SOHLH2 has been demonstrated the downregulation in various cancers and the involvement in tumor growth and metastasis. However, the function of SOHLH2 on tumor angiogenesis and the underlying molecular mechanisms have not been interrogated. IHC staining results revealed that SOHLH2 was negatively associated with microvessel density (MVD), tumor size, histology grade, and metastasis. Overexpression of SOHLH2 inhibited the angiogenic behavior of human umbilical vein endothelial cells (HUVEC) by a tumor cell–mediated paracrine signal, while knockdown of SOHLH2 promoted HUVEC angiogenic behavior. Ectopic SOHLH2 expression remarkably suppressed tumor growth and MVD in xenograft tumors, downregulated the expression of hypoxia inducible factor-1 alpha (HIF1α)-mediated proangiogenic genes in vivo and in vitro, while knockdown of SOHLH2 had an opposite result. Furthermore, we found that upregulation of HIF1α reversed SOHLH2-induced suppression of breast cancer angiogenesis, while KC7F2, the inhibitor of HIF1α, could attenuate the promotion of angiogenesis by SOHLH2 silencing. Using Chromatin immunoprecipitation and luciferase reporter assays, we validated that SOHLH2 could directly bind to HIF1α promoter and repress its transcriptional activity. Collectively, SOHLH2 suppresses breast cancer angiogenesis by downregulating HIF1α transcription and may be a potential biomarker for anti-angiogenesis therapy.

Implications:

SOHLH2 directly represses HIF1α–mediated angiogenesis and serves as an important inhibitor of angiogenesis in breast cancer.

Breast cancer is the most common malignancy and a major cause of cancer-induced mortality in females worldwide, bringing approximately 2,000,000 new cases and 600,000 deaths in 2018 (1). While the advancement and variety of clinical treatment, ranging from surgery following with adjuvant chemotherapies, endocrine therapy for specific receptors, and radiotherapy, the prognosis of breast cancer remains poor because of its recurrence and metastasis (2), and the therapeutic effect is also hampered because of drug resistance and the difficulty of individual treatment. The pathogenesis of breast cancer initiation and progression is still not fully illustrated. Hence, it is urgent to explore novel biomarkers involved in breast cancer progression and provide sensitive therapeutic targets to improve breast cancer patients' outcome.

Angiogenesis is a dynamic hypoxic physiologic or pathologic process formed new vessels from preexisting blood vascular beds, which is a key rate-limiting step for solid tissue growth, invasion, and metastasis (3). Angiogenesis is based on the regulation of many factors, including pro-angiogenic and anti-angiogenic factors in the tumor microenvironment, of which VEGF A (VEGFA) originated from tumor cell–mediated paracrine manner plays a significant role (4, 5). Bevacizumab, a mAb targeting VEGFA, is considered to be the first-line drug against tumor angiogenesis. Despite its indiscriminate targeting, the VEGF/VEGFR2 pathway in both healthy and tumor-associated cells, and its failing to effectively disrupt multiple of proangiogenic pathways, breast cancer still harbors poor prognosis (6). Therefore, we should take more enthusiasm to explore novel and sensitive biomarkers for anti-angiogenesis of breast cancer.

Hypoxia inducible factor-1 alpha (HIF1α) functions as an important promoter in solid tumor angiogenesis (7, 8). HIF1α belongs to HIF1 gene family. Low-level expression of HIF1α in normoxia is due to being hydroxylated and rapidly degraded via the ubiquitin-proteasome pathway (9). In the hypoxic tumor microenvironment, HIF1α protein, as hypoxia-responsive factor, could be accumulated and translocated into cell nucleus, and bind to HIF1β to activate rapidly transcriptional expression of the downstream genes, such as VEGFA, angiopoietin-like protein 4 (Angptl4), TGFβ, IL8, and erythropoietin (EPO; ref. 10). HIF1α regulates the transcription of these above target genes to change classical cellular metabolism signaling pathway for the adaption the hypoxic environment. Accumulating evidence has been confirmed that overactivation of HIF1α obviously promotes solid tumor angiogenesis, and could be a promising predictor of poor outcome for patients with cancer (11–14). Breast cancer is featured with hypervascularized solid tumor and blocking angiogenesis is an attractive way to minimize disease progression. Therefore, targeting HIF1α is generally regarded as a promising therapy strategy, with the potential for suppressing multiple pathways crucial for tumor angiogenesis (15). Hence, how to selectively block HIF1α expression and its downstream proangiogenic factors have increasingly attracted more researchers' attentions (16, 17).

A spermatogenesis- and oogenesis-specific bHLH transcription factor, SOHLH2, has been classified into bHLH transcription factor superfamily (18). The conserved bHLH domain could participate in regulation of cell biological behaviors in the manner of forming a functional homodime or heterodime transcriptional unit and targeting downstream genes. bHLH transcription factors mainly bind to the canonical E-box response element (CANNTG) located in promoter regions of target genes (19, 20). Our previous data showed SOHLH2 was expressed at higher level in normal tissues (21), whereas at lower level in numerous cancers, implying a possible role in tumorigenesis. We confirmed that SOHLH2 acted as a tumor suppressor in breast cancer (22, 23), ovarian cancer (24, 25), clear cell carcinoma of kidney, and colon cancer. In addition, SOHLH2 inhibited cell proliferation through wnt/β-catenin signaling (23) and suppressed epithelial–mesenchymal transition via the downregulation of IL8 in breast cancer (22). More recently, SOHLH2 was demonstrated that mitigated ovarian cancer cell malignant properties under hypoxia via negatively regulating the HIF1α/CA9 axis (26). Nevertheless, the regulation of SOHLH2 on breast cancer angiogenesis remains unknown.

In this study, IHC results of clinical breast cancer specimens revealed that SOHLH2 was negatively associated with microvessel density (MVD), suggesting SOHLH2 may repress the angiogenesis of breast cancer. We first found that SOHLH2 inhibited human umbilical vein endothelial cells (HUVEC) angiogenic behavior by tumor cell–mediated paracrine signaling pathway. Because of the prominent role of HIF1α on tumor angiogenesis, we hypothesis that SOHLH2 functions as a tumor suppressor to attenuate breast cancer angiogenesis through HIF1α-mediated proangiogenic signaling pathway.

Patient specimens and IHC

A total of 30 cases of primary human breast cancer specimens were collected from Qilu Hospital, Shandong University (Jinan, P.R. China). The histopathologic diagnoses were determined by expert pathologists, and the study complied with the Ethics Committee of Shandong University. The written informed consent was obtained from involved patients.

IHC was performed with procedures similar to that described previously (18). The immunostaining images were captured using an Olympus computerized image analysis system and mean optical density (MOD) values for each specimen were measured by Image-pro plus software.

Cell culture and reagents

Human breast cancer cells (MDA-MB-231 and MCF-7) and HUVECs were obtained from the Cell Banks of Type Culture Collection of Chinese Academy of Sciences (Shanghai, P.R. China) and the ATCC. MDA-MB-231 and MCF-7 grown in RPMI1640 and DMEM supplemented with 10% FBS (Biological Industries), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO2 humidified atmosphere. The number of cell line passages used in the experiments was 20 to 26 passages for MDA-MB-231, 16 to 23 for MCF-7, and 3 to 8 for HUVECs. All cell lines were authenticated by short tandem repreat analysis. Testing for Mycoplasma infection was performed using the MycoAlert Mycoplasma Detection Kit (Lonza) and the final inspection date was March 12, 2021. Lentivirus SOHLH2 or ShSOHLH2 were established (Genechem) and then transfected into breast cancer cells according to the manufacturer's guidelines. The stably cell lines were selected by using 1 μg/mL puromycin (Sigma-Aldrich) for 14 days. HUVECs were maintained at 37°C in endothelial cell medium (ECM) (ScienceCell) containing 10% FBS and endothelial cell growth supplement. Cobalt chloride (CoCl2) was purchased from Sigma-Aldrich and added into culture medium at a concentration of 100 μM for 24 hours. KC7F2 (APExBIO), the inhibitor of HIF1α, could block HIF1α-mediated proangiogenic signaling pathway at 10 μM for 12 hours.

Tumor cell conditioned medium and ELISA

Breast cancer cells were seeded at 3 × 105 per well in 6-well plate. After adhesion, cells were gently washed three times by PBS and continued to culture in fresh serum-free medium for 24 hours. The supernatants were collected and then centrifugated at 3,000 rpm for 10 minutes to remove cells and cell debris, stored at −80°C until used as tumor cell conditioned medium (TCM). VEGFA and Angptl4 in TCM were monitored by utilizing human VEGFA ELISA Kit (R&D Systems) and human Angptl4 ELISA Kit (Bioswamp) according to the manufacturer's instructions.

Cell counting kit-8 assay

To observe the effect of tumor paracrine factors on endothelial cell proliferation, cell counting kit-8 (CCK8) assay was carried out. HUVECs were digested and the cellular density was adjusted to 3 × 105/mL. A total of 100 μL cell suspension was seeded into a 96-well plate and incubated at 37°C in 5% CO2 humidified atmosphere for adhesion. The medium was replaced with TCM and continued to cultivate for 48 hours. HUVEC viability was monitored by Cell Counting Kit-8 (Dojindo Laboratories) at 450 nm absorbance. Each sample was repeated in triplicate.

Cell cycle and apoptosis analysis

To mimic the interaction between breast cancer cells and endothelial cells, co-culture system and flow cytometry were implemented to explore endothelial cell cycle and apoptosis. First, transwell inserts with 0.4-μM pores (Corning) were deposited in 6-well plates, the filters merely allowed the exchange of medium cytokines and inhibited cell migration. Second, HUVECs were seeded in the lower chamber and incubated at 37°C for cell adhesion, and then breast cancer cells were added into the inserts. After 48 hours of interaction, HUVECs were harvested for further endothelial function study.

For cell-cycle analysis, HUVECs were suspended by 70% ethanol and fixed at 4°C overnight, then incubated with PI-RNase staining solution (Solarbio Life Science) for 1 hour in the darkness. For cell apoptosis analysis, HUVECs were double stained by Annexin V-FITC and propidium iodide (PI) buffer for 10 minutes in the darkness (Vazyme). The flow cytometry (Beckman Coulter) was used to measure cell-cycle population and cell apoptosis, and finally these results were analyzed by FlowJo software.

Migration assay

To explore the function of tumor paracrine pathway on HUVEC migration, we performed transwell assay using chambers with 8-μm pore size filters (Corning). A total of 3 × 104 HUVECs suspended in 200 μL TCM were placed in the top chamber, while 500 μL medium containing 10% FBS was added to the low chamber. After 24 hours incubation, the top chambers were removed and the migration cells on the bottom surface were fixed with 4% formaldehyde for 20 minutes, stained by 0.5% crystal violet for 15 minutes and captured in five randomly chosen fields. Each sample was repeated in triplicate.

Tube formation assay

To assess whether tumor paracrine pathway affects endothelial cell function, 60 μL Matrigel was laid into a 96-well plate and polymerized at 37°C for 30 minutes. Then, 3 × 104 HUVECs were seeded onto Matrigel and incubated at 37°C in 5% CO2 humidified atmosphere. After observing cell adhesion, the medium was removed and replaced with TCM. After 4 hours incubation, the capillary-like tubes were photographed from at least five randomly chosen fields under an inverted microscope (Olympus). The number of formed tubular structures were quantified by Image J software.

Western blot analysis

To investigate the expression of proteins, cells were pelleted and lysed in RIPA lysis buffer (Solarbio Life Science) supplemented with 1% protease inhibitor cocktails (Sigma-Aldrich). Frozen tumor tissues were grinded and washed three times by PBS, then resuspended in RIPA lysis buffer. After 0.5 hours of incubation on ice, cellular debris was removed by centrifugation at 12,000 rpm for 10 minutes at 4°C. Proteins concentration was measured by using BCA protein Assay (Thermo Fisher Scientific). Total 30 μg proteins were separated by 12% SDS-polyacrylamide gel, transferred onto polyvinylidene fluoride membranes (Millipore Corp). After blocking with 5% fat-free milk for 2 hours at room temperature, then membranes were incubated with primary antibodies (1:5,000–1:10,000) overnight at 4°C, followed by peroxidase-conjugated secondary antibodies for 2 hours at room temperature. The immune complex was visualized by an enhanced chemiluminescence kit (Thermo Fisher Scientific). β-Actin antibody was used to monitor the loading amount. Antibody to SOHLH2 was purchased from Novus Biologicals), and other antibodies were obtained from Affinity Biosciences.

Quantitative real-time PCR

Total RNA was extracted using TRIzol reagent (Invitrogen) following the manufacturer's instructions. One microgram of total RNA was reverse transcribed using Reverse Transcriptase kit (Thermo Fisher Scientific). Quantitative real-time PCR (qPCR) was performed with Ultra SYBR Mixture (ComWin Biotech) on a CFX96 Real Time PCR Detection System (Bio-Rad).Each sample was practiced in triplicate. β-Actin was used as an internal control for all samples.

Immunofluorescence

Immunofluorescence staining was practiced with procedures similar to that described previously (22).

Luciferase reporter assay

Dual Luciferase Reporter Assay kit (Promega) was used to assess luciferase activity of HIF1α. HIF1α promoter firefly luciferase reporter plasmid containing nucleotides −2180 to +28 (27) was obtained from Addgene. MDA-MB-231–expressing higher SOHLH2 and MCF-7–silencing-SOHLH2 cells were seeded in 24-well plate. A total of 200 ng HIF1α plasmid and 17 ng Renilla luciferase plasmid were cotransfected using Lipofectamine 2000 (Invitrogen) when cell coverage reached 70%. The luciferase activity of HIF1α was monitored by a dual-luciferase reporter assay system (Promega). Data were presented as the ratio of firefly to Renilla values and normalized to vector control.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (CHIP) assay was carried out as described previously (28). MDA-MB-231–transfected SOHLH2 plasmid and its control group cells were prepared for CHIP assay following the manufacturer's instructions (Cell Signaling Technology). Briefly, chromatin fragments solutions were incubated overnight with 30 μL anti-SOHLH2 antibodies or the rabbit IgG at 4°C with rotation state. The precipitated DNAs were eluted, separated by magnetic separator, purified using spin columns, and finally analyzed by qPCR. The specific primer pairs for human HIF1α promoter were as follows: 5′-CTTTCCTCCGCCGCTAAACA-3′ and 5′-CTCGAGATCCAATGGCGAGC-3′.

Tumor xenograft model

For further exploring the effect of SOHLH2 in tumor angiogenesis, tumor xenograft model was established following the institutional instructions, and the experimental protocols were performed complied with the policy on the Ethical Use and Care of Animals (School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, P.R. China). All 6 or 7 weeks old female nude mice were purchased from Charles River Laboratories and raised under the pathogen-free mouse facility. Each group was randomly assigned to 4 nude mice. A total of 5 × 106/100 μL MDA-MB-231 stably expressing SOHLH2 or the control cells, MCF-7–ablated-SOHLH2 or short hairpin RNA control cells were injected into the fourth right mammary fat pad with 50% Matrigel (BD Biosciences). Tumor size was measured every 3 days for 4 weeks and tumor volume was calculated: volume (cm3) = (length × width2) × 0.5. All nude mice were euthanized and tumors were removed, weighted, and embedded in paraffin for IHC analysis.

Statistical analysis

Quantitative values were presented as mean ± SD. SPSS and Prism 7 (GraphPad Software) software were applied for data analysis. The comparison between groups was analyzed by χ2 test, Student t test, or one-way ANOVA. Bivariate relationship between measured variables was calculated by Spearman rank correlation coefficients. P < 0.05 was considered statistically significant.

SOHLH2 expression is negatively associated with MVD in human breast cancer tissues

To evaluate the role of SOHLH2 in breast cancer angiogenesis, IHC staining assay was used to analyze the correlation among SOHLH2 expression and MVD (as measured by CD31, vWF—the two vascular endothelial markers), and clinical characteristics in 30 cases of patients with breast cancer. SOHLH2 immunostaining localized in the cytoplasm and nuclei of tumor cells. Interestingly, SOHLH2 expression was rarely observed in the nuclei of advanced stage breast cancer tissues. We found that breast cancer tissues with higher SOHLH2 level were associated with lower CD31, vWF expression, while these tissues expressing lower SOHLH2 were accompanied by higher CD31, vWF expression (Fig. 1A). In addition, the Spearman test was performed to evaluate the association between SOHLH2 and CD31, and the coefficient was −0.772, showing a strongly negative relationship between SOHLH2 and MVD (Fig. 1B). These results indicated that SOHLH2 may suppress breast cancer angiogenesis.

Figure 1.

SOHLH2 expression is negatively correlated with MVD in human breast cancer. A, IHC staining of SOHLH2, CD31, and vWF in breast cancer samples. The representative images from two cases were shown. Original magnification: × 200 and × 400. Scale bar indicates 50 μm (top) and 20 μm (bottom). The immunostaining of SOHLH2, CD31, and vWF was calculated by MOD scores using Image Pro-plus software. The correlation of SOHLH2 and CD31 (B) was analyzed by the Spearman test. C,The expression of SOHLH2 in low histology grade tumor (n = 17) was significantly stronger than that in advanced histology grade tumor (n = 13). *, P < 0.05; **, P < 0.01.

Figure 1.

SOHLH2 expression is negatively correlated with MVD in human breast cancer. A, IHC staining of SOHLH2, CD31, and vWF in breast cancer samples. The representative images from two cases were shown. Original magnification: × 200 and × 400. Scale bar indicates 50 μm (top) and 20 μm (bottom). The immunostaining of SOHLH2, CD31, and vWF was calculated by MOD scores using Image Pro-plus software. The correlation of SOHLH2 and CD31 (B) was analyzed by the Spearman test. C,The expression of SOHLH2 in low histology grade tumor (n = 17) was significantly stronger than that in advanced histology grade tumor (n = 13). *, P < 0.05; **, P < 0.01.

Close modal

To further explore the regulation of SOHLH2 on breast cancer pathogenesis, we assigned breast cancer samples to two groups: SOHLH2 high (n = 17) and SOHLH2 low (n = 13), according to MOD scores of SOHLH2 IHC staining. Among the 30 cases of breast cancer samples, SOHLH2 was mostly higher level expression in grade I and II samples (76.47%), whereas the lower expression was found in grade III and IV samples (69.23%; Fig. 1C). The correlations between SOHLH2 expression and clinical pathologic characteristics of 30 cases of patients with breast cancer were exhibited in Supplementary Table S1. SOHLH2 expression was remarkably negatively associated with tumor size, providing more evidences to indicate that SOHLH2 represses the angiogenesis and leads to slow down the growth of breast cancer. Furthermore, SOHLH2 expression was higher in non-metastasis tissues compared with metastasis tissues.

SOHLH2 inhibits the angiogenic function of HUVECs by tumor cell–mediated paracrine mechanism

It is well known that tumor cells could secrete proangiogenic factors to promote the formation and stabilization of tumor neovascularization, mainly regulate endothelial cell behaviors. To further evaluate the effect of SOHLH2 on breast cancer angiogenesis and whether SOHLH2 has potential to regulate endothelial cell function through tumor cell–mediated paracrine pathway, we constructed co-cultural system or collected TCM to mimic the interaction between tumor cells and HUVECs under tumor microenvironment. We established ectopic SOHLH2 expression in MDA-MB-231 cells with lower endogenous SOHLH2 level and SOHLH2 knockdown in MCF-7 cells with higher endogenous SOHLH2 by lentivirus transfection using three independent siRNAs (Fig. 2A and B).

Figure 2.

The effects of SOHLH2 in breast cancer cells on the angiogenic behaviors of HUVECs. The gain-of-function and loss-of-function assays were carried to survey the function of SOHLH2 on tumor cell–induced angiogenic promotion. Established co-cultural system and collected TCM from indicated tumor cells were used to evaluate the effect of SOHLH2 on HUVECs. A, Schematic diagram of sample treatment, TCM collection, and co-cultural system construction. B, Western blot analysis of breast cancer cells transfected with SOHLH2 or control vector, and the quantitative results were shown in the right panel. C, Cell proliferation was determined by CCK-8 assay in co-cultural system for 48 hours. D, Flow cytometry was utilized to analyze the proliferation of HUVECs, the representative plots were shown. E, The quantification results of cell cycle were presented in the right panel. F, Flow cytometry was practiced to monitor the apoptosis of HUVECs in co-cultural system for 48 hours, and the percentage of apoptotic cells was measured. G, The quantification results of apoptotic cells were shown in the right panel. H, Transwell migration assay was performed to assess the migration capability of HUVECs treated with TCM from SOHLH2-MDA-MB-231 or si-SOHLH2 MCF-7 cells. The typical images were presented. I, The quantification results of migration cells were presented. J, Tube formation was evaluated in HUVECs cultured with TCM of SOHLH2-MDA-MB-231 or si-SOHLH2 MCF-7. K, Quantification results of tube number were presented in the right panel. Results were presented as mean ± SD of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

The effects of SOHLH2 in breast cancer cells on the angiogenic behaviors of HUVECs. The gain-of-function and loss-of-function assays were carried to survey the function of SOHLH2 on tumor cell–induced angiogenic promotion. Established co-cultural system and collected TCM from indicated tumor cells were used to evaluate the effect of SOHLH2 on HUVECs. A, Schematic diagram of sample treatment, TCM collection, and co-cultural system construction. B, Western blot analysis of breast cancer cells transfected with SOHLH2 or control vector, and the quantitative results were shown in the right panel. C, Cell proliferation was determined by CCK-8 assay in co-cultural system for 48 hours. D, Flow cytometry was utilized to analyze the proliferation of HUVECs, the representative plots were shown. E, The quantification results of cell cycle were presented in the right panel. F, Flow cytometry was practiced to monitor the apoptosis of HUVECs in co-cultural system for 48 hours, and the percentage of apoptotic cells was measured. G, The quantification results of apoptotic cells were shown in the right panel. H, Transwell migration assay was performed to assess the migration capability of HUVECs treated with TCM from SOHLH2-MDA-MB-231 or si-SOHLH2 MCF-7 cells. The typical images were presented. I, The quantification results of migration cells were presented. J, Tube formation was evaluated in HUVECs cultured with TCM of SOHLH2-MDA-MB-231 or si-SOHLH2 MCF-7. K, Quantification results of tube number were presented in the right panel. Results were presented as mean ± SD of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Close modal

The proliferation of endothelial cells is a key step of tumor angiogenesis. First, co-culture system between SOHLH2-MDA-MB-231 (si-SOHLH2 MCF-7) and HUVECs was maintained for 48 hours; CCK8 assay and flow cytometry were used to examine the function of SOHLH2 on proliferation of HUVECs in the manner of paracrine mechanism. The results showed that overexpression SOHLH2 significantly inhibited HUVEC proliferation by hampering cell-cycle transition from G1- to S-phase, whereas knockdown of SOHLH2 promoted HUVEC proliferation by inducing cell-cycle progression (Fig. 2C–E). Furthermore, the results of flow cytometry showed SOHLH2 slightly induced the apoptosis of HUVECs via tumor cell–mediated paracrine signaling pathway, while HUVEC apoptosis was repressed when SOHLH2 expression was silenced (Fig. 2F and G). In addition, the migration and tube formation of endothelial cells are also an indispensable part of tumor angiogenesis. The consequences of transwell migration assay showed forced expression of SOHLH2 in MDA-MB-231 cells attenuated the migration ability of HUVECs with treatment of TCM compared with the control group, whereas silencing SOHLH2 expression in MCF7 cells had an opposite effect on migration (Fig. 2H and I). TCM-mediated angiogenesis was also evaluated by tube formation on Matrigel. The results of tube formation demonstrated that enhanced SOHLH2 expression in MDA-MB-231 cells suppressed the tube formation of HUVECs, while SOHLH2 silenced in MCF7 cells promoted tube formation (Fig. 2J and K). Taken together, these data support the fact that SOHLH2 exerts the inhibition effect on angiogenic behavior of HUVECs through breast tumor cell–mediated paracrine signaling pathway.

SOHLH2 suppresses significantly tumor growth and angiogenesis in xenograft tumors

To further verified these above findings in vitro studies, mouse xenograft model was established, we orthotopically injected 5 × 106 MDA-MB-231 cells overexpressing SOHLH2 or MCF-7 cells silencing SOHLH2 and its respective control cells into the right fourth fat pad of nude mice. After 4 weeks of injection, the tumors were excised. The regulation of SOHLH2 on breast cancer angiogenesis in vivo was explored. Compared with the control group, the overexpression of SOHLH2 remarkably suppressed MDA-MB-231 cell tumorigenesis, while the silencing of SOHLH2 in MCF-7 cells accelerated tumor growth in vivo (Fig. 3A–C). These results were in line with the negative association SOHLH2 expression with tumor size in breast cancer clinical specimens. IHC results demonstrated that the correlation between the expression of SOHLH2 and CD31, vWF was negative, while the knockdown of SOHLH2 enhanced the percentage of CD31, vWF positive cells in xenografts, presenting the same trend with breast cancer tissues expressing MVD (Fig. 3D–F). These data indicate SOHLH2 represses breast cancer growth and tumor angiogenesis in xenografts.

Figure 3.

The suppression of SOHLH2 on tumor growth and angiogenesis in vivo. A, Images of tumors derived from the control and SOHLH2 overexpression MDA-MB-231 or the control siRNA and SOHLH2 knockdown MCF-7 groups were shown. n = 4. B, The quantitative results of tumor weight in ectopic SOHLH2 expression MDA-MB-231 xenografts and SOHLH2-ablated MCF-7 xenografts, compared with their control groups, respectively. C, Tumor growth curve was depicted over a 4-week period. D, The representative images of SOHLH2, CD31, vWF immunostaining in adjacent section of above four groups. Scale bar indicates 20 μm. E and F, On the basis of CD31 or vWF immunostaining results, the MVD in xenografts was assessed in at least three independent microscopic field within each tumor (n = 4 independent experiments). *, P < 0.05; **, P < 0.01.

Figure 3.

The suppression of SOHLH2 on tumor growth and angiogenesis in vivo. A, Images of tumors derived from the control and SOHLH2 overexpression MDA-MB-231 or the control siRNA and SOHLH2 knockdown MCF-7 groups were shown. n = 4. B, The quantitative results of tumor weight in ectopic SOHLH2 expression MDA-MB-231 xenografts and SOHLH2-ablated MCF-7 xenografts, compared with their control groups, respectively. C, Tumor growth curve was depicted over a 4-week period. D, The representative images of SOHLH2, CD31, vWF immunostaining in adjacent section of above four groups. Scale bar indicates 20 μm. E and F, On the basis of CD31 or vWF immunostaining results, the MVD in xenografts was assessed in at least three independent microscopic field within each tumor (n = 4 independent experiments). *, P < 0.05; **, P < 0.01.

Close modal

SOHLH2 downregulates HIF1α-mediated proangiogenic signaling pathway in breast cancer cells and xenografts

Tumor angiogenesis is a dynamic hypoxia-stimulated process, HIF1α plays an essential role in mediating tumor angiogenesis via targeting proangiogenic genes to regulate angiogenic procession. Previous RNA-sequencing analysis screened out a series of differentially expressed genes in SOHLH2-silenced MCF-7 cells compared with the control group, including HIF1α and its downstream proangiogenic factors (VEGF, IL8; data not shown). These data prompt us to explore the effects of SOHLH2 on HIF1α-mediated proangiogenic signaling pathway, the mRNA and protein levels of HIF1α and its downstream target genes were detected. The results of qPCR showed that decreased mRNA expression of HIF1α, VEGFA, Angptl4, EPO, IL8, and TGFβ were found in SOHLH2 overexpression MDA-MB-231 cells, whereas elevated expression of these genes was observed in SOHLH2-ablated MCF-7 cells (Fig. 4A and B). Western blot analysis showed SOHLH2 inhibited the protein levels of HIF1α, VEGFA, and Angptl4, while SOHLH2 silencing induced these protein level upregulation (Fig. 4C and D). As shown in Fig. 4E, the secretion of VEGFA was repressed by ectopic expression of SOHLH2 in MDA-MB-231 cells, while increased by the knockdown of SOHLH2 in MCF-7 cells. However, the secretion of Angptl4 from MDA-MB-231 cells overexpressing SOHLH2 was slightly reduced, and the reduction of SOHLH2 did not change its secretion. In addition, the results of immunofluorescent staining showed that SOHLH2 reduced the cytoplasm distribution of VEGFA and Angptl4 and an opposite effect was presented in SOHLH2 knockdown group (Supplementary Fig. S1). Collectively, these data suggest that HIF1α is required in SOHLH2 negative regulation of breast cancer angiogenesis.

Figure 4.

The suppression of SOHLH2 on HIF1α-mediated proangiogenic signaling pathway in vitro and in vivo. A and B, qPCR was used to assay the mRNA expression of HIF1α and its downstream target genes VEGFA, Angptl4, EPO, IL8, TGFβ in SOHLH2 overexpression MDA-MB-231 cells and SOHLH2 knockdown MCF-7 cells. C, Western blot analysis was used to detect the protein levels of HIF1α, VEGFA, Angptl4 in SOHLH2 overexpression MDA-MB-231 cells and SOHLH2 knockdown MCF-7 cells, the quantitative results were shown in D. E, The VEGFA and Angptl4 secretion level in the supernatant of indicated transfected breast cancer cells were measured by ELISA, the quantitative results were shown. F and G, qPCR was performed to assay the mRNA expression of HIF1α, and its downstream genes VEGFA, Angptl4, EPO, IL8, TGFβ in MDA-MB-231 and MCF-7 xenografts when SOHLH2 was upregulated or downregulated by lentivirus transfection. H and I, Western blot analysis was used to detect HIF-1α, VEGFA, Angptl4 protein expression in SOHLH2 overexpression MDA-MB-231 and SOHLH2–ablated MCF-7 xenografts, the quantitative results were presented. Data were presented as mean ± SD of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, non significant.

Figure 4.

The suppression of SOHLH2 on HIF1α-mediated proangiogenic signaling pathway in vitro and in vivo. A and B, qPCR was used to assay the mRNA expression of HIF1α and its downstream target genes VEGFA, Angptl4, EPO, IL8, TGFβ in SOHLH2 overexpression MDA-MB-231 cells and SOHLH2 knockdown MCF-7 cells. C, Western blot analysis was used to detect the protein levels of HIF1α, VEGFA, Angptl4 in SOHLH2 overexpression MDA-MB-231 cells and SOHLH2 knockdown MCF-7 cells, the quantitative results were shown in D. E, The VEGFA and Angptl4 secretion level in the supernatant of indicated transfected breast cancer cells were measured by ELISA, the quantitative results were shown. F and G, qPCR was performed to assay the mRNA expression of HIF1α, and its downstream genes VEGFA, Angptl4, EPO, IL8, TGFβ in MDA-MB-231 and MCF-7 xenografts when SOHLH2 was upregulated or downregulated by lentivirus transfection. H and I, Western blot analysis was used to detect HIF-1α, VEGFA, Angptl4 protein expression in SOHLH2 overexpression MDA-MB-231 and SOHLH2–ablated MCF-7 xenografts, the quantitative results were presented. Data were presented as mean ± SD of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, non significant.

Close modal

To further validate the prominent role of HIF1α on SOHLH2 regulation breast cancer angiogenesis, the changes of HIF1α-mediated proangiogenic factors were analyzed in xenografts. Consistent with the above described data in breast cancer cells, overexpression of SOHLH2 significantly inhibited the expression of HIF1α, VEGFA, Angptl4, EPO, IL8, and TGFβ at mRNA and protein levels, and knockdown of SOHLH2 enhanced the mRNA and protein expression of these proangiogenic genes in vivo (Fig. 4FI). The observation of IHC staining of HIF1α, VEGFA, and Angptl4 was coherent with the results of Western blot assay. More importantly, ectopic SOHLH2 expression in MDA-MB-231 xenografts decreased the nuclear HIF1α protein level, whist nuclear HIF1α protein level were enhanced when SOHLH2 was silence in MCF-7 cells bearing xenografts (Supplementary Fig. S2). Altogether, these data suggest HIF1α-mediated proangiogenic signaling pathway is involved in SOHLH2-induced suppression of tumor angiogenesis in vivo and in vitro.

HIF1α mediates the effects of SOHLH2 on breast cancer angiogenesis

To verify whether SOHLH2 directly regulates the transcription of HIF1α to reduce breast cancer angiogenesis, the HIF1α promoter was inserted into a PGL 4.20 plasmid and luciferase reporter assay was performed in forced SOHLH2 expression MDA-MB-231 cells and silenced SOHLH2 expression MCF-7 cells. The results demonstrated SOHLH2 significantly inhibited the transcriptional activity of HIF1α promoter, while knockdown of SOHLH2 stimulated the transcriptional activity of HIF1α promoter (Fig. 5A). CHIP assay was carried out to identify the presence of the interacting HIF1α promoter fragments with SOHLH2 in MDA-MB-231 cells. The results revealed that SOHLH2 could specifically bind to the promoter of HIF1α by anti-SOHLH2 antibody but not control IgG antibody (Fig. 5B). Above these results, we concluded that SOHLH2 repressed the breast cancer angiogenesis through directly inhibiting HIF1α transcription.

Figure 5.

HIF1α mediates the effect of SOHLH2 on angiogenesis of breast cancer. Forced SOHLH2 expression MDA-MB-231 cells were cultured with CoCl2 (100 μM) for 24 hours, silenced SOHLH2 expression MCF-7 cells were treated with KC7F2 (10 μM) for 12 hours. A, Luciferase report assay was used to detect the function of SOHLH2 on transcriptional activity of HIF1α in breast cancer cells. B, CHIP assay was performed in ectopic SOHLH2 expression MDA-MB-231 cells, qPCR was used to determine the binding of SOHLH2 to HIF1α promoter. C, HUVEC tube formation on Matrigel treated with TCM. CoCl2 enhanced the tube formation of HUVECs in ectopic SOHLH2 expression MDA-MB-231 cells. The quantitative results of tube number were shown in the right panel. D and E, Forced HIF1α expression stimulated tube formation in SOHLH2 overexpression MDA-MB-231 cells. KC7F2, the inhibitor of HIF1α, inhibited tube formation in SOHLH2-silenced MCF-7 cells. The typical images of tube formation were shown and quantitative results of tube number were presented. F–I, CoCl2 or HIF1α overexpression could reverse the inhibition effect of SOHLH2 on mRNA and protein levels of proangiogenic factors (VEGFA and Angptl4) in MDA-MB-231 cells. KC7F2, the inhibitor of HIF1α, could attenuate the upregulation protein levels of VEGFA, Angptl4 in SOHLH2-ablated MCF-7 cells. The quantitative results of protein expression were shown in the bottom panel of corresponding figures. All results were obtained from at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.

HIF1α mediates the effect of SOHLH2 on angiogenesis of breast cancer. Forced SOHLH2 expression MDA-MB-231 cells were cultured with CoCl2 (100 μM) for 24 hours, silenced SOHLH2 expression MCF-7 cells were treated with KC7F2 (10 μM) for 12 hours. A, Luciferase report assay was used to detect the function of SOHLH2 on transcriptional activity of HIF1α in breast cancer cells. B, CHIP assay was performed in ectopic SOHLH2 expression MDA-MB-231 cells, qPCR was used to determine the binding of SOHLH2 to HIF1α promoter. C, HUVEC tube formation on Matrigel treated with TCM. CoCl2 enhanced the tube formation of HUVECs in ectopic SOHLH2 expression MDA-MB-231 cells. The quantitative results of tube number were shown in the right panel. D and E, Forced HIF1α expression stimulated tube formation in SOHLH2 overexpression MDA-MB-231 cells. KC7F2, the inhibitor of HIF1α, inhibited tube formation in SOHLH2-silenced MCF-7 cells. The typical images of tube formation were shown and quantitative results of tube number were presented. F–I, CoCl2 or HIF1α overexpression could reverse the inhibition effect of SOHLH2 on mRNA and protein levels of proangiogenic factors (VEGFA and Angptl4) in MDA-MB-231 cells. KC7F2, the inhibitor of HIF1α, could attenuate the upregulation protein levels of VEGFA, Angptl4 in SOHLH2-ablated MCF-7 cells. The quantitative results of protein expression were shown in the bottom panel of corresponding figures. All results were obtained from at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Close modal

To further examine whether HIF1α could mediate the effect of SOHLH2 on the inhibition of tube formation and proangiogenic genes, CoCl2 (100 μM) was used to imitate the hypoxic tumor microenvironment for the upregulation of HIF1α protein level in ectopic SOHLH2 expression MDA-MB-231 cells. The results of tube formation on Matrigel showed that hypoxia distinctly abrogated SOHLH2-reduced tube formation. Conversely, KC7F2 (10 μM), the inhibitor of HIF1α, was added into SOHLH2 knockdown MCF-7 cells (Fig. 5C). We transfected HIF1α plasmid in the cells overexpressed SOHLH2 for avoiding the other effects of CoCl2, the repression of SOHLH2-induced tube formation was notably rescued upon HIF1α upregulation. KC7F2 remarkably attenuated the increased tube formation caused by the decrease of SOHLH2 expression via SOHLH2 siRNA (Fig. 5D and E). As expected, SOHLH2 on the inhibition mRNA and protein levels of VEGFA, Angptl4 was abolished by upregulation HIF1α, nonetheless the promotion of these protein by the knockdown of SOHLH2 was attenuated after treatment with KC7F2(Fig. 5F–I). Altogether, these data indicate that HIF1α and its downstream proangiogenic genes are important targets of SOHLH2 and mediate the function of SOHLH2-induced inhibition of breast cancer angiogenesis (Supplementary Fig. S3).

SOHLH2 suppresses the expression of HIF1α, VEGFA, and Angptl4 in human breast cancer tissues

All the above data showed that SOHLH2 could suppress HIF1α and HIF1α-inducible VEGFA, Angptl4 genes in vitro and in vivo to regulate the angiogenesis of breast cancer. To further clarify these results, the expression of SOHLH2 and HIF1α, VEGFA, Angptl4 was measured in human breast cancer tissues by IHC. As expected, SOHLH2 was negatively correlation with HIF1α, VEGFA, Angptl4 in clinical samples. The samples expressing higher SOHLH2 had a lower HIF1α, VEGFA, and Angptl4 expression, while the samples expressing lower SOHLH2 were accompanied by a higher expression of HIF1α, VEGFA, and Angptl4 (Fig. 6A and B). The results of Spearman test declared the strongly negative relationship among SOHLH2 and HIF1α, VEGFA, and Angptl4 in breast cancer samples. The coefficient of SOHLH2 and HIF1α, VEGFA, Angptl4 was −0.762, −0.774, and −0.710(Fig. 6C–E). On the basis of these above results, we confirmed that SOHLH2/HIF1α signaling pathway may play an important role in the pathogenesis of breast cancer angiogenesis.

Figure 6.

Correlation analysis of SOHLH2 and HIF-1α, VEGFA, Angptl4 expression in clinical breast cancer samples (A and B) Representative images of IHC staining of SOHLH2 and HIF1α, VEGFA, Angptl4 in two cases of breast cancer samples. These images were captured under ×100 (top) and ×400 (bottom) visual fields. Scale bars indicated 100 μm (top) and 20 μm (bottom). C–E, The scatter plot was used to show the correlation between SOHLH2 and HIF1α, VEGFA, Angptl4 expression.

Figure 6.

Correlation analysis of SOHLH2 and HIF-1α, VEGFA, Angptl4 expression in clinical breast cancer samples (A and B) Representative images of IHC staining of SOHLH2 and HIF1α, VEGFA, Angptl4 in two cases of breast cancer samples. These images were captured under ×100 (top) and ×400 (bottom) visual fields. Scale bars indicated 100 μm (top) and 20 μm (bottom). C–E, The scatter plot was used to show the correlation between SOHLH2 and HIF1α, VEGFA, Angptl4 expression.

Close modal

The initiation and progression of breast cancer are a complex process, interfering multiple overactivated oncogenes and inactivated tumor suppressors. SOHLH2, a bHLH transcription factor, is downregulated and involved in pathogenesis of many cancers, such as breast cancer, colon cancer, and ovarian cancer. It has been reported that SOHLH2 inhibits cancer cell proliferation, migration, invasion, and metastasis of breast cancer. In the current study, we found SOHLH2 expression was negatively associated with MVD, tumor size, histology grade, and metastasis in human breast cancer tissues. In addition, SOHLH2 inhibited tumor growth and angiogenesis in MDA-MB-231 tumor-bearing nude mice, while the opposite effects were observed in SOHLH2-silenced MCF-7 xenografts. Overall, these results offer a forceful clue that SOHLH2 may suppress breast cancer angiogenesis and provide a favorable biomarker target for anti-angiogenesis therapy.

Angiogenesis is well known to play an essential role in tumor progression, transporting enough blood and nutrients to solid tumors and resulting in unrestrained growth and metastasis (3). Accumulating evidences have been demonstrated that tumor cells could express and secrete proangiogenic factors to regulate endothelial cell proliferation, migration, and tube formation (29). Moreover, several studies have reported that the factors derived from endothelial cells stimulate the tumor cell proliferation and the aggressive phenotype of breast cancer cells (30–32). In view of the effects of bHLH transcription factors on cellular differentiation, cell-cycle arrest, and apoptosis (19), we first evaluated whether SOHLH2 affected endothelial cell functions through tumor cell–mediated paracrine pathway and eventually inhibited breast cancer angiogenesis. Co-culture system and TCM collection were used to mimic the interaction of breast cancer cells and HUVECs in tumor environment. We found ectopic SOHLH2 expression in MDA-MB-231 cells significantly suppressed HUVEC proliferation, migration, and tube formation on Matrigel, slightly enhanced HUVEC apoptosis, whereas ablated SOHLH2 in MCF-7 cells improved HUVEC angiogenic behavior. Among these proangiogenic factors secreted from tumor cells, VEGFA plays an important role in the cross-talk between tumor cells and endothelial cells (33). We subsequently measured VEGFA concentrations in TCM by ELISA assay. SOHLH2 repressed VEGFA secretion in MDA-MB-231 cells, while SOHLH2-silenced MCF-7 cells stimulated VEGFA secretion. These results provided more evidence that SOHLH2 inhibited breast cancer angiogenesis due to it negatively regulated endothelial cells behaviors through breast tumor cell–mediated paracrine signaling pathway.

HIF1α is a master regulator in initiation and progression of solid tumors, leading to high permeability pathologic vascular network formation (34). HIF1α is common and overexpression in many malignancy, such as brain (35), oropharynx (36), breast (37), cervix (38), ovary (39), and uterus (40) cancers, presenting remarkable association with poor prognosis. This prompts us to explore whether SOHLH2 regulates tumor angiogenesis through HIF1α-mediated proangiogenic signaling pathway. In the current study, we identified that HIF1α was involved in the inhibitory effect of SOHLH2 on breast cancer angiogenesis. Several articles have confirmed that Stra13/DEC1, a bHLH transcription factor, is tightly associated with HIF1α expression in many kinds of tumors (41, 42). Li and colleagues found that Stra13/DEC1 was an upstream regulator of HIF1α and stabilized HIF1α proteins (43). Consistent with these above data, SOHLH2 restrained the mRNA and protein levels of HIF1α and its downstream proangiogenic genes [VEGFA, Angptl4 (44), EPO, IL8, TGFβ] in vitro and in vivo; however, knockdown of SOHLH2 promoted HIF1α and its target genes mRNA and protein levels. These results hint us that SOHLH2 regulates HIF1α expression at transcriptional level. In clinical breast cancer specimens, higher SOHLH2 expression was accompanied by lower HIF1α, VEGFA, and Angptl4 expression, while lower SOHLH2 expression was associated with higher HIF1α, VEGFA, and Angptl4 expression, suggesting that SOHLH2/HIF1α signaling pathway plays an important role in the pathogenesis of breast cancer angiogenesis. Of note, we surprisingly observed that SOHLH2 was mostly expressed in the cytoplasm, but nuclear SOHLH2 was markedly reduced in breast cancer tissues with the advanced histology grade. Nevertheless, HIF1α nuclear translocation was more in the advanced histology grade tumor compared with low histology grade tumor. The results indicate that the advanced grade tumor cells may suppress the nuclear translocation of SOHLH2 by certain mechanism, which results in high HIF1α nuclear level. The underlying mechanism the nuclear translocation of SOHLH2 needs to be further discovered.

Furthermore, some researchers indicated HIF1α is a key stimulus for endothelial cell angiogenic property and vascular expansion (45). We therefore detected whether SOHLH2 inhibited of HUVEC behaviors through HIF1α signaling pathway. CoCl2 has been used in vitro to mimic hypoxic tumor condition, which stabilizes HIF1α by preventing its proteasomal degradation (46). HIF1α overexpression partially blocked SOHLH2-reduced tube formation and the inhibition of VEGFA and Angptl4 expression in MDA-MB-231 cells. Conversely, KC7F2, the inhibitor of HIF1α, remarkably mitigated the increase of tube formation and VEGFA and Angptl4 protein levels caused by the knockdown of SOHLH2 in MCF-7 cells. These results demonstrated HIF1α was necessary for SOHLH2 negatively regulation of endothelial cell behaviors and breast cancer angiogenesis.

Although it has been well illustrated that HIF1α overactivation under hypoxia is mainly obtained by post-transcriptional mechanisms, some articles have demonstrated that HIF1α mRNA transcription level can be regulated by several cytokines, growth factors, reactive oxygen species, and oncogene under normoxic condition (47, 48). bHLH transcription factors bind to the CANNTG in the promoter of many genes, regulating cellular differentiation, proliferation, and apoptosis. SIM2, a bHLH transcription factor, attenuated the mRNA and protein expression of HIF1α and its target genes in the angiogenesis of uterine cervical squamous cell carcinoma (49). Genetical overexpression of Stra13/DEC1 in lung cancer cells strongly downregulated HIF1α mRNA and protein levels (50). Indeed, SOHLH2 downregulated HIF1α expression at transcriptional level. The results of luciferase reporter and CHIP assays confirmed SOHLH2 repressed HIF1α transcriptional activity via directly binding to HIF1α promoter, the opposite effect was observed when SOHLH2 was downregulated. These data proved the hypothesis that SOHLH2 suppresses angiogenesis by downregulating HIF1α transcription in breast cancer. However, it takes more efforts to identify the specific E-box motifs bound by SOHLH2 in the future study.

In summary, we first demonstrate SOHLH2 inhibits breast cancer angiogenesis by tumor cell–mediated paracrine signaling pathway. SOHLH2 downregulates HIF1α transcription and suppresses breast cancer angiogenesis through HIF1α-mediated proangiogenic axis. We provide a new insight into the significance of SOHLH2 on tumor angiogenesis, and SOHLH2 may be a novel potential and effective target for the upcoming anti-angiogenesis therapy.

No disclosures were reported.

W. Cui: Investigation, writing–original draft. Y. Xiao: Conceptualization, investigation. R. Zhang: Investigation. N. Zhao: Investigation. X. Zhang: Investigation. F. Wang: Investigation. Y. Liu: Investigation. X. Zhang: Data curation, supervision, writing–review and editing. J. Hao: Data curation, supervision, writing–review and editing.

This work was supported by the National Natural Science Foundation of China (grant numbers 81874118 and 81672861), Shandong Department of Science and Technology Plan Project (grant number 2019GSF107013) and the Natural Science Foundation of Shandong Province (grant number ZR2017MH001).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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