Machine Learning Discoveries of TNF-X Synergy in ETC-1922159 Treated Colorectal Cancer Cells

1. Introduction

In the unpublished preprint Sinha [1], a frame work of a search engine was developed which can rank combinations of factors (genes/proteins) in a signaling pathway. Such combinations are of import due to the vast search space in which they exist and the difficulty to find them. The search engine facilitates in prioritizing the combinations as ranked biological hypotheses which the biologists might want to test in wet lab, to know if a synergistic combination is prevalent in a signaling pathway, in a direct or indirect manner. Interested readers are advised to go through unpublished preprints Sinha [1] and Sinha [2] for details regarding the search engine and the discoveries mentioned in there.

2. Materials and Methods

2.1. Combinatorial Search Problem and a Possible Solution

The issue of combinatorial search problem and a possible solution has been addressed in Sinha [3] and Sinha [2]. The details of the methodology of this manuscript have been explained in great detail in Sinha [3] & its application in Sinha [2]. Readers are requested to go through the same for gaining deeper insight into the working of the pipeline and its use of published data set generated after administration of 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.

Briefly, from Sinha [2], the pipleline works by computing sensitivity indicies for each of these unique combinations and then vectorising these indices to connote and form discriminative feature vector for each combination. Since each combination is unique, the training and the test data are same. In the training data, the combinations are arranged and ranks from 1 to n are assigned. The ranking algorithm then learns the patterns from these combinations/sensitivity index vectors. Next the learned model is used to rank the test data by generating the ranking score for each of the unique combination. Sorting these shuffled scores of test data leads to prioritization of the combinations. Joachims [4] show an example of applying learned model to training data (same as the test data) in https://www.cs.cornell.edu/people/tj/svm_light/svm_rank.html. Note that these combinations are now ranked and give the biologists a chance to narrow down their focus on crucial biological hypotheses in the form of combinations which the biologists might want to test. Analogous to the webpage search engine, where the click of a button for a few key-words leads to a ranked list of web links, the pipeline uses sensitivity indices as an indicator of the strength of the influence of factors or their combinations, as a criteria to rank the combinations.

3. Results & Discussion

3.1. Tumor Necrosis Factor Related Synergies

3.1.1. TNF - WNT Cross Family Analysis

Brooks et al. [5] observed TNF-$\alpha$ induced alterations in the Wnt signaling cascade as a potential mechanism for obesity-associated colorectal tumorigenesis. Effects of TNF inhibitors on parathyroid hormone and Wnt signaling antagonists in rheumatoid arthritis have been studies in Adami et al. [6]. A complex interaction between Wnt signaling and TNF-$\alpha$ in nucleus pulposus cells has been studied by Hiyama et al. [7]. Ma and Hottiger [8] study the crosstalk between Wnt/$\beta$-catenin and NF-$\kappa$B signaling pathway during inflammation. Roubert et al. [9] study the influence of tumor necrosis factor-$\alpha$ on the tumorigenic Wnt-signaling pathway in human mammary tissue from obese women. Jang et al. [10] observe that WNT/$\beta$-catenin pathway modulates the TNF-$\alpha$-induced inflammatory response in bronchial epithelial cells. These studies suggest already existing synergistic roles of WNTs and TNFs. In CRC cells affected with ETC-1922159, members of TNF and WNT family were found to be up regulated. Our search engine alloted high numerical valued ranks to some of the combinations between WNTs and TNFs. Table 1 shows rankings of TNF w.r.t to WNTs on the left half and vice verse on the right half.

On the left half, we found TNF-RSF1A/RSF10A/RSF10B/SF15 to be up regulated w.r.t WNT2B. These were reflected in rankings of 2170 (laplace) and 2127 (linear) for TNFRSF1A - WNT2B; 1861 (laplace), 2367 (linear) and 1800 (rbf) for TNFRSF10A - WNT2B; 2020 (laplace) and 1881 (rbf) for TNFRSF10B - WNT2B and 2476 (laplace) and 2073 (rbf) for TNFSF15 - WNT2B. TNF-RSF10A/RSF10D/RSF12A/SF10 were found to be up regulated w.r.t WNT4. These were reflected in rankings of 2509 (laplace) and 2460 (linear) for TNFRSF10A - WNT4; 2233 (linear) and 2126 (rbf) for TNFRSF10D - WNT4; 2294 (linear), 1775 (linear) and 2384 (rbf) for TNFRSF12A - WNT4 and 2451 (linear) and 1782 (rbf) for TNFSF10 - WNT4. TNF-RSF12A/SF10 were found to be up regulated w.r.t WNT7B. These were reflected in rankings of 2100 (laplace) and 1983 (rbf) for TNFRSF12A - WNT7B and 2462 (laplace) and 2179 (rbf) for TNFSF10 - WNT7B. TNFRSF21 were found to be up regulated w.r.t WNT9A. These were reflected in rankings of 1805 (laplace) and 1999 (linear) for TNFRSF21 - WNT9A.

On the left half, we found WNT2B to be up regulated w.r.t TNF-RSF10B/RSF10D/RSF14. These were reflected in rankings of 1797 (laplace) and 2056 (rbf) for TNFRSF10B - WNT2B; 1989 (linear) and 2130 (rbf) for TNFRSF10D - WNT2B and 1932 (laplace) and 2399 (rbf) for TNFRSF14 - WNT2B. WNT4 was upregulated w.r.t TNF-AIP3/RSF10B. These are refliected in rankings of 2336 (laplace), 2511 (linear) and 2342 (rbf) for TNFAIP3 - WNT4 and 2105 (linear) and 2264 (rbf) for TNFRSF10B - WNT4. WNT7B was upregulated w.r.t TNF, TNF-RSF1A/RSF14. These are reflected in rankings of 2511 (linear) and 2210 (rbf) for TNF - WNT7B; 2084 (laplace), 1975 (linear) and 2154 (rbf) for TNFRSF1A - WNT7B and 2079 (laplace) and 1928 (rbf) for TNFRSF14 - WNT7B. WNT9A was upregulated w.r.t TNF-AIP2/AIP3/RSF10A/RSF12A/SF10. These are reflected in rankings of 2125 (laplace) and 2437 (linear) for TNFAIP2 - WNT9A; 1764 (laplace) and 2460 (linear) for TNFAIP3 - WNT9A; 2259 (laplace) and 2413 (linear) for TNFRSF10A - WNT9A; 2345 (laplace) and 2466 (rbf) for TNFRSF12A - WNT9A and 2054 (laplace) and 2338 (linear) for TNFSF10 - WNT9A.

Table 2 shows the derived influences which can be represented graphically, with the following influences - • TNF w.r.t WNT with TNF-RSF1A/RSF10A/RSF10B/SF15 $<-$ WNT2B; TNF-RSF10A/RSF10D/RSF12A/SF10 $<-$ WNT4; TNF-RSF12A/SF10 $<-$ WNT7B and TNF-RSF21 $<-$ WNT9A; and • WNT w.r.t TNF with TNF-RSF10B/RSF10D/RSF14 $->$ WNT2B; TNF-AIP3/RSF10B $->$ WNT4; TNF, TNF-RSF1A/RSF14 $->$ WNT7B; and TNF-AIP2/AIP3/RSF10A/RSF12A/SF10 $->$ WNT9A.

3.1.2. MUC - TNF Cross Family Analysis

In a recent development in Sheng et al. [11] MUC13 promoted tumor necrosis factro (TNF)-induced NFkB activation by interacting with TNFR1 and the E3 ligase, cIAP1, to increase ubiquitination of RIPK1. Dharmani et al. [12] show that TNF-$\alpha$ and MUC2 (Mucin 2) play major roles in disease onset and progression in dextran sodium sulphate-induced colitis. TNF-$\alpha$ is also shown to induce mucin hypersecretion and MUC2 gene expression by human airway epithelial cells by Levine et al. [13]. Also, inhibition of TNF-$\alpha$ induced MUC5AC expression and production by wogonin through the inactivation of NF-$\kappa$B signaling in airway epithelial cells, as shown by Sikder et al. [14]. Similarly, neutrophil elastase induces MUC5AC production in human airway epithelial cells via a cascade involving protein kinase-C, reactive oxygen species, and TNF-$\alpha$- converting enzyme, as shown by Shao and Nadel [15]. TNF-$\alpha$ or transforming growth factor-$\alpha$ stimulation of human epithelial cells resulted in mucus secretion as measured by MUC5AC mRNA and protein (Lora et al. [16]). In earlier experiments by Fischer et al. [17], TNF-$\alpha$ was found to stimulate mucin secretion and cyclic GMP production by guinea pig tracheal epithelial cells in vitro. Similar earlier experiments by Lin et al. [18], induction of mucin gene expression in middle ear of rats by TNF-$\alpha$ was the potential cause for mucoid otitis media. Effects of TNF-$\alpha$ and IL-1$\beta$ on mucin, lysozyme, IL-6 and IL-8 in passage-2 normal human nasal epithelial cells have been stuided by Yoon et al. [19]. Also, Mercogliano et al. [20] show that TNF-$\alpha$ induced MUC4 expression elicits trastuzumab resistance in HER2-$+ive$ breast cancer. These findings suggest deep synergy between Mucin family and TNF family members. However, not all synergies might have been explored till now. A set of family members of MUC and TNFs were found to be UP regulated after ETC-1922159 treatment in CRC cells.

Table 2 and Table 3 show the additional range of TNFs and MUCs that might be engaged in CRC through the NFkB pathway, in the light of the recent findings of MUC13 and TNFRSF1A in Sheng et al. [11]. Table 2 shows the rankings of the TNF family w.r.t to MUCIN family and Table 3 shows the rankings of the MUCIN family w.r.t to TNF family. Followed by this are the derived influences from the majority votings of the rankings in the foregoing tables, which are depicted in Table 4.

Ranking TNF family w.r.t MUC family
Ranking of TNF family w.r.t MUC1				Ranking of TNF family w.r.t MUC3A
	laplace	linear	rbf		laplace	linear	rbf
MUC1 - TNF	112	72	88	MUC3A - TNF	1353	1659	1479
MUC1 - TNFAIP1	1193	1603	997	MUC3A - TNFAIP1	2178	1209	1347
MUC1 - TNFAIP2	716	405	2340	MUC3A - TNFAIP2	1075	1614	1158
MUC1 - TNFAIP3	2115	1636	1882	MUC3A - TNFAIP3	962	1020	2491
MUC1 - TNFRSF1A	1380	422	1390	MUC3A - TNFRSF1A	461	1708	189
MUC1 - TNFRSF10A	1009	2180	1095	MUC3A - TNFRSF10A	2237	1910	335
MUC1 - TNFRSF10B	1923	732	88	MUC3A - TNFRSF10B	450	1443	2040
MUC1 - TNFRSF10D	2303	2154	376	MUC3A - TNFRSF10D	1678	2049	102
MUC1 - TNFRSF12A	2019	2009	1700	MUC3A - TNFRSF12A	2349	1315	382
MUC1 - TNFRSF14	1955	1899	1429	MUC3A - TNFRSF14	956	1442	1953
MUC1 - TNFRSF21	337	477	968	MUC3A - TNFRSF21	1297	1492	1959
MUC1 - TNFSF10	1111	1592	1198	MUC3A - TNFSF10	891	257	798
MUC1 - TNFSF15	936	986	2391	MUC3A - TNFSF15	2285	795	1164
Ranking of TNF family w.r.t MUC4				Ranking of TNF family w.r.t MUC12
	laplace	linear	rbf		laplace	linear	rbf
MUC4 - TNF	1896	231	1355	MUC12 - TNF	1862	102	135
MUC4 - TNFAIP1	864	397	987	MUC12 - TNFAIP1	1386	479	942
MUC4 - TNFAIP2	73	1011	1087	MUC12 - TNFAIP2	1056	303	1587
MUC4 - TNFAIP3	1159	1751	179	MUC12 - TNFAIP3	2493	1259	1330
MUC4 - TNFRSF1A	179	71	16	MUC12 - TNFRSF1A	1709	1440	837
MUC4 - TNFRSF10A	1668	1892	1652	MUC12 - TNFRSF10A	598	531	363
MUC4 - TNFRSF10B	2024	1396	331	MUC12 - TNFRSF10B	409	1572	1297
MUC4 - TNFRSF10D	2503	2403	2356	MUC12 - TNFRSF10D	30	102	149
MUC4 - TNFRSF12A	1684	700	745	MUC12 - TNFRSF12A	298	882	153
MUC4 - TNFRSF14	1675	2029	1146	MUC12 - TNFRSF14	1749	2237	135
MUC4 - TNFRSF21	647	326	323	MUC12 - TNFRSF21	1795	607	2438
MUC4 - TNFSF10	936	2134	1957	MUC12 - TNFSF10	801	1795	2435
MUC4 - TNFSF15	1440	1180	1627	MUC12 - TNFSF15	1741	889	1098

Considering Table 2, TNF family w.r.t MUC1, we find TNFAIP3, TNFRSF-10D/12A/14 to be highly up regulated. These are reflected in the rankings of 2115 (laplace) and 1882 (rbf) for MUC1 - TNFAIP3; 2303 (laplace) and 2154 (linear) for MUC1 - TNFRSF10D; 2019 (laplace) and 2009 (linear) for MUC1 - TNFRSF12A; and 1955 (laplace) and 1899 (linear) for MUC1 - TNFRSF14. TNF family w.r.t MUC3A, we find TNFRSF-10A/10D to be highly up regulated. These are reflected in the rankings of 2237 (laplace) and 1910 (linear) for MUC3 - TNFRSF10A; 1678 (laplace) and 2049 (linear) for MUC3 - TNFRSF10D. TNF family w.r.t MUC4 we find TNFRSF10D/TNFSF10 to be highly up regulated. These are reflected in the rankings of 2503 (laplace), 2403 (linear) and 2356 (rbf) for MUC4 - TNFRSF10D and 2134 (laplace) and 1957 (linear) for MUC4 - TNFSF10. TNF family w.r.t MUC12 we find TNFRSF21/TNFSF10 to be highly up regulated. These are reflected in the rankings of 1795 (laplace) and 2438 (linear) for MUC12 - TNFRSF21 and 1795 (linear) 2435 (rbf) for MUC12 - TNFSF10. TNF family w.rt MUC13 we find TNFRSF10A/TNFRSF10D to be highly up regulated. These are reflected in the rankings of 2500 (laplace) and 1844 (rbf) for MUC13 - TNFRSF10A and 2263 (linear) and 2294 (rbf) for MUC13 - TNFRSF10D. TNF family w.r.t MUC17 we find TNFRSF-10A/10D/12A to be highly up regulated. These are reflected in the rankings of 2269 (laplace) 2364 (linear) and 2005 (rbf) for MUC17 - TNFRSF10A; 1798 (laplace) and 2302 (rbf) for MUC17 - TNFRSF10D and 2041 (laplace) and 2303 (linear) for MUC17 - TNFRSF12A. TNF family w.r.t MUC20 we find TNFAIP3/TNFSF15 to be highly up regulated. These are reflected in the rankings of 2205 (laplace) and 2136 (rbf) for MUC20 - TNFAIP3 and 2493 (laplace) and 2108 (rbf) for MUC20 - TNFSF15.

Considering Table 3, MUC1 w.r.t TNF family, we find TNFRSF1A to be highly up regulated. These are reflected in the rankings of 2344 (linear) and 2312 (rbf) for MUC1 - TNFRSF1A. MUC4 w.r.t TNF family, we find TNFAIP2 to be highly up regulated. These are reflected in the rankings of 1875 (laplace) and 1792 (linear) for MUC4 - TNFAIP2. MUC12 w.r.t TNF family we find TNFAIP1/TNFAIP2/TNFRSF21/TNFSF10 to be highly up regulated. These are reflected in the rankings of 2321 (laplace) and 2457 (rbf) for MUC12 - TNFAIP1; 1829 (linear) and 1913 (rbf) for MUC12 - TNFAIP2; 1975 (linear) and 1769 (rbf) for MUC12 - TNFRSF21; 2135 (linear) and 2255 (rbf) for MUC12 - TNFSF10. MUC12 w.r.t TNF family we find TNFRSF21/TNFSF10 to be highly up regulated. These are reflected in the rankings of 1795 (laplace) and 2438 (linear) for MUC12 - TNFRSF21 and 1795 (linear) and 2435 (rbf) for MUC12 - TNFSF10. MUC20 w.r.t TNF family we find TNFAIP1/TNFAIP2 to be highly up regulated. These are reflected in the rankings of 2266 (laplace) and 2057 (rbf) for MUC20 - TNFAIP1 and 2404 (linear) and 2157 (rbf) for MUC20 - TNFAIP2.

One can also interpret the results of the Table 4 graphically, with the following influences - • TNF family w.r.t MUC family with MUC1 $->$ TNFAIP3/TNFRSF-10D/12A/14; MUC3A $->$ TNFRSF-10A/10D; MUC4 $->$ TNFRSF10D/TNFSF10; MUC12 $->$ TNFRSF21/TNFSF10; MUC13 $->$ TNFRSF-10A/10D; MUC17 $->$ TNFRSF-10A/10D/12A; MUC20 $->$ TNFAIP3/TNFSF15 and • MUC family w.r.t TNF family with MUC1 $<-$ TNFRSF1A; MUC4 $<-$ TNFAIP2; MUC12 $<-$ TNFAIP1/TNFAIP2/TNFRSF21/TNFSF10 and MUC13 $<-$ TNFAIP1/TNFAIP2.

3.1.3. STEAP4 - TNF Cross Family Analysis

STEAP4 or six transmembrane epithelial antigen of prostate 4, resides in the golgi apparatus and functions as a metalloreductase with the capacity to reduce insoluble ferric ions $F{e}^{3+}$ to soluble ferrous ions $F{e}^{2+}$. Emerging role of STEAP4 in metabolism and homeostasis of cellular iron and copper in metabolism and homeostasis of cellular iron and copper has been studied in Scarl et al. [21]. STEAP4 was first identified as a novel gene induced by TNF-$\alpha$ during adipose differentiation by Moldes et al. [22]. Zhang et al. [23] observe that STEAP4 was up-regulated by LPS at a very early time point, consistent with reports that STEAP4 could be up-regulated by tumor necrosis factor-alpha. Tanaka et al. [24] show that STEAP4 is expressed on monocytes/neutrophils, and is regulated by TNF antagonist in patients with rheumatoid arthritis. Also, Tanaka et al. [25] show STEAP4 is a tumor necrosis factor alpha-induced protein that regulates IL-6, IL-8, and cell proliferation in synovium from patients with rheumatoid arthritis. Gauss et al. [26] observe that the STEAP4 expression in adipocytes is normally induced by nutritional stress, leptin, and proinflammatory cytokines, including TNF-$\alpha$, interleukin-1$\beta$, and interleukin-6. ZHANG et al. [27] show that the downregulation of STEAP4, a highly-expressed TNF-$\alpha$-inducible gene in adipose tissue, is associated with obesity in humans 1. Liang et al. [28] show that STEAP comprises a novel inflammatory nexus in patients with pustular skin disorders. They show that in primary human keratinocytes STEAP4 expression was induced by TNF-$\alpha$, IL-1$\beta$, IL-36$\alpha$, IL-36$\gamma$, IL-17A, and IL-17A combined with TNF-$\alpha$ or IL-22. Gomes et al. [29] further show the TNF STEAP interactions while studying the structure of STEAP proteins and its applications to cancer therapy. Such interactions point to the existing synergy between STEAP4 and TNF-$\alpha$. In CRC cells treated with ETC-1922159, both TNF members and STEAP4 were found to be up regulated. Our search engine alloted the dual combinations with numerically high ranked values thus pointing to the possible synergies that might be existing in the cells and may not have been explored. Table 6 shows the rankings of each with the other. On the left we found, TNF, TNF-AIP1/AIP2/AIP3/RSF10B/RSF10D/SF10 to be up regulated w.r.t STEAP4. These are reflected in rankings of 1914 (linear) and 2130 (rbf) for TNF - STEAP4; 2189 (laplace) and 1910 (rbf) for TNFAIP1 - STEAP4; 2002 (linear) and 1840 (rbf) for TNFAIP2 - STEAP4; 1882 (linear) and 2197 (rbf) for TNFAIP3 - STEAP4; 2210 (laplace), 1717 (linear) and 1827 (rbf) for TNFRSF10B - STEAP4; 2192 (linear) and 1797 (rbf) for TNFRSF10D - STEAP4; and 2083 (laplace) and 1773 (rbf) for TNFSF10 - STEAP4. On the right we found, STEAP4 to be up regulated w.r.t TNF-RSF10A/RSF10D/RSF14. These are reflected in rankings of 1796 (linear) and 2026 (rbf) for TNFRSF10A - STEAP4; 2339 (linear) and 2405 (rbf) for TNFRSF10D - STEAP4; and 1956 (linear) and 2256 (rbf) for TNFRSF14 - STEAP4.

Table 5. 2^nd order combinatorial hypotheses between TNF and STEAP4 family.

Ranking TNF family vs STEAP4 family
Ranking of TNF family w.r.t STEAP4				Ranking of STEAP4 w.r.t IL family
	laplace	linear	rbf		laplace	linear	rbf
TNF - STEAP4	1579	1914	2130	TNF - STEAP4	1116	1482	999
TNFAIP1 - STEAP4	2189	1293	1910	TNFAIP1 - STEAP4	691	611	1105
TNFAIP2 - STEAP4	1172	2002	1840	TNFAIP2 - STEAP4	228	1747	2463
TNFAIP3 - STEAP4	1458	1882	2197	TNFAIP3 - STEAP4	159	727	219
TNFRSF1A - STEAP4	803	75	1086	TNFRSF1A - STEAP4	1483	408	1966
TNFRSF10A - STEAP4	239	1949	339	TNFRSF10A - STEAP4	1512	1796	2026
TNFRSF10B - STEAP4	2210	1717	1827	TNFRSF10B - STEAP4	565	571	248
TNFRSF10D - STEAP4	510	2192	1797	TNFRSF10D - STEAP4	1018	2339	2405
TNFRSF12A - STEAP4	757	338	1497	TNFRSF12A - STEAP4	1495	1430	581
TNFRSF14 - STEAP4	1323	1512	792	TNFRSF14 - STEAP4	1363	1956	2256
TNFRSF21 - STEAP4	1643	1920	165	TNFRSF21 - STEAP4	1646	802	160
TNFSF10 - STEAP4	2083	544	1773	TNFSF10 - STEAP4	845	675	2468
TNFSF15 - STEAP4	631	1296	1020	TNFSF15 - STEAP4	1558	600	784

Table 6. 2^nd order combinatorial hypotheses between STEAP4 and TNF

Table 5. 2^nd order combinatorial hypotheses between TNF and STEAP4 family.

Ranking TNF family vs STEAP4 family
Ranking of TNF family w.r.t STEAP4				Ranking of STEAP4 w.r.t IL family
	laplace	linear	rbf		laplace	linear	rbf
TNF - STEAP4	1579	1914	2130	TNF - STEAP4	1116	1482	999
TNFAIP1 - STEAP4	2189	1293	1910	TNFAIP1 - STEAP4	691	611	1105
TNFAIP2 - STEAP4	1172	2002	1840	TNFAIP2 - STEAP4	228	1747	2463
TNFAIP3 - STEAP4	1458	1882	2197	TNFAIP3 - STEAP4	159	727	219
TNFRSF1A - STEAP4	803	75	1086	TNFRSF1A - STEAP4	1483	408	1966
TNFRSF10A - STEAP4	239	1949	339	TNFRSF10A - STEAP4	1512	1796	2026
TNFRSF10B - STEAP4	2210	1717	1827	TNFRSF10B - STEAP4	565	571	248
TNFRSF10D - STEAP4	510	2192	1797	TNFRSF10D - STEAP4	1018	2339	2405
TNFRSF12A - STEAP4	757	338	1497	TNFRSF12A - STEAP4	1495	1430	581
TNFRSF14 - STEAP4	1323	1512	792	TNFRSF14 - STEAP4	1363	1956	2256
TNFRSF21 - STEAP4	1643	1920	165	TNFRSF21 - STEAP4	1646	802	160
TNFSF10 - STEAP4	2083	544	1773	TNFSF10 - STEAP4	845	675	2468
TNFSF15 - STEAP4	631	1296	1020	TNFSF15 - STEAP4	1558	600	784

Table 6. 2^nd order combinatorial hypotheses between STEAP4 and TNF

One can also interpret the results of the Table 4 graphically, with the following influences - • TNF w.r.t STEAP4 with TNF, TNF-AIP1/AIP2/AIP3/RSF10B/RSF10D/SF10 $<-$ STEAP4 and • STEAP4 w.r.t TNF with TNF-RSF10A/RSF10D/RSF14 $->$ STEAP4.

Unexplored combinatorial hypotheses
TNF w.r.t STEAP4
TNF, TNF-AIP1/AIP2/AIP3/RSF10B/RSF10D/SF10	STEAP4
STEAP4 w.r.t TNF
TNF-RSF10A/RSF10D/RSF14	STEAP4

3.1.4. TNF - UBE2 Cross Family Analysis

Fu et al. [30] show that the ubiquitin conjugating enzyme UBE2L3 regulates TNF$\alpha$-induced linear ubiquitination. They show by western blotting of HOIL-1L immunoprecipitates demonstrates that endogenous HOIL-1L interacts with endogenous UBE2L3 in vivo and these associations are stable following TNF$\alpha$ stimulation. Through various hypotheses, the authors show the interaction of UBE2L3 with TNF. In conclusion, the authours state that increased UBE2L3 expression enhances NF-$\kappa$B activation, and increased levels of NF-$\mathsf{\kappa }$B activity are linked to inflammatory and autoimmune diseases. Li et al. [31] show that TNF-$\alpha$ increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UBCH2/E2_20k. Shembade et al. [32] show that IL-1$\beta$ or TNF induce late depletion of UBE2D3 (UBCH5C) and UBE2N (UBC13) in mouse embryonic fibroblasts. These studies show a definite synergy between UBE family and TNFs. In CRC cells treated with ETC-1922159, both TNF members and UBE2 were found to be up regulated. Our search engine alloted the dual combinations with numerically high ranked values thus pointing to the possible synergies that might be existing in the cells and may not have been explored. Table 7 and Table 8 shows the rankings of each with the other.

On the left side is the ranking of UBE2 family w.r.t TNF family. We found UBE2A to be up regulated w.r.t TNF-AIP1/RSF1A/RSF10A/RSF10B/RSF10D/RSF12A/RSF14/ RSF21/SF15. These are reflected in rankings of 2357 (linear) and 2455 (rbf) for TNFAIP1 - UBE2A; 2457 (laplace) and 2020 (rbf) for TNFRSF1A - UBE2A; 2164 (laplace) and 2126 (linear) for TNFRSF10A - UBE2A; 2284 (laplace) and 1901 (linear) for TNFRSF10B - UBE2A; 1989 (laplace) and 2291 (linear) for TNFRSF10D - UBE2A; 2484 (laplace) and 2427 (linear) for TNFRSF12A - UBE2A; 2301 (laplace), 2180 (linear) and 2323 (rbf) for TNFRSF14 - UBE2A; 2419 (laplace) and 2035 (linear) for TNFRSF21 - UBE2A; 1768 (laplace) and 1942 (rbf) for TNFSF15 - UBE2A. UBE2B to be up regulated w.r.t TNF-RSF10A/RSF10B/RSF10D/RSF14/RSF21. These are reflected in rankings of 2132 (laplace) and 2184 (rbf) for TNFRSF10A - UBE2B; 2399 (laplace) and 2000 (linear) for TNFRSF10B - UBE2B; 1959 (laplace) and 2232 (rbf) for TNFRSF10D - UBE2B; 2297 (linear) and 2373 (rbf) for TNFRSF14 - UBE2B; and 1986 (laplace) and 1754 (rbf) for TNFRSF21 - UBE2B. UBE2F to be up regulated w.r.t TNF, TNF-AIP1/RSF1A/RSF10A/RSF10B/RSF12A/SF15. These are reflected in rankings of 2162 (laplace), 2484 (linear) and 2500 (rbf) for TNF - UBE2F; 1732 (laplace), 2239 (linear) and 2003 (rbf) for TNFAIP1 - UBE2F; 1980 (laplace), 2255 (linear) and 1872 (rbf) for TNFRSF1A - UBE2F; 2085 (laplace), 2218 (linear) for TNFRSF10A - UBE2F; 2432 (laplace), 2011 (linear) and 2144 (rbf) for TNFRSF10B - UBE2F; 2458 (laplace) and 2336 (linear) for TNFRSF12A - UBE2F; 1910 (laplace) and 2353 (rbf) for TNFSF15 - UBE2F; UBE2H to be up regulated w.r.t TNF-RSF12A/RSF14/RSF21. These are reflected in rankings of 1950 (laplace), 1793 (linear) and 1851 (rbf) for TNFRSF12A - UBE2H; 2297 (laplace) and 2385 (rbf) for TNFRSF14 - UBE2H; and 2022 (laplace) and 2231 (rbf) for TNFRSF21 - UBE2H; UBE2J1 to be up regulated w.r.t TNF, TNF-AIP1/RSF1A/RSF10A/RSF10B/RSF12A/RSF14/RSF21/SF15. These are reflected in rankings of 2308 (linear) and 2336 (rbf) for TNF - UBE2J1; 2292 (linear) and 1756 (rbf) for TNFAIP1 - UBE2J1; 1992 (laplace) and 2268 (rbf) for TNFRSF1A - UBE2J1; 1893 (laplace), 2090 (linear) and 2363 (rbf) for TNFRSF10A - UBE2J1; 1913 (laplace) and 1838 (rbf) for TNFRSF10B - UBE2J1; 2401 (laplace) and 1901 (linear) for TNFRSF12A - UBE2J1; 2277 (laplace), 2347 (linear) and 1943 (rbf) for TNFRSF14 - UBE2J1; 1976 (laplace), 2333 (linear) for TNFRSF21 - UBE2J1; and 2021 (laplace), 2013 (linear) and 2515 (rbf) for TNFSF15 - UBE2J1; UBE2Z to be up regulated w.r.t TNF, TNF-AIP1/AIP2/RSF1A/RSF10A/RSF10D/RSF12A/SF10/SF15. These are reflected in rankings of 2264 (laplace), 2505 (linear) and 2479 (rbf) for TNF - UBE2Z; 2055 (linear) and 2332 (rbf) for TNFAIP1 - UBE2Z; 2404 (laplace) and 2139 (rbf) for TNFAIP2 - UBE2Z; 2473 (laplace), 2194 (linear) and 2405 (rbf) for TNFRSF1A - UBE2Z; 2234 (laplace) and 2152 (linear) for TNFRSF10A - UBE2Z; 2264 (laplace), 2360 (linear) and 2278 (rbf) for TNFRSF10D - UBE2Z; 2207 (laplace) and 2149 (linear) for TNFRSF12A - UBE2Z; 2374 (linear) and 2235 (rbf) for TNFSF10 - UBE2Z; and 2081 (laplace) and 2102 (rbf) for TNFSF15 - UBE2Z;

One the right side is the ranking of TNF family w.r.t UBE2 family. We found TNF-RSF10A to be up regulated w.r.t UBE2A. This is reflected in rankings of 2116 (laplace) and 2376 (rbf) for TNFRSF10A - UBE2A. TNF-RSF10A/RSF12A/SF10/SF15 were up regulated w.r.t UBE2B. These are reflected in rankings of 2318 (linear) and 2265 (linear) for TNFRSF10A - UBE2B; 1940 (laplace); 1868 (linear) and 1758 (linear) for TNFRSF12A - UBE2B; 2208 (laplace); 2326 (linear) and 2470 (linear) for TNFSF10 - UBE2B; and 2055 (laplace) and 1964 (linear) for TNFSF15 - UBE2B. TNF-RSF14 were up regulated w.r.t UBE2F. These is reflected in rankings of 2324 (laplace) and 1924 (linear) for TNF-RSF14 - UBE2F. TNF-SF15 were up regulated w.r.t UBE2H. These is reflected in rankings of 2416 (linear) and 1960 (rbf) for TNF-SF15 - UBE2H. TNF-RSF10A/RSF10D/RSF14 were up regulated w.r.t UBE2J1. These are reflected in rankings of 2379 (laplace), 2213 (linear) and 2135 (rbf) for TNFRSF10A - UBE2J1; 2393 (laplace) and 2102 (rbf) for TNFRSF10D - UBE2J1; and 2133 (linear) and 2313 (rbf) for TNFRSF14 - UBE2J1. TNF-RSF10A/RSF14 were up regulated w.r.t UBE2Z. These are reflected in rankings of 2410 (laplace) and 2103 (laplace) for TNFRSF10A - UBE2Z and 1779 (laplace) and 2100 (rbf) for TNFRSF14 - UBE2Z.

One can also interpret the results of the Table 9 graphically, with the following influences - • UBE2 w.r.t TNF with TNF-AIP1/RSF1A/RSF10A/RSF10B/RSF10D/RSF12A/RSF14/ RSF21/SF15 $->$ UBE2A; TNF-RSF10A/RSF10B/RSF10D/RSF14/RSF21 $->$ UBE2B; TNF, TNF-AIP1/RSF1A/RSF10A/RSF10B/RSF12A/SF15 $->$ UBE2F; TNF-RSF12A/RSF14/RSF21 $->$ UBE2H; TNF, TNF-AIP1/RSF1A/RSF10A/RSF10B/RSF12A/RSF14/RSF21/SF15 $->$ UBE2J1; and TNF, TNF-AIP1/AIP2/RSF1A/RSF10A/RSF10D/RSF12A/SF10/SF15 $->$ UBE2Z • TNF w.r.t UBE2 with TNF-RSF10A $<-$ UBE2A; TNF-RSF10A/RSF12A/SF10/SF15 $<-$ UBE2B; TNF-RSF14 $<-$ UBE2F; TNF-SF15 $<-$ UBE2H; TNF-RSF10A/RSF10D/RSF14 $<-$ UBE2J1; TNF-RSF10A/RSF14 $<-$ UBE2Z.

3.1.5. TNF - BCL Cross Family Analysis

Tamatani et al. [33] observe that tumor necrosis factor induces BCL-2 and BCL-x expression through NF$\kappa$B activation in primary hippocampal neurons. The role of Bcl-2 Expression in EGF Inhibition of TNF-$\alpha$/IFN-$\gamma$-induced Villous Trophoblast Apoptosis has been studied by Ho et al. [34]. Genestier et al. [35] show that tumor necrosis factor-$\alpha$ up-regulates BCL-2 expression and decreases calcium-dependent apoptosis in human B cell lines. In breast carcinoma cells, Bcl-x and Bcl-2 inhibit TNF and FAS-induced apoptosis and activation of phospholipase A2 (Jäättelä et al. [36]). Kim et al. [37] show that TNF-$\alpha$-induced ROS production triggering apoptosis is directly linked to Romo1 and BCL-X_L. Kuwata et al. [38] showed that IL-10-inducible BCL-3 negatively regulates LPS-induced TNF-$\alpha$ production in macrophages. Esche et al. [39] showed that tumor necrosis factor-$\alpha$-promoted expression of BCL-2 and inhibition of mitochondrial cytochrome c release mediate resistance of mature dendritic cells to melanoma-induced apoptosis. These studies show a definite synergy between BCL family and TNFs. In CRC cells treated with ETC-1922159, both TNF members and BCL were found to be up regulated. Our search engine alloted the dual combinations with numerically high ranked values thus pointing to the possible synergies that might be existing in the cells and may not have been explored. Table 10 and Table 11 show the rankings of each with the other.

On the left side is the ranking of BCL family w.r.t TNF family. We found BCL2L2 to be up regulated w.r.t TNF, TNF-AIP1/RSF1A/RSF10B/RSF10D/RSF12A/RSF14/RSF21/ SF10/SF15. These are reflected in rankings of 1822 (laplace), 1926 (linear) and 2359 (rbf) for TNF - BCL2L2; 2266 (laplace), 2478 (linear) and 1847 (rbf) for TNFAIP1 - BCL2L2; 2311 (linear) and 1920 (rbf) for TNFRSF1A - BCL2L2; 2478 (laplace) and 2239 (rbf) for TNFRSF10B - BCL2L2; 2278 (linear) and 2237 (rbf) for TNFRSF10D - BCL2L2; 1945 (laplace) and 2484 (rbf) for TNFRSF12A - BCL2L2; 2358 (laplace) and 2310 (rbf) for TNFRSF14 - BCL2L2; 2292 (laplace) and 1850 (linear) for TNFRSF21 - BCL2L2; 2438 (laplace) and 2013 (rbf) for TNFSF10 - BCL2L2 and 2443 (linear) and 2350 (rbf) for TNFSF15 - BCL2L2; BCL2L3 was up regulated w.r.t TNF, TNF-AIP1/RSF1A/RSF10A/RSF10D/RSF12A/RSF14/RSF21/ SF10/SF15. These are reflected in rankings of 2437 (laplace), 2482 (linear) and 2482 (rbf) for TNF - BCL2L13; 1863 (laplace) and 2386 (linear) for TNFAIP1 - BCL2L13; 1962 (linear) and 2489 (rbf) for TNFRSF1A - BCL2L13; 2055 (linear) and 2499 (rbf) for TNFRSF10A - BCL2L13; 2204 (laplace), 2159 (linear) and 2343 (rbf) for TNFRSF10D - BCL2L13; 2183 (laplace), 2509 (linear) for TNFRSF12A - BCL2L13; 1852 (laplace), 1974 (linear) and 2339 (rbf) for TNFRSF14 - BCL2L13; 2280 (laplace), 2424 (linear) and 2301 (rbf) for TNFRSF21 - BCL2L13; 2429 (linear) and 1803 (rbf) for TNFSF10 - BCL2L13; and 2438 (linear) and 2252 (rbf) for TNFSF15 - BCL2L13; BCL3 was up regulated w.r.t TNFRSF10B. This is reflected in rankings of 2427 (laplace) and 1868 (rbf). BCL6 was up regulated w.r.t TNF, TNF-AIP1/AIP2/RSF1A/RSF10A/RSF10D/RSF21. These are reflected in rankings of 2271 (laplace), 2071 (linear) and 1810 (rbf) for TNF - BCL6; 2135 (laplace) and 2158 (linear) for TNFAIP1 - BCL6; 2340 (laplace) 1808 (rbf) for TNFAIP2 - BCL6; 1771 (linear) and 2503 (rbf) for TNFRSF1A - BCL6; and 1831 (linear) and 2096 (rbf) for TNFRSF10A - BCL6; and 2213 (laplace) and 2188 (rbf) for TNFRSF10D - BCL6; and 2071 (linear) and 2335 (rbf) for TNFRSF21 - BCL6; BCL10 was up regulated w.r.t TNF-RSF10D/RSF12A. These are reflected in rankings of 1831 (laplace) and 2040 (rbf) for TNFRSF10D - BCL10; and 2015 (laplace) and 1883 (rbf) for TNFRSF12A - BCL10;

On the right side is the ranking of TNF family w.r.t BCL family. We found TNF-RSF10A/RSF10B/RSF10D/RSF12A/RSF14/SF10 to be up regulated w.r.t BCL2L1. These are reflected in rankings of 2273 (laplace) and 1928 (linear) for TNFRSF10A - BCL2L1; 2252 (linear) and 2217 (rbf) for TNFRSF10B - BCL2L1; 1868 (laplace), 2420 (linear) and 2392 (rbf) for TNFRSF10D - BCL2L1; 1923 (laplace) and 1936 (rbf) for TNFRSF12A - BCL2L1; 2350 (linear) and 2414 (rbf) for TNFRSF14 - BCL2L1 and 2115 (laplace) and 2299 (linear) for TNFSF10 - BCL2L1; TNFRSF10B was up regulated w.r.t BCL2L2. This is reflected in rankings of 2153 (laplace) and 1983 (rbf) for TNFRSF10B - BCL2L2; TNFRSF14 was up regulated w.r.t BCL2L13. This is reflected in rankings of 2459 (laplace) and 2381 (linear) for TNFRSF14 - BCL2L13; TNF-RSF10A/SF10D/SF10 were up regulated w.r.t BCL3. These are reflected in rankings of 2388 (laplace) and 2493 (linear) for TNFRSF10A - BCL3; 2213 (laplace) and 1972 (rbf) for TNFRSF10D - BCL3 and 1926 (laplace) and 2107 (rbf) for TNFSF10 - BCL3; TNFRSF12A was up regulated w.r.t BCL6. This is reflected in rankings of 2200 (linear) and 1910 (rbf) for TNFRSF12A - BCL6; TNF-AIP1/SF10/SF15 was up regulated w.r.t BCL9L. This is reflected in rankings of 2470 (linear) and 1802 (rbf) for TNFAIP1 - BCL9L; 1812 (laplace), 1796 (linear) and 2095 (rbf) for TNFSF10 - BCL9L; and 1939 (laplace), 2114 (linear) and 2405 (rbf) for TNFSF15 - BCL9L; TNFRSF14 was up regulated w.r.t BCL10. This is reflected in rankings of 1894 (linear) and 2227 (rbf) for TNFRSF14 - BCL10;

One can also interpret the results of the Table 12 graphically, with the following influences - • BCL w.r.t TNF with TNF, TNF-AIP1/RSF1A/RSF10B/RSF10D/RSF12A/RSF14/RSF21/SF10/SF15 $->$ BCL2L2; TNF, TNF-AIP1/RSF1A/RSF10A/RSF10D/RSF12A/ RSF14/RSF21/SF10/SF15 $->$ BCL2L13; TNFRSF10B $->$ BCL3; TNF, TNF-AIP1/AIP2/RSF1A/RSF10A/RSF10D/RSF21 $->$ BCL6; TNF-RSF10D/RSF12A $->$ BCL10; • TNF w.r.t BCL with TNF-RSF10A/RSF10B/RSF10D/RSF12A/ RSF14/SF10 $<-$ BCL2L1; TNF-RSF10B $<-$ BCL2L2; TNF-RSF14 $<-$ BCL2L13; TNF-RSF10A/SF10D/SF10 $<-$ BCL3; TNF-RSF12A $<-$ BCL6; TNF-AIP1 $<-$ BCL9L; TNF-SF10/SF15 $<-$ BCL9L and TNF-RSF14 $<-$ BCL10.