Expression and secretion of inhibin is regulated by hypoxia in ovarian xenograft tumors cell lines and in patients
We and others have previously demonstrated increased expression of inhibinα mRNA and protein in a broad spectrum of cancers leading to increased angiogenesis in vitro and in vivo impacting metastasis17,19,36. Based on the potential role of inhibins’ in cancer angiogenesis, we tested the impact of hypoxia, a key regulator of angiogenesis, on INHA expression. A panel of ovarian cancer cell lines representing a broad spectrum of ovarian cancer subtypes, including HEY, OV90, OVCAR5 of high grade serous origin, PA1 a teratocarcinoma cell line of the ovary, and ID8ip2 a mouse ovarian cancer cell line, were grown for twelve or twenty-four hours under either hypoxic conditions (0.2% O2) or normoxic control tissue culture conditions (17-21%). Changes to INHA were evaluated by semi quantitative RT-PCR with VEGFA as a positive control (Fig. 1Ai-ii). We find a 3-6 times increase in INHA expression across all four cell lines (HEY: 4.87-times, OVCAR5: 4.4-times, PA1: 5.28, OV90: 3.1-times, ID8ip2: 4-times, Fig. 1Ai). All cell lines showed maximum INHA increases after 24hrs of hypoxia growth except for OVCAR5 which increased INHA expression within 12hrs under hypoxia. VEGFA was evaluated side by side as a positive control and representative of the hypoxia response37 in all four cell lines and was elevated 2-6-times (HEY: 3.5-times, OVCAR5: 3.1-times, PA1: 5.18-times, OV90: 2.5-times, Fig. 1Aii). HIF-1α stabilization in all cell lines confirmed by westerns indicated an active response to hypoxia in indicated cell lines (Fig. 1Aiii).
INHA translates into the protein inhibinα which can be secreted as a free monomer or can dimerize with INHBA or INHBB to produce dimeric functional inhibin A or inhibin B12. Thus, total inhibin ELISA, which detects all three, was used to test if the changes in INHA mRNA resulted in alterations to secreted protein. We find that conditioned media collected from HEY and OV90 exposed to hypoxia increased total inhibin protein secretion as well, (4.2-times in HEY and 3.8-times OV90, Fig. 1B). These data suggest that INHA mRNA and functional secreted inhibin protein, is increased by hypoxia.
Since total inhibin protein, reflecting either inhibin A/B and free inhibinα, increased in response to hypoxia (Fig. 1B), we evaluated mRNA changes in INHBA and INHBB subunits in HEY and OV90 cells. While INHA was increased three to five-times in response to hypoxia (Fig. 1A), INHBA and INHBB levels were unchanged in the two cell lines evaluated (Fig. 1C), indicating that changes in inhibin protein levels (Fig. 1B) were largely related to increases in inhibinα. The INHA response to hypoxia was also more robust in tumor cells as compared to endothelial cells (HMEC-1) grown under hypoxia (0.2% O2) for 24hrs exhibited (Supp Fig. 1) indicating that inhibinα increases in response to hypoxia occur more significantly in tumor cells.
To evaluate other pathologically relevant hypoxic conditions pertinent to ovarian cancer growth and metastasis, we evaluated hypoxia and INHA expression in cells grown in spheroids under anchorage independence, an environment that is often hypoxic38. PA1 and OVCA420 cells were chosen due to their ability to form spheroids39,40. Cells were grown on poly-hema coated plates for either 72hrs (PA1) or 48hrs (OVCA420). Under such 3D conditions, where HIF-1α was stabilized (Fig. 1Di), INHA was increased 7.8-times in PA1 and 4.6-times in OVCA420 when compared to 2D growth conditions in a dish (Fig. 1Dii).
Previous studies have established that in healthy pre-menopausal women, inhibin levels cycle across the menstrual cycle reaching a peak of 65.6 pg/mL, while in post-menopausal women, total serum inhibin levels are below 5 pg/mL41. Ovarian cancer patients are commonly postmenopausal42 and tumor tissues can display higher inhibin levels19. We thus wanted to assess if the peritoneal ascites fluid of advanced ovarian cancer patients, which has been shown to be a hypoxic environment43 and contains disseminated ovarian cancer spheroids22, also displays detectable or elevated inhibin levels. To test if inhibin protein is secreted and detectable in clinical ascites, total inhibin ELISA was performed on a cohort of 25 patient ascites (Methods). We find total inhibin levels in the range of 6.7 to 120.53pg/mL in the ascites fluid with increasing concentrations found in higher stages of disease (Fig. 1E).
We next evaluated if INHA expression was elevated in vivo with increasing xenograft tumor size. 5 million HEY cells were subcutaneously implanted and harvested at varying tumor sizes. Tumors greater than 500mm3 were found to be hypoxic based on pimonidazole staining (4.8-times, Fig. 1Fi and Supp Fig. 2A). INHA expression was increased 9.8 times in tumors greater than 500mm3 than in tumors less than 500mm3 (Fig. 1Fii). INHA expression was also significantly correlated with tumor size (Supp Fig. 2B). To further examine the potential clinical relevance of inhibinα expression in response to hypoxia, we analyzed the TCGA/PanCancer Atlas patient data set from cBioportal35,44 and obtained hypoxia scores from two different hypoxia gene signatures (Buffa and Winter)45,46. The signatures consisted of 51 (Buffa) and 99 (Winter) hypoxia related genes from a large meta-analysis of breast and head and neck squamous cell cancer that were independently verified for prognostic value45,46. Using these signatures, inhibinα (INHA) expression was significantly correlated with both hypoxia Buffa (r=0.1961, p=0.0221) and Winter hypoxia (r=0.223, p=0.009) scores in the ovarian cancer data set (Fig. 1Gi-ii). Analysis of breast cancer data revealed a similar trend as INHA expression was significantly correlated (r=0.2026, p=0.0165) with the Buffa hypoxia score (Fig. 1Giii). Taken together, these data strongly indicate that inhibinα mRNA and protein expression are increased under hypoxia conditions in ovarian cancer cell lines, xenograft tumors and in patients.
INHA is a direct HIF-1 target under hypoxia
Hypoxia inducible factors (HIFs) are key transcriptional regulators of the hypoxia adaptive response and increase expression of critical pro-angiogenic genes21. To test whether HIF proteins are regulators of INHA expression, we first utilized cobalt chloride (CoCl2), a well characterized chemical stabilizer of HIF’s47. HIF-1α was stabilized in PA1 and OVCAR5 cells treated with 100µM of CoCl2 for either 6, 12, or 24 hrs (Fig. 2Ai). We find that INHA expression was significantly increased; 10-times in OVCAR5 after 12hrs and 11.5-times in PA1 cells after 24hrs of CoCl2 treatment (Fig. 2Aii). Maximum increases in INHA expression with CoCl2 occurred at the same time points as exposure to hypoxia (12hrs for OVCAR5 and 24hrs for PA1, Fig. 1Ai). VEGFA, used as a positive control increased 4.8 and 4.3-times at 12hrs and 2.7 and 3.7-times at 24hrs in both OVCAR5 and PA1, respectively (Fig. 2Aii). To test if INHA could be a direct hypoxia target leading to increased inhibinα expression, we evaluated the effect of reducing the levels of HIF-1β/ARNT which is the binding partner for all HIF’s48. Stable ARNT knockdown cells were generated in HEY cells (Methods). We find that control HEY cells increase INHA levels 2.8-times under 0.2% hypoxia (Fig. 2B). However, shRNA ARNT lead to a 2.7-times reduction in hypoxia induced increase in INHA mRNA levels (Fig. 2B) indicating direct contributions of HIFs’ to the regulation of inhibin.
To determine the roles of the HIF-1 and HIF-2 heterodimeric transcriptions factors, that both require ARNT48, in the transcriptional regulation of INHA we used siRNA to knockdown the levels of HIF-1α and HIF-2α (Methods). HEK293 cells were used as they express relatively equal levels of both HIF isoforms (Fig. 2Ci). HEK293 cells with either control or HIF1/2α siRNAs were exposed to hypoxia for 24hrs and efficacy of HIF1/2α knockdown was confirmed by immunoblotting (Fig. 2Ci). Notably, siRNA to HIF-1α (siHIF-1α; Fig. 2Cii) decreased hypoxia induced INHA expression 1.8-times as compared to scramble controls (siScr; Fig. 2Cii). However, siRNA to HIF-2α resulted in a smaller (1.25-times) and non-significant reduction in INHA expression compared to siScr when exposed to hypoxia (Fig. 2Cii). These data suggest that increases in INHA under hypoxia were more significantly impacted by HIF-1 as compared to HIF-2 .
In silico, analysis of the INHA gene, which is located at Chr:2q35 revealed two hypoxia response element (HRE) consensus sites within 2Kb of the promoter, GGCGTGG and CGCGTGG, at -144 and -1789 bp from the transcription start site (TSS) (Supp Fig. 3A) respectively. These HRE sites conform precisely to the (G/C/T)(A/G)CGTG(G/C) consensus sequence48. Two hypoxia ancillary sequences (HAS) (CAGGG and CACGG) were also found directly flanking the proximal HRE sequence at -169 and -173 bp from the TSS, respectively. One HAS sequence (CACGT) was found flanking the distal HRE sequence at -1761 bp from TSS (Supp Fig. 3A). A previously well characterized CREB binding site (CRE) is designated for reference (Supp Fig. 3A).
To test direct interactions between HIF-1 and the INHA promoter, chromatin immunoprecipitation (ChIP) was performed using OVCAR5 and OV90 cells. Primers were designed to amplify the region including the HRE site closest to the transcription start site (HRE1) and chromatin shear size optimized accordingly (Methods). We find that exposure to hypoxia led to a 4-times increase in enrichment of HIF-1 binding to INHA’s HRE site in OVCAR5 and 3-times in OV90 (Fig. 2D). The second HRE site is GC rich which lead to modest amplification. Despite this, a 2-times increase in HIF-1 enrichment at this site in OV90 cells was observed (Supp Fig. 3B) which was however not statistically significant.
Given the poor enrichment of HIF-1 at the distal promoter site (Fig. S3B), we next evaluated if the proximal promoter was sufficient to increase INHA levels under hypoxia and if this was dependent on HIF-1. To achieve this, we utilized 547 base pairs of the INHA promoter, containing the first HRE site (Fig. S3A), in a luciferase reporter assay (Fig. 2E). The effect of HIF-1 on INHA promoter activity, was evaluated in HEK293 cells exposed to hypoxia (0.2% O2) for 24hrs and compared to cells under normoxia (Fig. 2Ei), or in the presence or absence of HIF-1 ODD (pcDNA3-HA-HIF1aP402A/P564A) (Fig. 2Eii) that prevents degradation of the HIF1α subunit26. We find that in un-transfected or control vector expressing cells (pcDNA3.1), INHA promoter luciferase activity is increased two times in response to hypoxia (Fig. 2Ei) that was mimicked by stabilization of HIF-1α (HIF-1 ODD) under normoxia conditions (Fig. 2Eii). These data point to a central role for HIF-1 in regulating INHA expression under hypoxia.
INHA has been previously reported to be regulated by other factors particularly the cAMP response element binding (CREB) family member in multiple systems8. The CREB family of transcription factors can act downstream of the hypoxia response49. To thus test whether cAMP was involved in regulating INHA expression under hypoxia, we utilized forskolin (Fsk), an activator of cAMP previously shown to induce INHA expression and the PKA inhibitor H89 previously shown to inhibit forskolin induced INHA expression50. Treatment of ID8ip2 cells with Fsk increased INHA expression 5.2-times under hypoxia compared to just 2-times under normoxia (Supp Fig. 3C). This relationship appeared to be additive and not synergistic as addition of the PKA inhibitor, H89, was not able to reduce hypoxia induced INHA expression (Supp Fig. 3C). Taken together, these data implicate HIF-1 as being the key transcriptional factor responsible for increase of INHA in hypoxia.
Inhibin promotes hypoxia induced angiogenesis and stimulates endothelial cell migration and vascular permeability
Hypoxia is a key driver of endothelial cell migration and blood vessel permeability within the tumor leading to alterations in angiogenesis37. To determine the overall contribution of inhibin to hypoxia induced angiogenesis in vivo, we utilized an in vivo Matrigel plug assay. Conditioned media (CM) from HEY tumor cells exposed to normoxia or hypoxia was used to stimulate angiogenesis into the plugs and a well-established anti-inhibinα antibody, R1 (recognizing the junction between the αN region, and αC region)51 was used to block inhibin in the CM with IgG as a control. We find that CM from hypoxia grown cells increased hemoglobin in the plugs 2.9-times compared to CM from normoxia grown cells (Fig. 3Ai-ii). Anti-inhibinα in the hypoxic CM fully reduced the hemoglobin content in the plug (2.1-times suppression, Fig. 3Ai-ii) indicating that inhibin is required for hypoxia induced blood vessel formation in vivo.
Since blood vessel flow is an indication of endothelial cell functionality52, we sought to define the specific effects of increased inhibinα on hypoxia induced endothelial cell biology, specifically endothelial cell chemotaxis and vascular permeability. To determine the impact on endothelial chemotaxis to hypoxic CM, CM from either hypoxia (24 hrs, 0.2% O2) or normoxia grown OV90 or HEY cells were used as a chemoattractant to measure migration of human microvascular endothelial cells (HMEC-1; Fig. 3B). Two anti-inhibinα antibodies, R1 and a second well established antibody PO23 (recognizing the C-terminus of the αC region)51, were used with IgG controls to test the effect of blocking/sequestering hypoxia produced inhibinα. We find that CM from hypoxia grown tumor cells significantly increased migration of endothelial cells (IgG, Fig. 3B) and incubation of hypoxic CM with anti-inhibinα R1 significantly suppressed hypoxia induced endothelial migration (2.1 and 1.6-times for OV90 and HEY conditioned media respectively, Fig. 3Bi-ii). Anti-inhibinα PO23 was also able to significantly suppress CM stimulated endothelial migration (1.5 and 1.75-times for OV90 and HEY CM, respectively, Fig. 3Bi-ii). Similar to the effects of hypoxic CM, recombinant inhibin A was also able to stimulate HMEC-1 migration to similar extents as VEGF A at equimolar amounts (Fig. 3Biii).
We next evaluated the effect of CM from hypoxic tumor cells on changes to permeability across an endothelial monolayer using a trans-well permeability assay that measures solute (FITC-dextran) flux across endothelial monolayers. Permeability was monitored across a four-hour time course and CM from hypoxic tumor cells was used to induce permeability across the HMEC-1 monolayer. Effect of inhibin in the CM was evaluated either in the presence of anti-inhibinα (PO23 and R1) or IgG control (Fig. 3Ci-ii). We find that both inhibinα antibodies (R1 and PO23) significantly decreased solute flux induced by hypoxic CM from two tumor cell lines, albeit with moderate differences in the kinetics and time to inhibition (Fig. 3Ci-ii). Specifically, significant inhibition of permeability was seen beginning at two hours for CM treated with PO23 and three hours for R1. PO23 was moderately more effective than R1 as it effectively reduced permeability within 1 hour (Fig. 3Ci-ii). Recombinant inhibin was also able to induce endothelial cell permeability to similar extents as LPS (Fig. 3D), an established permeability inducing factor43. Since perturbations to the endothelial barrier are critical to invasion and extravasation of cancer cells during metastasis53, we tested whether inhibin induced vascular permeability facilitates tumor cell extravasation. To test this, we used a trans-endothelial cell migration assay to mimic the process. HEY tumor cells infected with GFP adenovirus to distinguish them from migrated non-GFP endothelial cells were plated on top of a non-GFP endothelial cell monolayer that was then either pre-treated with 1nM inhibin A for 4 hours or left untreated. We find that HEY GFP tumor cells, were 2.9-times more invasive across the inhibin treated monolayer than untreated conditions (Fig. 3Eii-iii). All together, these data implicate inhibin as a robust contributor to hypoxia mediated angiogenesis, vascular permeability and thereby tumor cell extravasation across the vascular endothelium.
Inhibin promotes vascular permeability through increased VE-cadherin trafficking.
Endothelial permeability is regulated through changes in junctional proteins which are maintained through contacts with the actin cytoskeleton54. VE-cadherin is a critical junctional protein involved in regulating endothelial cell permeability54. To delineate the mechanism of inhibin’s effects on vascular permeability, we first evaluated the effect of inhibin on endothelial cell junctions and the actin cytoskeleton through immunofluorescent staining of VE-cadherin and actin (Fig. 4A). Examination of the actin cytoskeleton revealed significant contractile actin staining, with a significant increase in stress fiber formation after 30 minutes of inhibin A treatment (two times increase, Fig. 4Ai-ii). VEGF A treatment was used as a comparison that also led to similar changes in actin stress fiber formation (Fig. 4A). VE-cadherin localization also appeared to be reduced qualitatively at the cell-cell junctions after 30 minutes of inhibin treatment as compared to untreated cells, suggestive of perturbation of the endothelial cell barrier at the level of the cytoskeleton (Fig. 4A). Loss of VE-cadherin at the cell junctions was also observed in VEGF A treated cells (Fig. 4A). However, total VE-cadherin levels were unchanged in response to inhibin as evaluated over a time course of 60 minutes (Supp Fig. 4) indicating no change in the total pool of VE-cadherin in response to inhibin A. Actin contractility and stress fiber assembly is regulated through phosphorylation of myosin light chain (MLC)54. In accordance, we find that phosphorylation of MLC-2 (Ser19) increased within 5 minutes of inhibin A treatment and was sustained across a 60-minute time course (Fig. 4Bi-ii). Based on the qualitative changes in VE-cadherin in response to inhibin A treatment (Fig. 4A), we tested whether alterations in VE-cadherin at the cell-cell junctions were due to inhibin induced VE-cadherin internalization (Fig. 4C). To determine this, HMEC-1 membrane localized VE-cadherin was labeled at 4°C with an anti-VE-cadherin antibody recognizing the extra-cellular domain. HMEC-1 cells were washed with acid to remove membrane bound anti-VE-cadherin leaving only any internalized VE-cadherin that may have been labeled at 4°C prior to treatment with inhibin A or VEGF A (Fig. 4Ci). Stripping of cell surface VE-cadherin was verified by cell surface immunostaining of VE-cadherin with little to no internalized VE-cadherin detected (Fig. 4Cii,iv). Cells were then either left untreated or treated for 30 minutes with inhibin A at 37oC and VE-cadherin evaluated by immunofluorescence (Fig. 4Ciii). We find that inhibin A increased the internalized VE-cadherin pool compared to untreated cells 1.4-times (Fig. 4Cv) and to similar extents as VEGF A (1.6-times, Fig. 4Cv). These results indicate that inhibin induces rapid changes in the actin cytoskeleton and trafficking of VE-cadherin from the cell junctions of endothelial cells.
Inhibin’s effects on vascular permeability are mediated by ALK1 and CD105/endoglin that form a stable complex at the cell surface in response to inhibin
Previously, we demonstrated that inhibin’s effects on angiogenesis and endothelial cell signaling were dependent on the TGFβ receptors ALK1 and endoglin19. To evaluate if ALK1 and endoglin are required for inhibin’s influence on vascular permeability, we treated HMEC-1 cells with TRC105, a humanized endoglin monoclonal antibody 55, or with ALK1-Fc, a human chimeric ALK1 protein56. At four hours, treatment with (i) TRC105 and (ii) ALK1-Fc decreased inhibin A induced permeability by 2.2 and 1.5-times, respectively (Fig. 5A, Supp Fig. 5 for full time course) indicating both ALK1 and endoglin are required for inhibin’s effects on endothelial cell permeability.
We next evaluated if internalization of VE-cadherin by inhibin was dependent on endoglin using mouse embryonic endothelial cells (MEEC) that are either wild type (WT) or null for endoglin expression51 (Supp Fig. 6). Cell surface biotinylation of VE-cadherin was used to quantitatively assess VE-cadherin internalization. Towards this, cell surface proteins were labeled with Sulfo-NH-SS biotin and allowed to internalize for 30 minutes at 37°C in the presence or absence of inhibin followed by stripping of cell surface biotin, immunoprecipitation with neutravidin resin and immunoblotting to detect internalized biotin labeled VE-cadherin (Fig. 5Bi). Treatment with inhibin A increased internalized VE-cadherin 1.9-times in MEEC WT compared to control (Fig. 5Bi), similar to extents seen by immunofluorescence in HMEC-1 cells (Fig. 4C). However, in the absence of endoglin in MEEC ENG-/- cells inhibin A did not change the internalized VE-cadherin pool (Fig. 5Bii). This data indicates that endoglin is essential for inhibins effects on VE-cadherin.
Based on the significant dependency of inhibin’s effects on endothelial cell permeability and VE-cadherin internalization on endoglin and ALK1 respectively (Fig. 5A,B), we evaluated biophysically, in a sensitive and quantitative manner, the extent of the endoglin-ALK1 interaction in response to inhibin. We utilized a patch/FRAP (fluorescence recovery after photobleaching) methodology to measure interactions between endoglin and ALK1 at the surface of live cells. This method differentiates between stable and transient interactions as described in detail previously57. Herein, one receptor carrying an extracellular epitope tag is patched and immobilized through cross-linking with a double layer of IgGs. The effects of this immobilization on the lateral diffusion of a co-expressed, differently-tagged receptor labeled exclusively with Fab’ fragments are then measured by FRAP (Methods). Stable complex formation between the two co-expressed receptors (complex lifetimes longer than the characteristic FRAP fluorescence recovery time) reduces the mobile fraction (Rf) of the Fab’-labeled receptor, since bleached Fab’-labeled receptors associated with immobilized receptors do not appreciably dissociate from the immobile patches during the FRAP measurement. On the other hand, transient complexes (short complex lifetimes) would reduce the apparent lateral diffusion coefficient (D), since each Fab’-labeled receptor molecule can undergo multiple association-dissociation cycles during the FRAP measurement57. For these studies, COS7 cells were transfected with myc-ALK1, HA-endoglin or co-transfected with both, and subjected to patch/FRAP experiments in the absence or presence of 4 nM of inhibin A (Fig. 5C). Fig. 5Ci-iii depict representative FRAP curves showing the lateral diffusion of myc-ALK1 (Fig. 5Ci), IgG-crosslinked and immobilized HA-endoglin (Fig. 5Cii), and myc-ALK1 co-transfected with HA-endoglin followed by IgG cross-linking of HA-endoglin in the presence of inhibin (Fig. 5Ciii). Average values derived from multiple independent experiments are shown in (Rf in Fig. 5Cv, D values in Fig. 5Cv). Singly expressed myc-ALK1 had lateral mobility resembling other TGF-β superfamily receptors34, which was insensitive to inhibin treatment (Fig. 5Ci and iv). Immobilization of HA-endoglin (Fig. 5Cii and iv) reduced Rf of myc-ALK1 by about 45%, and the presence of inhibin increased this reduction significantly (from 45–70% reduction) (Fig. 5Ciii and iv). Under all these conditions, the lateral diffusion coefficient (D) of myc-ALK1 was not significantly affected (Fig. 5Cv), indicating that endoglin and ALK1 form stable complexes at the plasma membrane which are enhanced and stabilized by inhibin.
Previous studies indicate that inhibinα may bind to ALK458, an established Type I receptor for the Activin family of proteins8. We thus employed patch/FRAP to determine the interactions between endoglin and ALK4 and to examine whether inhibin A enhanced these interactions. To this end, we expressed HA-ALK4, myc-endoglin or both in COS7 cells, and subjected them to patch/FRAP studies on the lateral diffusion of HA-ALK4 without and with IgG cross-linking of myc-endoglin, and with or without inhibin A. In the absence of inhibin A, endoglin and ALK4 exhibited significant stable interactions, as demonstrated by the reduction in Rf of HA-ALK4 upon immobilization of myc-endoglin (40% reduction in Rf, with no effect on the D value) (Fig. 5Di-ii). However, in contrast to the observations with endoglin-ALK1 complexes, the interactions between endoglin and ALK4 were weakened in the presence of inhibin A (the reduction in Rf decreased to 20%) (Fig. 5Di). Taken together, these results indicate that inhibin shifts the balance of endoglin complexes from interactions with ALK4 to interactions with ALK1, both of which (endoglin and ALK1) are required for inhibin mediated vascular permeability.
Inhibin promotes hypoxia induced tumor growthin vivothrough alterations in permeability and angiogenesis
The significance of hypoxia in ovarian cancer is well documented and we previously demonstrated increased ascites accumulation in tumor bearing mice in the presence of inhibin19. To precisely define the contribution of inhibin to hypoxia induced tumor growth and angiogenesis, we first evaluated the effects of pre-exposure to hypoxia on tumor growth in a subcutaneous model in vivo, a model that allows for quantitative analysis of the vasculature in tumors59. HEY pLKO.1 control vector (shControl) cells were pre-exposed to hypoxia (0.2% O2) for 24hrs or kept under normoxia followed by injection into the right flank of Ncr nude mice. Tumors were measured throughout and harvested after 30 days (n=10 mice). HEY cells pre-exposed to hypoxia produced rapid growing tumors compared to those that originated from normoxia grown cells (Fig. 6A, purple versus black line). In parallel, we utilized two methods to perturb inhibin: 1) shRNA knockdown of INHA in HEY cells (Supp Fig. 7A,B) and 2) intraperitoneal administration of anti-inhibinα antibody (R1). R1 is a human antibody51 and consistent with this no overall toxicity was noted in pilot toxicity studies that utilized daily injections of R1 (Supp Fig. 7C). shINHA cells exposed to hypoxia maintained their knockdown to INHA at the end of the study (Supp Fig. 7B) and produced tumors with significantly slower growth rates than shControl hypoxia tumors (Fig. 6A, blue versus black lines). In complementary findings, hypoxia exposed tumor cells had significantly reduced tumor growth upon receiving treatment with the R1 antibody when compared to tumors in mice that received vehicle only (Fig. 6A, red versus blue line, n=6 for R1 treated mice). The group receiving anti-inhibinα (R1) grew at a similar rate as the shINHA hypoxia tumors (Fig. 6A, red versus blue line). In mice with shINHA tumors, treatment with R1 further reduced tumor growth albeit moderately compared to vehicle shINHA (Fig. 6A, blue versus green line). These data indicate that perturbation of inhibin through shRNA targeting and anti-inhibin antibody treatment reduces tumor growth. We hence sought to determine the effect of shINHA on the angiogenic cytokine profile of the tumors using a proteome array of 55 different human angiogenesis targets. We find that the most up-regulated proteins in control tumors compared to shINHA tumors were a subset of pro-angiogenic cytokines IL8 (2.5-times) and EGF (2.1-times) (Fig. 6Bi) indicating a proangiogenic profile of the tumor cells in the presence of inhibin. In contrast, the shINHA hypoxia tumors showed increases in proteins including ADAMTS-1 (1.6-times) and Pentraxin-3 (1.3-times), indicating an anti- angiogenic profile in shINHA tumor cells as both have been demonstrated to be anti-angiogenic60,61. Activin A and endoglin were also found to be elevated in shINHA tumors (Fig. 6Bi). To complement the human tumor array, we analyzed changes in the mouse angiogenic proteome as well to delineate any host differences in response to shControl and shINHA tumor cells. We find that host cells also upregulated significantly more pro-angiogenic proteins, including CXCL1662, PIGF-263, and NOV64 in shControl tumors compared to shINHA tumors ( Fig. 6Bii). Taken together, these data suggest that altering inhibin in the tumors results in a change in the balance of angiogenic factors leading to a significant reduction in pro-angiogenic factors and slower overall tumor growth.
To rule out whether the reduction in tumor growth in shINHA cells was due to slower proliferation of tumor cells, growth rate of HEY shINHA and HEY shControl was evaluated in culture under hypoxia for 3 days. No significant change was observed (Supp Fig. 7D) suggesting that the major effect of inhibin on tumor growth are likely through effects on the tumor vasculature due to the effects of hypoxia regulated inhibin on angiogenesis and vascular permeability in vitro (Fig. 4,5). We thus determined the effect of inhibin on the tumor vasculature and associated permeability changes as a contributing factor to the altered tumor growth in shINHA and antibody treated hypoxia tumors (Fig. 6A). To this end, HEY shControl or shINHA cells pre-exposed to hypoxia for 24hrs were injected subcutaneously into the right flank of Ncr nude mice (n=4 mice, Fig. 6B) and tumors in all groups were harvested upon reaching 700-850mm3 (Fig. 6Ci) to eliminate any tumor size effects on angiogenesis. These tumors (Fig. 6Ci) were evaluated for changes in vascular permeability by visualization of a rhodamine-dextran dye that leaks from the blood vessels into the tumors when administered into mice prior to sacrifice. We find that rhodamine-dextran was present at 5.5-times higher levels in shControl tumors compared to shINHA tumors indicating higher vascular permeability within the tumors in the presence of inhibin (Fig. 6Cii-iii). To further characterize the differences in the vasculature between shControl and shINHA tumors, blood vessels were stained with CD-31 to evaluate vessel number and size (Fig. 6D). We find an increase in the total number of blood vessels in shControl tumors compared to shINHA tumors (Fig. 6Di,iii). Quantitation of the size of the vessels revealed significantly smaller vessels in shControl tumors as compared to the shINHA tumors (Fig. 6Dii,iii). These data together demonstrate that reducing inhibin in the tumor decreases vascular leakiness, alters vessel size and numbers and promotes more normalized vasculature in the tumors.