HMGA2 promotes colorectal cancer angiogenesis via dual regulation of Sema3A and VEGFA

Background: HMGA2 encodes a small non histone chromatin-associated protein that has no intrinsic transcriptional activity, but can modulate transcription by altering the chromatin architecture. HMGA2 was found overexpressed in a variety of epithelial and mesenchymal tumors and promoted invasion and metastasis in most malignant epithelial tumors. A recent study showed that P53 inhibited CRC progression by targeting HMGA2. However, the mechanism by which HMGA2 affect angiogenesis in CRC has not been clarified. Methods: The expression of HMGA2 was analyzed by IHC, WB and bio infomatic analysis. Cbioportal and mexpress online tools were applied to explore the CNV and methylation of HMGA2 in CRC patients. Single cell data from GEO was used to examine the specific cell type that contribute to the high HMGA2 expression in CRC. Lentivirus was used to knock down HMGA2 in CRC cells and HUVECs was used to study angiogenesis. Results: In the current study, we first detected the expression pattern of HMGA2 in CRC patients and evaluated its clinical values and CNV amplification could possibly contribute to the up regulation of HMGA2 in CRC patients. By analyzing CRC single cell data we found that HMGA2 was specifically up regulated in the colorectal epithelial cells. Furthermore, knocking down of HMGA2 suppresses angiogenesis via dual regulation of VEGF-A and SEMA3A in CRC through inactivating VEGRR2 pathway in HUVECs. Conclusions: HMGA2 might be a promising prognostic marker and target for treating advanced CRC patients. HMGA2 KD inhibited theses effects. Furthermore, we showed that HMGA2 promoted CRC angiogenesis by dual regulating Sema3A and VEGFA. It is well known that VEGFA inducing tumor angiogenesis via banding with tyrosine VEGFR2 on the vascular endothelial cells and SEMA3A can inhibit the binding of VEGFA to NP1 which is able to inhibit in vitro angiogenesis . Thus, HMGA2 might be a promising target due to its multiple roles in compromising CRC angiogenesis.

But the adverse effects of increased risk of stroke and arterial events, gastrointestinal bleeding, gastrointestinal perforation, delayed wound healing was increase after use anti-angiogenesis therapy was confined with its side effects [8,9]. Therefore new therapeutic strategies of tumor angiogenesis would be needed to improve patient survival.
Carcinogenesis is the pathological alteration of epithelial/mesenchymal cells under the stimulation of carcinogenic factors, including inflammation, chemicals, and radiation [10] .
HMGA2 encodes a small non histone chromatin-associated protein that has no intrinsic transcriptional activity, but can modulate transcription by altering the chromatin architecture [11,12]. HMGA2 was found overexpressed in a variety of epithelial and mesenchymal tumors [13][14][15] and promoted invasion and metastasis in most malignant epithelial tumors [16,17]. A recent study showed that P53 inhibited CRC progression by targeting HMGA2 [18] However, the mechanisms by which HMGA2 affect angiogenesis in CRC has not been clarified.
In the current study, we first detected the expression pattern of HMGA2 in CRC patients and evaluated its clinical values and CNV amplification could possibly contribute to the up regulation of HMGA2 in CRC patients. By analyzing CRC single cell data we found that HMGA2 was specifically up regulated in the colorectal epithelial cells. Furthermore, knocking down of HMGA2 suppresses angiogenesis via dual regulation of VEGF-A and SEMA3A in CRC through inactivating VEGRR2 pathway in HUVECs.

HMGA2 is overexpressed due to copy number amplification in CRC patients and associates with worsen prognosis
To investigate the potential role of HMGA2 in colorectal cancer, we first detected the expression pattern of HMGA2 in CRC patients' sample and cell lines. HMGA2 expression was markedly higher in CRC patients' sample and cell lines as detected by IHC and WB (Fig. 1A, B ,D) and this observation was further confirmed by analyzing the TCGA colorectal cancer cohort (Fig.   1C). Thus we speculated whether this abnormal expression could reflect clinical progression. We collected 154 CRC patients' sample with clinical information and found that patients featured by low differentiation and advanced clinical stage showed high expression of HMGA2( Fig. 2 A, B) which was further confirmed using public datasets (Fig. 2C). High HMGA2 expression showed worse prognosis (P < 0.05; Fig. 1D, E). Tumor angiogenesis is a vital supporter of tumor invasion and metastasis, which is responsible for the high mortality and poor prognosis [19]. The expression of CD31 could partially reflect the formation of micro vascular and denoted increased angiogenesis [18] and we found that HMGA2 protein level was positively correlated with CD31 expression in serial sections of CRC tissues from 154 cases and TCGA CRC datasets (Fig. 2F).
Due to the heterogeneity origins of CRC [20] we analyzed a CRC single cell data set (including 9 CRC patient samples) to unveil which type of cell contribute to the high expression of HMGA2. After tSNE reduction and cell type annotation (Fig. 3A, B) we found that colorectal epithelial cells showed high expression of HMGA2 among 7 types of cells (Fig. 3C). To further investigate the possible mechanisms contributed to the abnormal expression of HMGA2 in CRC.
We analyzed online data sets and found CNV was significantly disrupted in CRC (Fig. 4A) and gain of copy number denoted high expression of HMGA2 by analyzing TCGA data sets (Fig. 4 B-D), even though promoter methylation was observed however it was not correlated with gene expression (Fig. 4E). Thus the above results manifested that overexpression of HMGA2 was associated with undesired disease progression which could possible attribute to angiogenesis. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 20 October 2021

HMGA2 knock down inhibits CRC induced HUVECs metastasis in vitro and tumor formation in vivo
Previous study on HMGA2 in CRC was confined to CRC cells per se [21,22]. However, accumulating studies emphasized the micro environment in modulating the progression of tumors [23]. Thus we collected the Conditional medium (CM) from scrambled or HMGA2 shRNA lentivirus infected cells and treated HUVECs as previously described [3]. The results showed that HMGA2 shRNA CM from two CRC cell lines (Fig. 5A  . Previous studies showed that VEGF-A was a major factor that regulates angiogenesis by activating the tyrosine kinase receptor VEGFR-2 [24], and SEMA3A competitively bond to VEGFR-2 to suppress tumor angiogenesis [25]. Then we detected the expression of VEGFA and Sema3A in CRC patient tissues. We found VEGF-A level was lower while SEMA3A was up-regulated in HMGA2 low expression patients vice versa (Fig. 7D). We further found that HMGA2 was positively correlated with VEGFA and negatively correlated with Sema3A when analyzing TCGA COAD data sets (Fig. 7E).
To confirm the direct regulation of HMGA2 on VEGFA and SMA3A, we analyzed the protein expression by knocking down HMGA2 in HCT116 cells. Since previous study showed that HMGA2 could repress TGFβ signaling [26] we first confirmed this observation in our model ( Fig.   8A) and subsequently we found that HMGA2 knock down up regulated SEMA3A and inhibited VEGFA (Fig. 8A). Based on our observation that HMGA2 KD increased VEGF-A and reduced SEMA3A levels in the supernatant of HCT-116 and HT-29 colon cancer cells, we evaluated the activation of VEGFR2 pathway in HUVECs by detecting total and phosphorylation expression of VEGF-R2, ERK, and Akt in HUVECs which stimulated with conditioned medium. We found that HMGA2 KD conditioned medium from HCT-116 decrease the phosphorylation protein expression of VEGFR-2, ERK, and Akt, which are important regulator of endothelial cell function such as cell migration, endothelium-dependent relaxation, and angiogenesis (Fig. 8B). These data suggested that HMGA2 promoted angiogenesis in CRC by enhancing VEGFA and suppressing SEMA3A simultaneously, which activated VEGFR-2 on the surface of HUVECs to promote metastasis.

Patient samples
Surgically resected specimens were collected from 154 CRC cases and paired adjacent normal tissues at Third Military Medical University and Fu Ling Central Hospital from 2004 to 2007. The specimens were fixed with 4% neutral buffered paraformaldehyde and embedded in paraffin; after staining with hematoxylin and eosin, the specimens were analyzed by a senior pathologist. A semi-automated tissue microarrayer (Beecher Instruments, Sun Prairie, WI, USA) was used to construct tissue microarrays (needle diameter: 1 mm) by punching out one or two tissue cores from each specimen to obtain two paraffin blocks containing 154 CRC specimens and paired adjacent normal tissues. The study protocol was approved by the Ethical Committee of medium. All media were purchased from Gibco (Grand Island, NY, USA) and were supplemented with 10% fetal bovine serum (FBS) (Gibco). Cells were cultured at 37℃ and 5% CO2.

Conditioned medium preparation and treatment for HUVECs
Equal numbers of HCT-116 and HT-29 cells infected with lentivirus containing scrambled or HMGA2 shRNA were seeded in 100-mm dishes and allowed to attach. The medium was centrifuged at 1000 rpm for 3 min to remove any cell contaminants and termed as Conditional medium (CM). As for the treatment of HUVECs, CM collected from scrambled or HMGA2 shRNA HCT116 cells were subsequently subjected to the culture of HUVECs.

Bioinformatics analysis and public data base
TCGA COAD raw counts data was download use GDC client from https://portal.gdc.cancer.gov/. Raw data was processed in R (version 4.0.2) using Dseq2 and EdgeR package and figures were plotted using ggplot2.
Single cell analysis: The single cell data set of 9 CRC patients was retrieved from GEO GSE166555 and standard pipeline was applied for data processing using R. All clusters of cells in CRC were annotated by singleR and CellMarker according to the composition of the marker genes.

Statistical analysis
Statistical analyses were performed using SPSS v.17.0 software (SPSS Inc., Chicago, IL, USA). Kaplan-Meier survival plots and the log-rank test were used to compare patient survival rates. Differences between experimental groups and controls were assessed with the Student's test.
P<0.05 was considered statistically significant.

Discussion
Angiogenesis is the fundamental step of tumors transition from dormant to malignant state and it is recognized as one of the hallmarks of cancer. since its critical role in tumor progression, invasion, and metastasis; therefore, angiogenesis represents a rational target for therapeutic intervention [27]. A recent study showed epigenetic regulation contributed to the expression of HMGA2 in COAD [28], however our data for the first time indicated that CNV was another factor in regulating HMGA2 expression in COAD. Different strategies for angiogenesis intervention are based on modulating any of the key steps of the angiogenic process, including endothelial cell proliferation, protease secretion, cell-matrix adhesion, migration, and invasion. Rooted in the belief that blocking vessel supply starves tumors to death [29], it has become increasingly accepted that blocking tumor angiogenesis as much as possible would provide cancer patients with maximal survival benefit. It is well knowns that vascular endothelial growth factor (VEGFA) inducing tumor angiogenesis [30], it is a secreted factor via banding with tyrosine kinase VEGF receptor 2 (VEGFR2) on the vascular endothelial cells. Indeed ，the monoclonal anti-VEGF antibody bevacizumab [31,32]and the second-generation multitargeted receptor tyrosine kinase inhibitors (RTKIs) sunitinib [33] and sorafenib [34] have prolonged the life of numerous cancer patients.
However, drug resistance would be inevitably established out of unknown mechanisms. Thus, uncover novel pathways in CRC would provide potential target to inhibit tumor angiogenesis.
HMGA2 is an architectural transcription factor or organize assembly on enhancers of a variety of genes and overexpressed in a variety of epithelial and mesenchymal tumors. Since angiogenesis was closely related to metastasis, HMGA2 promotes invasion and metastasis in most malignant epithelial tumors. We first analyzed the expression of HMGA2 in colorectal cancer. Our experiments confirmed that HMGA2 was over expressed in human colorectal cancer cell and patient tissue, which also associated with poor prognosis. However, the relationship between HMGA2 and angiogenesis in CRC has not been previously reported. In the current study HMGA2 was found positively correlated with the density of CD31 in colorectal cancer patient tissues.
Furthermore, we established HMGA2-deficient HCT-116 and HT-29 cell lines and conditioned medium from the HMGA2 KD and scramble cells were applied to culture HUVECs. We found that the conditioned medium from negative controls cells promote the migration, invasion while Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 20 October 2021 HMGA2 KD inhibited theses effects. Furthermore, we showed that HMGA2 promoted CRC angiogenesis by dual regulating Sema3A and VEGFA. It is well known that VEGFA inducing tumor angiogenesis via banding with tyrosine VEGFR2 on the vascular endothelial cells [35] and SEMA3A can inhibit the binding of VEGFA to NP1 which is able to inhibit in vitro angiogenesis [25]. Thus, HMGA2 might be a promising target due to its multiple roles in compromising CRC angiogenesis.