differentially expressed in celecoxib treated hereditary nonpolyposis colon cancer patient cells 187 .GCFC2 was also found to be down regulated after the treatment of ETC-1922159 in colorectal cancer cells 51 .Our pipeline assigned a low rank of 226 regarding this down regulation.
Interferon regulatory factor 8 (IRF8) is a transcription factor that promotes regulation of lineage commitment.It has been known to have an inverse relation with colorectal cancer metastasis 188 via promotion of apoptosis and had been found to be suppressed in colorectal cancer cases.Deficiency in IRF8 promotes inflammation-mediated colon tumorigenesis 189 .Given this case, mutations in IRF8 could lead to tumorigenesis and administration of ETC-1922159 might have caused the inhibition of tumor growth where mutations IRF8 would have been present.Our pipeline suggests the down regulation of probable mutated IRF8 in treated colorectal cancer cells with a low rank of 227.
PAX-interacting protein 1 (PAXIP1-AS2) is known to play role in genomic stability and chromatin condensation, and has been found to play a role in colorectal cancer case 190 .After treatment of ETC-1922159, PAXIP1-AS2 was found to be down regulated in colorectal cancer and our pipeline assigned a low rank of 228.
Nucleoporin 160 (NUP160) 191 is one of the proteins that make up for the nuclear pore complex 192 which helps in nucleoplasmic transport.In a proteomics approach Albrethsen et al. 193 report down regulation of NPC which involves NUP160, indicating cellular and nuclear crisis.Mutations in NUP160 and overall NPC might be a play a role in colon cancer.However, Shitashige et al. 194 show that NPC plays major role in regulating Wnt pathway.Consistent with these, our pipeline assigned a low rank of 236 for the down regulation of NUP160 in colorectal cancer cells treated with ETC-1922159.
CD3EAP encodes DNA-directed RNA polymerase I subunit RPA34.CD3EAP (CD3e antigen, epsilon polypeptide associated protein) is also known by the name of ASE-1 (Anti Sense ERCC1) 195 increased polymorphisms of which have been associated with increased risk of colorectal adenomas and carcinoma in a Norwegian cohort 196 .However, in a Danish study CD3EAP was not found to play any role in colorectal cancer 197 .CD3EAP polymorphisms has been found to be associated in chronic atrophic gastritis also 198 .After administration of ETC-1922159 CD3EAP was found to be down regulated and our pipeline assigned this down regulation of CD3EAP with a low rank of 256.However, not much research work has been done on SNP variations of CD3EAP in colorectal cancer case.
XPOT encodes protein exportin-t, a necessary component that is used for the export of tRNA from the nucleus to the cytoplasm via GTP-bound RAN 199 & 200 .We earlier saw that CRM1 (chromosomal region maintenance 1 also XPO1/exportin 1) has been found to play a major role in the export process from nucleus to the cytoplasm while dealing with PABPC1L.The crystal structure of CRM1 suggests binding with RAN protein along with GTP, allowing for a conformational change that facilitates binding to different cargo proteins through a nuclear export signal (NES) 201 & 202 .Inhibition of CRM1 pathway has been found to arrest the transport of various oncoproteins and retention of various tumor suppressor factors 203 & 204 .XPOT work in a similar fashion as CRM1 in binding with RAN-GTP for the export of mature tR-NAs 199 .In colorectal cancer cases (MSI), XPOT was found to be mutated 205 .After the treatment of colorectal cancer cells by ETC-1922159 51 , XPOT was found to be down regulated and our pipeline shows assigns this down regulation with a low rank of 276.Probably, the colorectal cancer cases contain mutated versions of XPOT that might lead to transfer of oncoproteins and ETC-1922159 might act as an inhibitor for mutated XPOT.SNHG16 (snoRNA host gene 16) has been demonstrated to be significantly up regulated in adenomas and all stages of CRC 206 .Christensen et al. 206 report positive correlation with Wnt regulated factors like ASCL2 which is known for contributing to stemness.Also, silencing of SNHG16 has been found to affect lipid metabolism and increase apoptotic cell death.Based on these, after the ETC-1922159 treatment in colorectal cancer cells, SNHG16 was found to be down regulated and our pipeline allocates the same with a rank of 280.
Transforming growth factor β 1 or TGFβ1 207 is encoded by TGFB1 and found to play multiple roles in processes like cell proliferation, growth, differentiation and apoptosis.It is known to be the most abundant isoform of TGFβ family and has been found to be highly expressed in colorectal cancer case 208 , 209 & 210 .Consistent with these findings, TGFB1 was found to be down regulated after the ETC-1922159 treatment of colorectal cancer cells 51 .In silico, our pipeline indicated the down regulation with a rank of 298.
TBGR4 or Transforming growth factor beta regulator 4 is a part of FASTKD family of proteins that is involved in regulating the energy balance of mitochondria under stress and cell cycle progression 211 .TBGR4 has been found to be implicated in colorectal cancer 212 .After ETC-1922159 treatment 51 , TBGR4 was down regulated and our pipeline indicated this with a low rank of 336.DNASE2 (Deoxyribonuclease II, lysosomal) is known for engaging in the break down of DNA during apoptosis 213 .DNASE2 was found to be down regulated after the treatment of ETC-1922159 in colorectal cancer cells and our pipeline points to this with a rank of 354.Not much is known about the role of CAAP1 (Caspase activity and apoptosis inhibitor 1) in colorectal cancer and it was found to be down regulated after the treatment of ETC-1922159 51 .Our pipeline assigned a low rank of 410.ELAC2 (Zinc phosphodiesterase ELAC protein 2) is involved in the maturation of tRNA within the mitochondria.ELAC2 has been found to play a role in prostate cancer 214 , while its role in colorectal cancer is still ongoing.After the administration of ETC-1922159 drug, ELAC2 was found to be down regulated and our pipeline shows this down regulation with a low rank of 424.
SMC1A (Structural maintenance of chromosomes 1A) belongs to the family of the SMC proteins that are used for the cohesion of the sister chromatids 215 & 216 .Over expression of SMC1A has been found to be a poor prognostic marker in colorectal cancer cases 217 & 218 .SMC1A is known to recruit TAF (tumor associated fibroblasts) for promotions of invasiveness and formation of fibroblasts which assist in tumorigenesis 219 .Consistent with these, our pipeline assigned a low rank of 438 to the observed 51 down regulation of SCM1A after the administration of ETC-1922159.
Selenium is known to be anticarcinogenic in nature and has been found to prevent cancer via the Selenium binding proteins 220 & 221 .Selenium-binding protein 1 is encoded by SE-LENBP1.In colorectal cancer cases, SELENBP1 has been found to be down regulated 222 .Given the above scenario, administration of ETC-1922159 showed down regulation of SELENBP1 51 and our pipeline assigns a relatively low rank of 516.SELENBP1 should be down regulated in colorectal cancer cells, which not being the case, indicates mutations in SELENBP1 would have been present in CRC samples used in Madan et al. 51 and the administration of the drug led to down regulation of mutated SELENBP1.Or, the authors hypothesize that the ETC-1922159 drug is not effective on wild type SELENBP1 and thus the observed data on SELENBP1 might need further testing.This is due to the fact that suppression of SELENBP1 has been found to be a late event in colorectal cancer 223 .However, when we look across the ranking of the other tables the ranking of SELENBP1 has been found to be associated with a very high ranking on majority basis and this points to the fact that SELENBP1 should be up regulated to suppress the cancer cells as it is anticarcionogenic in nature and the effect of ETC-1922159 on SELENBP1 is not that potent.Finally, higer ranks also suggest that these combinations might not be of importance.Further chemical analysis might reveal information about ETC-1922159 on SELENBP1.
Reduced HUGL1, a homologue of LGL tumor suppressor, is found to contribute to progression of colorectal cancer 224 .LGL has been found to arrest G1 cell cycle via formation of a complex involving LGL-VPRBP-DDB1 225 & 226 .Yamashita et al. 225 show that depletion of depletion of VPRBP leads to rescue of over proliferation of LGL-depleted cells.Mutations in VPRBP might lead to cell proliferation in colorectal cancer cases and ETC-1922159 administration show down regulation of VPRBP.Reversibly, down regulation of wild type VPRBP leads to phase progression.Our pipeline shows a down regulation with a rank of 656 for the mutated version, however, across different tables, the majority voting points to higher rank.This high rank indicates the wild type VPRBP to be work reversibly and thus point to inhibition of progression of cell proliferation.Also, higher ranks also mean that these combinations might not be important in one specific condition while it might be in another.Further tests are needed for VPRBP.
Carbonyl reductase 1 is encoded by CBR1 gene 227 .It has been found to show protective role against cellular damage from oxidative stress and apoptosis 228 and 229 .It has been found to be highly regulated in colorectal cancer cases and known to build Doxorubicin resistance in human gastrointestinal cancers 230 .Consistent with these, CBR1 was found to be down regulated after the ETC-1922159 treatment and our pipeline shows indicates this with a low rank of 1918.
Lysyl-tRNA synthetase is an enzyme that is encoded by KARS.Mutations have been found in KARS in colorectal cancer cases 231 .However, our pipeline showed a high rank for SCO1-WNT10-KARS with an assignment of 2793.This indicates that the combi- ADP-ribosylation factor-like protein 9 or ARL9 belongs to the family of ARL 232 and not much has been studied regarding it's role in colorectal cancer.However, it was found to be down regulated after the ETC-1922159 treatment and our pipeline indicates a high rank of 3196 along with SCO1-WNT10B.This indicates that this combination might not be useful for investigation.Nevertheless, biologists might want to confirm negative results also in wet lab.
ODF2 or Outer dense fibre protein 2 has been implicated in fertility 233 .Not much research work as been found in context of the role of ODF2 in colorectal cancer case 234 , however, for the combination with SCO1-WNT10B, the pipeline showed a high rank of 4512, indicating not much importance.Also, other combinations (see other tables) show similar ranking with not much importance.
PDE7A encodes high affinity cAMP-specific 3',5'-cyclic phosphodiesterase 7A 235 and is found to be highly expressed in colorectal cancer case 236 .Contrary to this, our pipeline assigned a high rank of 4728, indicating that this combination with SCO1-WNT10B is of not much significance after the ETC-1922159 treatment.However, the rankings of the other combinations of the PDE7A are consistent with the down regulation of ETC-1922159 treatment (see the other tables).
Centrosomal protein of 78 kDa or CEP78 is found to be a tumor suppressor and low expression of the same is associated with poor prognosis of colorectal cancer patients 237 .Also, note that CEP78 controls centrosome homeostasis by inhibiting VPRBP associated complex 238 .It was found to be down regulated after the treatment of ETC-1922159.Probably the mutated versions of CEP78 might have been present in the colorectal cancer cells, before the treatment.Also, the pipeline assigns a very high rank (4844) and thus indicates the non significance of the perhaps mutated CEP78 role.On the other hand, low rankings of CEP78 have been found to be consistent with down regulation of other dual combinations (see other tables).This might indicate that the mutated versions of CEP78 might be playing essential role in colorectal cancer and do get sown regulated on treatment with drug.
RETNLB or resistin-like-β has been found to highly expressed in colorectal cancer 239 , however, down regulation of the same after ETC-15922159 was assigned a very high rank of 4850 along with WNT10B-SCO1 combination by our pipeline.This indicates to the non significance of the combination which biologist might want to overlook.

FZD7-WNT10B-X combinations
Hitherto, we observed the behaviour of the different genes in context of the dual combination of WNT10B-SCO1.We shift our attention to another important combination and see how the respective genes are behaving in context of the dual combination WNT10B-FZD7.FZD7 has been found to be highly expressed in colorectal cancer cells 105 & 106 .Ater the ETC-1922159 treatment, FZD7 was found to be highly suppressed.Further ranked confirmation in the 3 rd order combination of SCO1-WNT10B-FZD7 is depicted by a very low priority of 39 in table 2 and it correlates to the ranking in table 1.
Contrary to this, most the 3 rd order combinations of the different genes listed in table 1 with WNT10B-SCO1 showed opposite ranking behaviour to that with WNT10B-FZD7 as shown in table 2. Many of these combinations are now ranked extremely high along with FZD7-WNT10B.FZD7-WNT10B combination is itself found to be upregulated in colorectral cancer cases and both were down regulated after the treatment of ETC-1922159.Interestingly the 3 rd order combinations were found to show very high ranks indicating that these would be highly regulated after the drug treatment, which might not be true.These high ranks point to the fact that the combination of the genes with WNT10B-FZD7 are not of importance after the drug treatment as the low ranked combinations WNT10B-SCO1-X.The reversal of ranks with WNT10B-FZD7 for many of the genes show that the pipeline is pointing to the ineffectiveness of the combination after the ETC-1922159 drug treatment.These combinations might not be of interest (i.e WNT10B-FZD7-X) as the X genes associated with WNT10B-SCO1 have been found to be down regulated and the pipeline assigned low ranks to them.
The assignment of high ranks by the pipeline recommend the biologists to safely ignore these combinations.Note that many of these rankings are ≥ 2425 (i.e 1 2 × 4850 3 rd order combinations) which point to the non significance of the combinations.Those that have ranks ≤ 2425 are of value and the biologists might want to have a look at these WNT10B-FZD7-X combinations what have been found to be down regulated after the ETC-1922159 treatment.Finally, note that these rankings are not a hard and fast rule and give a guideline to the biologists of what might be of significance.Combinations lying on the border line (near to 2425) can also be tested.Also, it is not that each and every combination will have an exact reversal.In some cases there will be different behaviour and the biologists might want to tally the rankings across the tables also.For example WNT10B-SCO1-ODF2 and WNT10B-FZD10-ODF2 are of no importance due to high ranks but the combination WNT10B-ODF2-RRM1 and WNT10-B-ODF2-XX172 are of significance (see tables 3 and 4 Table 2 3 rd order interaction ranking using HSIC for radial basis function kernel.Total number of 3 rd order interactions in a set of 100 genes -161700.4851 3 rd order combinations for WNT10B associated work.Rankings for FDZ7-WNT10B-X have been tabulated.

RRM1-WNT10B-X combinations
Earlier, we observed the behaviour of RRM1, while explaining its 3 rd order combination with SCO1-WNT10B.To reiterate, RRM1 (Ribonucleoside-diphosphate reductase large subunit) 134 helps in the formation of deoxyribonucleotides prior to DNA synthesis.The role of RRM1 is also known from its association with PABC1L (see earlier discussion in the section of SCO1-WNT10B-X combinations).PABPC1L (as recorded by 51 ) or the cytoplasmic 1-like poly(A)-binding protein 60 , belong to a family of multifunctional proteins PABPs which regulate and stabilize the mRNA translation.They are observed to be helpful in the transportation of the mRNA also from the nucleus and there exists a nucleic version of PABP also 61 .PABPC1 contains four non identical RRMs that are joined with the main PABC domain and separated by a linker.We know that PABC family is down regulated after the treatment of ETC-1922159 and expect that RRM1 should also be down regulated after the drug administration.This is confirmed by experiments in 51 .Additionally, wild type RRM1 is known to be highly expressed in colorectal cancer 135 & 136 .
We found the ranking behaviour of many of the genes (X) along with WNT10B and RRM1 to follow a pattern similar to SCO1-WNT10B-X rankings.Again, not every combination will have ex- Table 3 3 rd order interaction ranking using HSIC for radial basis function kernel.Total number of 3 rd order interactions in a set of 100 genes -161700.4851 3 rd order combinations for WNT10B associated work.Rankings for RRM1-WNT10B-X have been tabulated.
actly similar ranking.However, the pattern of ranking in table 3 matches similar to that of 1, except for the fact that the rankings for RRM1-WNT10B-X are more spread out in comparison to the rankings of SCO1-WNT10B-X which is more concentrated near the lowest rank of 1.We also find that a majority of the rankings for RRM1-WNT10B-X fall below 2425 (i.e 1 2 × 4850 3 rd order combinations) which clearly indicate the down regulation at 3 rd order level after the administration of the drug.

XX172-WNT10B-X combinations
Hitherto, we concentrated our attention on the combinations which contained two known factors in a 3 rd order combination, namely, SCO1-WNT10B-X, FZD7-WNT10B-X and RRM1-WNT10B-X.The area were the pipeline needs to be tested is the zone where we are confronted with unknown factors that have been recorded to be down regulated after the administration of ETC-1922159.We choose XX172, a down regulated component after the drug was administered and generated the rankings of XX172 along with WNT10B and a factor X (known/unknown).Remarkably, the pattern of ranking for XX172-WNT10B-X are sim-RANKING USING HSIC -RBF KERNEL W.R.T WNT10B-XX172 Table 4 3 rd order interaction ranking using HSIC for radial basis function kernel.Total number of 3 rd order interactions in a set of 100 genes -161700.4851 3 rd order combinations for WNT10B associated work.Rankings for XX172-WNT10B-X have been tabulated.
ilar to FZD7-WNT10B-X.Note that many of these rankings are ≥ 2425 (i.e 1 2 × 4850 3 rd order combinations) which point to the non significance of the combinations.Those that have ranks ≤ 2425 are of value and the biologists might want to have a look at these WNT10B-FZD7-X combinations what have been found to be down regulated after the ETC-1922159 treatment.Table 4 shows the rankings of XX172-WNT10B with a range of 100 recorded down regulated genes after the administration of the drug.
It might be a possibility that XX172 shows similar behaviour of up regulation along with WNT10B as FZD7 in colorectal cancer case.Similar to WNT10B-FZD7 combination, the majority of the recorded genes might not be correlating with the functionality of WNT10B-XX172.Both WNT10B and FZD7 were found to be down regulated, however, their combination with the X showed very high ranking indicating that the factor X was not in synchronization with WNT10B-FZD7.Similar is the case with WNT10B-XX172 dual combination.

Conclusion
Third order combinations related to SCO1, RRM1, FZD7 and XX172, each in conjugation with WNT10B and range of 100 down regulated genes affected after ETC-1922159 treatment have been ranked.These rankings reveal the hitherto unknown/untested/unexplored combinations in the Wnt pathway that might be playing a major role directly or indirectly in colorectal cancer case.SCO1-WNT10B-X and RRM1-WNT10B-X showed similar ranking behaviour with a majority of combinations being down regulated and assigned a low priority rank.Contrary to this, a majority of FZD7-WNT10-B-X and XX172-WNT10B-X combinations showed no synchronization after being assigned a high priority indicating up regulation, which is not the case.Similar ranking pattern of unknown XX172 and FZD7 with WNT10B-X possibly points to the correlated behaviour with the WNT10B.These higher and lower ranks are guidelines for oncologists/biologists to navigate through the dense and vast combinatorial forest of search space to explore unknown and untested biological hypotheses in the Wnt pathway apropos a subtype of colorectal cancer.
fixed.The dissimilarity between density functions is measured with Csiszar f-divergences.Estimation is performed through kernel density estimation and the function kde of the package ks. 242and 243 2. sensiHSIC -conducts a sensitivity analysis where the impact of an input variable is defined in terms of the distance between the input/output joint probability distribution and the product of their marginals when they are embedded in a Reproducing Kernel Hilbert Space (RKHS).This distance corresponds to HSIC proposed by 244 and serves as a dependence measure between random variables.
3. soboljansen -implements the Monte Carlo estimation of the Sobol indices for both first-order and total indices at the same time (all together 2p indices), at a total cost of (p+2) × n model evaluations.These are called the Jansen estimators. 245and 246 4. sobol2002 -implements the Monte Carlo estimation of the Sobol indices for both first-order and total indices at the same time (all together 2p indices), at a total cost of (p+2) ×n model evaluations.These are called the Saltelli estimators.This estimator suffers from a conditioning problem when estimating the variances behind the indices computations.This can seriously affect the Sobol indices estimates in case of largely non-centered output.To avoid this effect, you have to center the model output before applying "sobol2002".Functions "soboljansen" and "sobolmartinez" do not suffer from this problem. 247 sobol2007 -implements the Monte Carlo estimation of the Sobol indices for both first-order and total indices at the same time (all together 2p indices), at a total cost of (p+2) × n model evaluations.These are called the Mauntz estimators.248 6. sobolmartinez -implements the Monte Carlo estimation of the Sobol indices for both first-order and total indices using correlation coefficients-based formulas, at a total cost of (p + 2) × n model evaluations.These are called the Martinez estimators.
7. sobol -implements the Monte Carlo estimation of the Sobol sensitivity indices.Allows the estimation of the indices of the variance decomposition up to a given order, at a total cost of (N + 1) × n where N is the number of indices to estimate. 249

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 18 October 2017 doi:10.20944/preprints201710.0127.v1 nation
might not be of value after the treatment of ETC-1922159.Combination of KARS with other components showed consistent behaviour and was found to be down regulated and assigned proper rank (see other tables).