MEK inhibition overcomes resistance to EphA2-targeted therapy in uterine cancer.

BACKGROUND: Our pilot clinical study of EphA2 inhibitor (dasatinib) plus paclitaxel and carboplatin showed interesting clinical activity in endometrial cancer with manageable toxicity. However, the underlying mechanisms of dasatinib resistance in uterine cancer are unknown. Here, we investigated potential mechanisms underlying resistance to EphA2 inhibitors in uterine cancer and examined the anti-tumor activity of EphA2 inhibitors alone and in combination with a MEK inhibitor. METHODS: We evaluated the antitumor activity of EphA2 inhibitors plus a MEK inhibitor using in vitro and in vivo orthotopic models of uterine cancer. RESULTS: EphA2 inhibitor induced MAPK in dasatinib-resistant uterine cancer cells (HEC-1A and Ishikawa) and BRAF/CRAF heterodimerization in HEC-1A cells. EphA2 inhibitor and trametinib significantly increased apoptosis in cancer cells resistant to EphA2 inhibitors compared with controls (p < 0.01). An in vivo study with the orthotopic HEC-1A model showed significantly greater antitumor response to combination treatment compared with dasatinib alone (p < 0.01). Combination treatment increased EphrinA1 and BIM along with decreased pMAPK, Jagged 1, and c-MYC expression in dasatinib-resistant cells. In addition, Spearman analysis using the TCGA data revealed that upregulation of EphA2 was significantly correlated with JAG1, MYC, NOTCH1, NOTCH3 and HES1 expression (p < 0.001, r = 0.25-0.43). Specifically, MAP3K15 and the NOTCH family genes were significantly related to poor clinical outcome in patients with uterine cancer. CONCLUSIONS: These findings indicate that the MAPK pathway is activated in dasatinib-resistant uterine cancer cells and that EphrinA1-mediated MEK inhibition overcomes dasatinib resistance. Dual targeting of both EphA2 and MEK, combined with chemotherapy, should be considered for future clinical development.

Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma.

Stress can alter immunological, neurochemical and endocrinological functions, but its role in cancer progression is not well understood. Here, we show that chronic behavioral stress results in higher levels of tissue catecholamines, greater tumor burden and more invasive growth of ovarian carcinoma cells in an orthotopic mouse model. These effects are mediated primarily through activation of the tumor cell cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway by the beta(2) adrenergic receptor (encoded by ADRB2). Tumors in stressed animals showed markedly increased vascularization and enhanced expression of VEGF, MMP2 and MMP9, and we found that angiogenic processes mediated the effects of stress on tumor growth in vivo. These data identify beta-adrenergic activation of the cAMP-PKA signaling pathway as a major mechanism by which behavioral stress can enhance tumor angiogenesis in vivo and thereby promote malignant cell growth. These data also suggest that blocking ADRB-mediated angiogenesis could have therapeutic implications for the management of ovarian cancer.

Enhancing oral delivery of plant-derived vesicles for colitis.

Plant-derived vesicles (PDVs) are attractive for therapeutic applications, including as potential nanocarriers. However, a concern with oral delivery of PDVs is whether they would remain intact in the gastrointestinal tract. We found that 82% of cabbage PDVs were destroyed under conditions mimicking the upper digestive tract. To overcome this limitation, we developed a delivery method whereby lyophilized Eudragit S100-coated cabbage PDVs were packaged into a capsule (Cap-cPDVs). Lyophilization and suspension of PDVs did not have an appreciable impact on PDV structure, number, or therapeutic effect. Additionally, packaging the lyophilized Eudragit S100-coated PDVs into capsules allowed them to pass through the upper gastrointestinal tract for delivery into the colon better than did suspension of PDVs in phosphate-buffered saline. Cap-cPDVs showed robust therapeutic effect in a dextran sulfate sodium-induced colitis mouse model. These findings could have broad implications for the use of PDVs as orally delivered nanocarriers of natural therapeutic plant compounds or other therapeutics.

Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L.

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) pathway is a powerful tool to combat tumor growth and progression; however, drug resistance frequently emerges. We identify CD5L (CD5 antigen-like precursor) as an important gene upregulated in response to antiangiogenic therapy leading to the emergence of adaptive resistance. By using both an RNA-aptamer and a monoclonal antibody targeting CD5L, we are able to abate the pro-angiogenic effects of CD5L overexpression in both in vitro and in vivo settings. In addition, we find that increased expression of vascular CD5L in cancer patients is associated with bevacizumab resistance and worse overall survival. These findings implicate CD5L as an important factor in adaptive resistance to antiangiogenic therapy and suggest that modalities to target CD5L have potentially important clinical utility.

FOXM1 mediates Dox resistance in breast cancer by enhancing DNA repair.

Transcription factors are direct effectors of altered signaling pathways in cancer and frequently determine clinical outcomes in cancer patients. To uncover new transcription factors that would determine clinical outcomes in breast cancer, we systematically analyzed gene expression data from breast cancer patients. Our results revealed that Forkhead box protein M1 (FOXM1) is the top-ranked survival-associated transcription factor in patients with triple-negative breast cancer. Surprisingly, silencing FOXM1 expression led breast cancer cells to become more sensitive to doxorubicin (Dox). We found that FOXM1-dependent resistance to Dox is mediated by regulating DNA repair genes. We further demonstrated that NFκB1 interacts with FOXM1 in the presence of Dox to protect breast cancer cells from DNA damage. Finally, silencing FOXM1 expression in breast cancer cells in a mouse xenograft model significantly sensitized the cells to Dox. Our systematic approaches identified an unexpected role of FOXM1 in Dox resistance by regulating DNA repair genes, and our findings provide mechanistic insights into how FOXM1 mediates resistance to Dox and evidence that FOXM1 may be a promising therapeutic target for sensitizing breast cancer cells to Dox.

Targeting CCR2+ macrophages with BET inhibitor overcomes adaptive resistance to anti-VEGF therapy in ovarian cancer.

PURPOSE: Tumor-associated macrophages (TAMs) are known to contribute to adaptive resistance to anti-vascular endothelial growth factor (VEGF) antibody (AVA) therapy in ovarian cancer. BET (bromodomain and extra-terminal domain) inhibitors (BETi) may have unique roles in targeting TAMs. Our objective was to examine the effects of BETi on TAMs, especially in the context of enhancing the efficacy of AVA therapy. METHODS: We conducted a series of in vitro (MTT assay, apoptosis, flow cytometry, and RNA sequencing) and in vivo (xenograft ovarian cancer model) experiments to determine the biological effects of BETi combined with AVA in ovarian cancer. For statistical analysis, a two-tailed Student's t test (equal variance) or ANOVA was used for multiple groups' comparison, and p < 0.05 was considered significant. RESULTS: BETi resulted in a dose-dependent decrease in cell viability and induced apoptosis (p < 0.01) in ovarian cancer cells (SKOV3ip1, OVCAR5, and OVCAR8). Treatment with BETi significantly increased apoptosis in THP-1 monocytes and macrophages (PMA-differentiated THP-1; p < 0.01). Furthermore, BETi selectively induced greater apoptosis in M2-like macrophages (PMA and IL-4, IL-13-differentiated THP-1) (31.3%-36.1%) than in M1-like macrophages (PMA and LPS-differentiated THP-1) (12.4%-18.5%) (p < 0.01). Flow cytometry revealed that the percentage of M1-like macrophages (CD68+/CD80+) was significantly increased after treatment with low-dose BETi (ABBV-075 0.1 µM; p < 0.05), whereas the percentage of CD68+/CCR2+ macrophages was significantly decreased (p < 0.001); these findings suggest that BETi may selectively inhibit the survival of CCR2+ macrophages and re-polarize the macrophages into an M1-like phenotype. RNA-seq analysis revealed that BETi selectively targeted macrophage infiltration-related cytokines/chemokines in ovarian cancer (adjusted p < 0.05 and Log2 fold change ≥ 1.5). Finally, using in vivo ovarian cancer models, compared with control or monotherapy, the combination of BETi (ABBV-075) and bevacizumab resulted in greater inhibition of tumor growth and macrophage infiltration (p < 0.05) and longer survival of tumor-bearing mice (p < 0.001). CONCLUSIONS: Our findings indicate a previously unrecognized role for BETi in selectively targeting CCR2+ TAMs and enhancing the efficacy of AVA therapy in ovarian cancer.

Endothelial p130cas confers resistance to anti-angiogenesis therapy.

Anti-angiogenic therapies, such as anti-VEGF antibodies (AVAs), have shown promise in clinical settings. However, adaptive resistance to such therapies occurs frequently. We use orthotopic ovarian cancer models with AVA-adaptive resistance to investigate the underlying mechanisms. Genomic profiling of AVA-resistant tumors guides us to endothelial p130cas. We find that bevacizumab induces cleavage of VEGFR2 in endothelial cells by caspase-10 and that VEGFR2 fragments internalize into the nucleus and autophagosomes. Nuclear VEGFR2 and p130cas fragments, together with TNKS1BP1 (tankyrase-1-binding protein), initiate endothelial cell death. Blockade of autophagy in AVA-resistant endothelial cells retains VEGFR2 at the membrane with bevacizumab treatment. Targeting host p130cas with RGD (Arg-Gly-Asp)-tagged nanoparticles or genomic ablation of vascular p130cas in p130casflox/floxTie2Cre mice significantly extends the survival of mice with AVA-resistant ovarian tumors. Higher vascular p130cas is associated with shorter survival of individuals with ovarian cancer. Our findings identify opportunities for new strategies to overcome adaptive resistance to AVA therapy.

Molecular Profiles of Serum-Derived Extracellular Vesicles in High-Grade Serous Ovarian Cancer.

Patients with high-grade serous ovarian cancer (HGSC) who have no visible residual disease (R0) after primary surgery have the best clinical outcomes, followed by patients who undergo neoadjuvant chemotherapy (NACT) and have a response enabling interval cytoreductive surgery. Clinically useful biomarkers for predicting these outcomes are still lacking. Extracellular vesicles (EVs) have been recognized as liquid biopsy-based biomarkers for early cancer detection and disease surveillance in other disease settings. In this study, we performed extensive molecular characterization of serum-derived EVs and correlated the findings with therapeutic outcomes in patients with HGSC. Using EV-DNA whole-genome sequencing and EV-RNA sequencing, we identified distinct somatic EV-DNA alterations in cancer-hallmark genes and in ovarian cancer genes, as well as significantly altered oncogenic pathways between the R0 group and NACT groups. We also found significantly altered EV-RNA transcriptomic variations and enriched pathways between the groups. Taken together, our data suggest that the molecular characteristics of EVs could enable prediction of patients with HGSC who could undergo R0 surgery or respond to chemotherapy.

Stress effects on FosB- and interleukin-8 (IL8)-driven ovarian cancer growth and metastasis.

A growing number of studies indicate that chronic stress can accelerate tumor growth due to sustained sympathetic nervous system activation. Our recent findings suggest that chronic stress is associated with increased IL8 levels. Here, we examined the molecular and biological significance of IL8 in stress-induced tumor growth. Norepinephrine (NE) treatment of ovarian cancer cells resulted in a 250-300% increase in IL8 protein and 240-320% increase in its mRNA levels. Epinephrine treatment resulted in similar increases. Moreover, NE treatment resulted in a 3.5-4-fold increase in IL8 promoter activity. These effects were blocked by propranolol. Promoter deletion analyses suggested that AP1 transcription factors might mediate catecholamine-stimulated up-regulation of IL8. siRNA inhibition studies identified FosB as the pivotal component responsible for IL8 regulation by NE. In vivo chronic stress resulted in increased tumor growth (by 221 and 235%; p < 0.01) in orthotopic xenograft models involving SKOV3ip1 and HeyA8 ovarian carcinoma cells. This enhanced tumor growth was completely blocked by IL8 or FosB gene silencing using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoliposomes. IL8 and FosB silencing reduced microvessel density (based on CD31 staining) by 2.5- and 3.5-fold, respectively (p < 0.001). Our findings indicate that neurobehavioral stress leads to FosB-driven increases in IL8, which is associated with increased tumor growth and metastases. These findings may have implications for ovarian cancer management.

Assessment of In Vivo siRNA Delivery in Cancer Mouse Models.

RNA interference (RNAi) has rapidly become a powerful tool for target discovery and therapeutics. Small interfering RNAs (siRNAs) are highly effective in mediating sequence-specific gene silencing. However, the major obstacle for using siRNAs for cancer therapeutics is their systemic delivery from the administration site to target cells in vivo. This chapter describes approaches to deliver siRNA effectively for cancer treatment and discusses in detail the current methods to assess pharmacokinetics and biodistribution of siRNAs in vivo.

Immune microenvironment composition in high-grade serous ovarian cancers based on BRCA mutational status.

PURPOSE: An in-depth analysis of the tumor microenvironment of ovarian cancer is needed. The purpose of this study was to elucidate the architecture of the immune microenvironment of high-grade serous ovarian cancers (HGSCs) with or without BRCA1 and BRCA2 mutations. METHODS: A cohort of highly annotated HGSC patients with known germline BRCA1 and BRCA2 status was selected, and pretreatment tumor tissue specimens were analyzed with a multiplexed staining technique aimed at detecting lymphocytes, macrophages, and fibroblasts in the whole tumor area and in specific regions including epithelium, stroma, and perivascular areas. RESULTS: BRCA1- or BRCA2-mutated tumors showed a more immunogenic microenvironment, characterized by a higher abundance of CD8+ and PD-L1+ cells, than did tumors with wild-type BRCA1 and BRCA2. High numbers of PD-L1+ and PD-L1+CD8+ cells were prognostic for event-free survival (hazard ratio [HR]: 0.41, 95% CI 0.21-0.79, p = 0.008 and HR 0.49, 95% CI 0.26-0.91, p = 0.025, respectively), as were high numbers of epithelial PD-L1+ and FAP+PD-L1+ cells (HR 0.52, 95% CI 0.28-0.96, p = 0.037 and HR 0.27, 95% CI 0.08-0.87, p = 0.029) and CD8+ cells (HR 0.51, 95% CI 0.28-0.93, p = 0.027). CONCLUSIONS: This study reveals substantial differences between the immune microenvironment composition of germline BRCA-mutated and BRCA wild-type HGSC.

Gain-of-function p53 protein transferred via small extracellular vesicles promotes conversion of fibroblasts to a cancer-associated phenotype.

Tumor and stromal interactions consist of reciprocal signaling through cytokines, growth factors, direct cell-cell interactions, and extracellular vesicles (EVs). Small EVs (≤200 nm) have been considered critical messengers of cellular communication during tumor development. Here, we demonstrate that gain-of-function (GOF) p53 protein can be packaged into small EVs and transferred to fibroblasts. GOF p53 protein is selectively bound by heat shock protein 90 (HSP90), a chaperone protein, and packaged into small EVs. Inhibition of HSP90 activity blocks packaging of GOF, but not wild-type, p53 in small EVs. GOF p53-containing small EVs result in their conversion to cancer-associated fibroblasts. In vivo studies reveal that GOF p53-containing small EVs can enhance tumor growth and promote fibroblast transformation into a cancer-associated phenotype. These findings provide a better understanding of the complex interactions between cancer and stromal cells and may have therapeutic implications.

CD63-mediated cloaking of VEGF in small extracellular vesicles contributes to anti-VEGF therapy resistance.

Despite wide use of anti-vascular endothelial growth factor (VEGF) therapy for many solid cancers, most individuals become resistant to this therapy, leading to disease progression. Therefore, new biomarkers and strategies for blocking adaptive resistance of cancer to anti-VEGF therapy are needed. As described here, we demonstrate that cancer-derived small extracellular vesicles package increasing quantities of VEGF and other factors in response to anti-VEGF therapy. The packaging process of VEGF into small extracellular vesicles (EVs) is mediated by the tetraspanin CD63. Furthermore, small EV-VEGF (eVEGF) is not accessible to anti-VEGF antibodies and can trigger intracrine VEGF signaling in endothelial cells. eVEGF promotes angiogenesis and enhances tumor growth despite bevacizumab treatment. These data demonstrate a mechanism where VEGF is partitioned into small EVs and promotes tumor angiogenesis and progression. These findings have clinical implications for biomarkers and therapeutic strategies for ovarian cancer.

Estrous cycle modulates ovarian carcinoma growth.

PURPOSE: The effects of reproductive hormones on ovarian cancer growth are not well understood. Here, we examined the effects of estrous cycle variation and specific reproductive hormones on ovarian cancer growth. EXPERIMENTAL DESIGN: We investigated the role of reproductive hormones in ovarian cancer growth using both in vivo and in vitro models of tumor growth. RESULTS: In vivo experiments using the HeyA8 and SKOV3ip1 ovarian cancer models showed that tumor cell inoculation during proestrus significantly increased tumor burden (251-273%) compared with injection during the estrus phase. Treatment of ovariectomized mice with 17beta-estradiol resulted in a 404% to 483% increase in tumor growth compared with controls. Progestins had no significant effect, but did block estrogen-stimulated tumor growth. Tumors collected from mice sacrificed during proestrus showed increased levels of vascular endothelial growth factor (VEGF) and microvessel density compared with mice injected during estrus. HeyA8, SKOV3ip1, and mouse endothelial (MOEC) cells expressed estrogen receptor alpha and beta and progesterone receptor at the protein and mRNA levels, whereas 2774 ovarian cancer cells were estrogen receptor-negative. In vitro assays showed that 17beta-estradiol significantly increased ovarian cancer cell adhesion to collagen in estrogen receptor-positive, but not in estrogen receptor-negative cells. Additionally, 17beta-estradiol increased the migratory potential of MOEC cells, which was abrogated by the mitogen-activated protein kinase (MAPK) inhibitor, PD 09859. Treatment with 17beta-estradiol activated MAPK in MOEC cells, but not in HeyA8 or SKOV3ip1 cells. CONCLUSION: Our data suggest that estrogen may promote in vivo ovarian cancer growth, both directly and indirectly, by making the tumor microenvironment more conducive for cancer growth.

Surgical stress promotes tumor growth in ovarian carcinoma.

PURPOSE: Surgical stress has been suggested to facilitate the growth of preexisting micrometastases as well as small residual tumor postoperatively. The purpose of this study was to examine the effects of surgical stress on ovarian cancer growth and to determine underlying mechanisms responsible for increased growth. EXPERIMENTAL DESIGN: To mimic the effects of surgery, we did a laparotomy or mastectomy under isoflurane inhalation on athymic nude mice 4 days after i.p. tumor cell injection. Propranolol infusion via Alzet pumps was used to block the influence of sympathetic nervous system activation by surgical stress. RESULTS: In both HeyA8 and SKOV3ip1 models, the mice in the laparotomy and mastectomy groups had significantly greater tumor weight (P < 0.05) and nodules (P < 0.05) compared with anesthesia only controls. There was no increase in tumor weight following surgery in the beta-adrenergic receptor-negative RMG-II model. Propranolol completely blocked the effects of surgical stress on tumor growth, indicating a critical role for beta-adrenergic receptor signaling in mediating the effects of surgical stress on tumor growth. In the HeyA8 and SKOV3ip1 models, surgery significantly increased microvessel density (CD31) and vascular endothelial growth factor expression, which were blocked by propranolol treatment. CONCLUSION: These results indicate that surgical stress could enhance tumor growth and angiogenesis, and beta-blockade might be effective in preventing such effects.

Therapeutic Targeting of ATP7B in Ovarian Carcinoma.

PURPOSE: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. EXPERIMENTAL DESIGN: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). RESULTS: ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC(50) levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH(2)-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. CONCLUSION: These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance.

Functional significance of VEGFR-2 on ovarian cancer cells.

Vascular endothelial growth factor receptor (VEGFR) has recently been discovered on ovarian cancer cells, but its functional significance is unknown and is the focus of this study. By protein analysis, A2780-par and HeyA8 ovarian cancer cell lines expressed VEGFR-1 and HeyA8 A2774, and SKOV3ip1 expressed VEGFR-2. By in situ hybridization (ISH), 85% of human ovarian cancer specimens showed moderate to high VEGFR-2 expression, whereas only 15% showed moderate to high VEGFR-1 expression. By immunofluorescence, little or no VEGFR-2 was detected in normal ovarian surface epithelial cells, whereas expression was detected in 75% of invasive ovarian cancer specimens. To differentiate between the effects of tumor versus host expression of VEGFR, nude mice were injected with SKOV3ip1 cells and treated with either human VEGFR-2 specific antibody (1121B), murine VEGFR-2 specific antibody (DC101) or the combination. Treatment with 1121B reduced SKOV3ip1 cell migration by 68% (p < 0.01) and invasion by 72% (p < 0.01), but exposure to VEGFR-1 antibody had no effect. Treatment with 1121B effectively blocked VEGF-induced phosphorylation of p130Cas. In vivo treatment with either DC101 or 1121B significantly reduced tumor growth alone and in combination in the SKOV3ip1 and A2774 models. Decreased tumor burden after treatment with DC101 or 1121B correlated with increased tumor cell apoptosis, decreased proliferative index, and decreased microvessel density. These effects were significantly greater in the combination group (p < 0.001). We show functionally active VEGFR-2 is present on most ovarian cancer cells. The observed anti-tumor activity of VEGF-targeted therapies may be mediated by both anti-angiogenic and direct anti-tumor effects.

Biobehavioral influences on matrix metalloproteinase expression in ovarian carcinoma.

PURPOSE: Stromal cells in the tumor microenvironment, such as macrophages, play an active role in tumor growth and angiogenesis. However, little is known about relationships of biobehavioral factors with angiogenic cytokines and matrix metalloproteinases (MMP) produced by stromal cells. This study examined distress, MMPs, and angiogenic cytokines in ovarian cancer patients and in vitro. EXPERIMENTAL DESIGN: Patients suspected of ovarian cancer completed preoperative questionnaires. At surgery, 56 were confirmed to have epithelial ovarian cancer. Tumor samples were analyzed for macrophage (CD68(+)) and tumor cell levels of MMP-2, MMP-9, and vascular endothelial growth factor. In vitro stimulation of isolated macrophage cells by the stress hormones norepinephrine and cortisol was done to assess effects on MMP-9. RESULTS: Depressed patients showed significant elevations of MMP-9 in CD68(+) cells, adjusting for stage (P<0.0001). Patients with higher levels of current stress (P=0.01), life stress over the last 6 months (P=0.004), and general negative affect (P=0.007) also showed significantly greater MMP-9 in CD68(+) cells. In contrast, higher social support was associated with lower levels of MMP-9 (P=0.023) and vascular endothelial growth factor (P=0.036) in tumor cells. In vitro analyses showed that macrophage MMP-9 production could be directly enhanced (up to a 2-fold increase) by the stress hormones norepinephrine and cortisol. CONCLUSIONS: Ovarian cancer patients with elevated depressive symptoms, chronic stress, and low social support showed elevations in MMP-9 in tumor-associated macrophages. Direct in vitro enhancement of stromal MMP-9 production by stress hormones was also shown. These findings may have implications for patient outcomes in ovarian cancer.