Morgan Taylor

Tumour angiogenesis regulation by the miR-200 family

The miR-200 family is well known to inhibit the epithelial-mesenchymal transition, suggesting it may therapeutically inhibit metastatic biology. However, conflicting reports regarding the role of miR-200 in suppressing or promoting metastasis in different cancer types have left unanswered questions. Here we demonstrate a difference in clinical outcome based on miR-200s role in blocking tumour angiogenesis. We demonstrate that miR-200 inhibits angiogenesis through direct and indirect mechanisms by targeting interleukin-8 and CXCL1 secreted by the tumour endothelial and cancer cells. Using several experimental models, we demonstrate the therapeutic potential of miR-200 delivery in ovarian, lung, renal and basal-like breast cancers by inhibiting angiogenesis. Delivery of miR-200 members into the tumour endothelium resulted in marked reductions in metastasis and angiogenesis, and induced vascular normalization. The role of miR-200 in blocking cancer angiogenesis in a cancer-dependent context defines its utility as a potential therapeutic agent.

Hypoxia-mediated downregulation of miRNA biogenesis promotes tumour progression

Cancer-related deregulation of miRNA biogenesis has been suggested, but the underlying mechanisms remain elusive. Here we report a previously unrecognized effect of hypoxia in the downregulation of Drosha and Dicer in cancer cells that leads to dysregulation of miRNA biogenesis and increased tumour progression. We show that hypoxia-mediated downregulation of Drosha is dependent on ETS1/ELK1 transcription factors. Moreover, mature miRNA array and deep sequencing studies reveal altered miRNA maturation in cells under hypoxic conditions. At a functional level, this phenomenon results in increased cancer progression in vitro and in vivo, and data from patient samples are suggestive of miRNA biogenesis downregulation in hypoxic tumours. Rescue of Drosha by siRNAs targeting ETS1/ELK1 in vivo results in significant tumour regression. These findings provide a new link in the mechanistic understanding of global miRNA downregulation in the tumour microenvironment. MicroRNAs play important roles in the maintenance of cellular homeostasis through the post-transcriptional regulation of gene expression. Here, the authors implicate loss of the miRNA biogenesis factor Drosha and altered miRNA maturation in tumour progression under hypoxic conditions.

Role of Focal Adhesion Kinase in Regulating YB–1–Mediated Paclitaxel Resistance in Ovarian Cancer

BACKGROUND We previously found focal adhesion kinase (FAK) inhibition sensitizes ovarian cancer to taxanes; however, the mechanisms are not well understood. METHODS We characterized the biologic response of taxane-resistant and taxane-sensitive ovarian cancer models to a novel FAK inhibitor (VS-6063). We used reverse-phase protein arrays (RPPA) to identify novel downstream targets in taxane-resistant cell lines. Furthermore, we correlated clinical and pathological data with nuclear and cytoplasmic expression of FAK and YB-1 in 105 ovarian cancer samples. Statistical tests were two-sided, and P values were calculated with Student t test or Fisher exact test. RESULTS We found that VS-6063 inhibited FAK phosphorylation at the Tyr397 site in a time- and dose-dependent manner. The combination of VS-6063 and paclitaxel markedly decreased proliferation and increased apoptosis, which resulted in 92.7% to 97.9% reductions in tumor weight. RPPA data showed that VS-6063 reduced levels of AKT and YB-1 in taxane-resistant cell lines. FAK inhibition enhanced chemosensitivity in taxane-resistant cells by decreasing YB-1 phosphorylation and subsequently CD44 in an AKT-dependent manner. In human ovarian cancer samples, nuclear FAK expression was associated with increased nuclear YB-1 expression (χ²) = 37.7; P < .001). Coexpression of nuclear FAK and YB-1 was associated with statistically significantly worse median overall survival (24.9 vs 67.3 months; hazard ratio = 2.64; 95% confidence interval = 1.38 to 5.05; P = .006). CONCLUSIONS We have identified a novel pathway whereby FAK inhibition with VS-6063 overcomes YB-1-mediated paclitaxel resistance by an AKT-dependent pathway. These findings have implications for clinical trials aimed at targeting FAK.

FAK regulates platelet extravasation and tumor growth after antiangiogenic therapy withdrawal

Recent studies in patients with ovarian cancer suggest that tumor growth may be accelerated following cessation of antiangiogenesis therapy; however, the underlying mechanisms are not well understood. In this study, we aimed to compare the effects of therapy withdrawal to those of continuous treatment with various antiangiogenic agents. Cessation of therapy with pazopanib, bevacizumab, and the human and murine anti-VEGF antibody B20 was associated with substantial tumor growth in mouse models of ovarian cancer. Increased tumor growth was accompanied by tumor hypoxia, increased tumor angiogenesis, and vascular leakage. Moreover, we found hypoxia-induced ADP production and platelet infiltration into tumors after withdrawal of antiangiogenic therapy, and lowering platelet counts markedly inhibited tumor rebound after withdrawal of antiangiogenic therapy. Focal adhesion kinase (FAK) in platelets regulated their migration into the tumor microenvironment, and FAK-deficient platelets completely prevented the rebound tumor growth. Additionally, combined therapy with a FAK inhibitor and the antiangiogenic agents pazopanib and bevacizumab reduced tumor growth and inhibited negative effects following withdrawal of antiangiogenic therapy. In summary, these results suggest that FAK may be a unique target in situations in which antiangiogenic agents are withdrawn, and dual targeting of FAK and VEGF could have therapeutic implications for ovarian cancer management.

Platelets reduce anoikis and promote metastasis by activating YAP1 signaling

Thrombocytosis is present in more than 30% of patients with solid malignancies and correlates with worsened patient survival. Tumor cell interaction with various cellular components of the tumor microenvironment including platelets is crucial for tumor growth and metastasis. Although it is known that platelets can infiltrate into tumor tissue, secrete pro-angiogenic and pro-tumorigenic factors and thereby increase tumor growth, the precise molecular interactions between platelets and metastatic cancer cells are not well understood. Here we demonstrate that platelets induce resistance to anoikis in vitro and are critical for metastasis in vivo. We further show that platelets activate RhoA-MYPT1-PP1-mediated YAP1 dephosphorylation and promote its nuclear translocation which induces a pro-survival gene expression signature and inhibits apoptosis. Reduction of YAP1 in cancer cells in vivo protects against thrombocytosis-induced increase in metastasis. Collectively, our results indicate that cancer cells depend on platelets to avoid anoikis and succeed in the metastatic process.Platelets have been associated with increased tumor growth and metastasis but the mechanistic details of this interaction are still unclear. Here the authors show that platelets improve anoikis resistance of cancer cells and increase metastasis by activating Yap through a RhoA/MYPT-PP1 pathway.

XPO1/CRM1 Inhibition Causes Antitumor Effects by Mitochondrial Accumulation of eIF5A

Purpose: XPO1 inhibitors have shown promise for cancer treatment, and yet the underlying mechanisms for the antitumor effects are not well understood. In this study, we explored the usefulness of selective inhibitors of nuclear export (SINE) compounds that are specific inhibitors of XPO1. Experimental Design: We used proteomic analysis in XPO1 inhibitor–treated ovarian cancer cell lines and examined antitumor effects in ovarian and breast cancer mouse models. We also studied the effects of XPO1 inhibitor in combination with chemotherapeutic agents. Results: XPO1 inhibitor treatment substantially increased the percentage of apoptotic cells (60%) after 72 hours of incubation. XPO1 inhibitor promoted the accumulation of eIF5A in mitochondria, leading to cancer cell death. Topotecan showed the greatest synergistic effect with XPO1 inhibitor. XPO1 inhibitors prevented the translocation of IGF2BP1 from the nucleus to the cytoplasm, thereby permitting the localization of eIF5A in the mitochondria. This process was p53, RB, and FOXO independent. Significant antitumor effects were observed with XPO1 inhibitor monotherapy in orthotopic ovarian (P < 0.001) and breast (P < 0.001) cancer mouse models, with a further decrease in tumor burden observed in combination with topotecan or paclitaxel (P < 0.05). This mitochondrial accumulation of eIF5A was highly dependent on the cytoplasmic IGF2BP1 levels. Conclusions: We have unveiled a new understanding of the role of eIF5A and IGF2BP1 in XPO1 inhibitor–mediated cell death and support their clinical development for the treatment of ovarian and other cancers. Our data also ascertain the combinations of XPO1 inhibitors with specific chemotherapy drugs for therapeutic trials. Clin Cancer Res; 21(14); 3286–97. ©2015 AACR.

Antitumor and antiangiogenic effects of aspirin-PC in ovarian cancer

To determine the efficacy of a novel and safer (for gastrointestinal tract) aspirin (aspirin-PC) in preclinical models of ovarian cancer, in vitro dose–response studies were performed to compare the growth-inhibitory effect of aspirin-PC versus aspirin on three human (A2780, SKOV3ip1, and HeyA8) and a mouse (ID8) ovarian cancer cell line over an 8-day culture period. In the in vivo studies, the aspirin test drugs were studied alone and in the presence of a VEGF-A inhibitor (bevacizumab or B20), due to an emerging role for platelets in tumor growth following antiangiogenic therapy, and we examined their underlying mechanisms. Aspirin-PC was more potent (vs. aspirin) in blocking the growth of both human and mouse ovarian cancer cells in monolayer culture. Using in vivo model systems of ovarian cancer, we found that aspirin-PC significantly reduced ovarian cancer growth by 50% to 90% (depending on the ovarian cell line). The efficacy was further enhanced in combination with Bevacizumab or B20. The growth-inhibitory effect on ovarian tumor mass and number of tumor nodules was evident, but less pronounced for aspirin and the VEGF inhibitors alone. There was no detectable gastrointestinal toxicity. Both aspirin and aspirin-PC also inhibited cell proliferation, angiogenesis, and increased apoptosis of ovarian cancer cells. In conclusion, PC-associated aspirin markedly inhibits the growth of ovarian cancer cells, which exceeds that of the parent drug, in both cell culture and in mouse model systems. We also found that both aspirin-PC and aspirin have robust antineoplastic action in the presence of VEGF-blocking drugs. Mol Cancer Ther; 15(12); 2894–904. ©2016 AACR.

Sustained Adrenergic Signaling Promotes Intratumoral Innervation through BDNF Induction

Mounting clinical and preclinical evidence supports a key role for sustained adrenergic signaling in the tumor microenvironment as a driver of tumor growth and progression. However, the mechanisms by which adrenergic neurotransmitters are delivered to the tumor microenvironment are not well understood. Here we present evidence for a feed-forward loop whereby adrenergic signaling leads to increased tumoral innervation. In response to catecholamines, tumor cells produced brain-derived neurotrophic factor (BDNF) in an ADRB3/cAMP/Epac/JNK-dependent manner. Elevated BDNF levels in the tumor microenvironment increased innervation by signaling through host neurotrophic receptor tyrosine kinase 2 receptors. In patients with cancer, high tumor nerve counts were significantly associated with increased BDNF and norepinephrine levels and decreased overall survival. Collectively, these data describe a novel pathway for tumor innervation, with resultant biological and clinical implications.Significance: Sustained adrenergic signaling promotes tumor growth and metastasis through BDNF-mediated tumoral innervation. Cancer Res; 78(12); 3233-42. ©2018 AACR.

Abstract 5048: Platelet FAK is a critical regulator of tumor growth after withdrawal of anti-angiogenic therapy

Objective: In most clinical trials, anti-angiogenic therapies have offered only modest improvements in progression-free survival without impacting overall survival. Following cessation of anti-angiogenic therapy, concerns have been raised about a possible rebound in tumor growth, but the underlying mechanisms are poorly understood. Therefore, in this study we aimed at comparing tumor growth and the effects on tumor microenvironment after therapy withdrawal compared to continuous treatment with anti-angiogenic agents. Methods: Mice were intraperitoneally injected with human or mouse ovarian cancer cells and were treated with the anti-angiogenic drugs pazopanib, bevacizumab or B20 for either a short-term with subsequent withdrawal or continuous therapy until necropsy. Immunohistochemical staining was used to evaluate platelet infiltration into the tumor microenvironment, tumor angiogenesis and vascular leakage. To assess the significance of focal adhesion kinase (FAK) in the process of platelet infiltration and tumor rebound after withdrawal of therapy, we either used the FAK inhibitor GSK2256098 or a mouse model with platelet-specific FAK deletion. Results: Cessation of therapy with pazopanib, bevacizumab and the cross-human and murine anti-VEGF antibody B20 was associated with up to 4-fold increased tumor growth in mouse models of ovarian cancer when compared to continuous treatment. Tumor outgrowth was associated with significant tumor hypoxia, increased tumor angiogenesis and vascular leakage. More importantly, we found 380% increased hypoxia-induced ADP production and 3-fold increased platelet infiltration into tumors where anti-angiogenic therapy was withdrawn. Lowering platelet levels significantly inhibited tumor rebound after withdrawal of anti-angiogenic therapy. Interestingly, FAK in platelets regulated their migration into tumor microenvironment and FAK knock-out specifically in platelets completely prevented the rebound tumor growth. Additionally, combined therapy with the FAK inhibitor GSK2256098 and the anti-angiogenic agents pazopanib and bevacizumab led to up to 5-fold reduced orthotopic tumors and likewise inhibited negative effects of withdrawal of anti-angiogenic therapy. Conclusions: Collectively, our results characterize a previously unknown role for platelets in the tumor microenvironment and provide a potential therapeutic benefit for FAK inhibitors in preventing rebound in tumor growth following discontinuation of anti-angiogenic agents. Additionally, dual targeting of FAK and VEGF could have important therapeutic implications for ovarian cancer management. Citation Format: Monika Haemmerle, Justin Bottsford-Miller, Sunila Pradeep, Morgan L. Taylor, Hyun-Jin Choi, Rebecca L. Stone, Min Soon Cho, Alpa M. Nick, Gabriel Lopez-Berestein, Vahid Afshar-Khargan, Anil K. Sood. Platelet FAK is a critical regulator of tumor growth after withdrawal of anti-angiogenic therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5048.

Abstract 1062: Sustained adrenergic signaling promotes intratumoral innervation through BDNF induction

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Mounting clinical and preclinical evidence supports a key role of sustained adrenergic signaling in the tumor microenvironment as a driver of tumor growth and progression. However, the mechanisms by which adrenergic neurotransmitters are delivered to the tumor microenvironment are not well understood. Sustained adrenergic signaling resulted in increased tumor growth and was correlated with a significant increase in intratumoral nerve density and norepinephrine in several orthotopic models of disease. These effects were completely abrogated by hexamethonium bromide (ganglionic blocker), but not by removal of the adrenal glands. To elucidate mechanism by which tumor cells induce nerve growth, we analyzed gene expression changes and found BDNF to be a key factor upregulated by catecholamine treatment. Treatment with different ADRB agonist and blockers determined that ADRB3 is required for the induction of BDNF. Use of cAMP and Epac agonist/antagonist demonstrated that this axis is required for catecholamine induced BDNF expression. Further analyses revealed that JNK exerts its control of BDNF expression through the regulation of AP-1. In various orthotopic models of disease, chronic stress significantly increased tumor growth and intratumoral innervation, an effect completely blocked by BDNF siRNA-DOPC nanoliposomes. Moreover, BNDF overexpression was sufficient to induce tumor growth and intratumoral innervation. Silencing of TrkB, BDNF receptor, on the host by murine BDNF siRNA-Chitosan nanoparticles blocked the effects of sustained adrenergic signaling on tumor growth and innervation. Additionally, inhibition of host TrkB activity in a transgenic TrkB Kinase-Switch mouse model inoculated with ovarian cancer cells showed that adrenergic induced tumor growth and innervation is mediated by the host TrkB. In cancer patients, high tumor nerve density was significantly associated with increased BDNF and norepinephrine levels, and worse overall survival. Collectively, these data describe a novel pathway for tumor neo-innervation with resultant biological and clinical implications. Citation Format: Guillermo N. Armaiz-Pena, Archana S. Nagaraja, Julie K. Allen, Nouara C. Sadaoui, Rajesha Rupaimoole, sherry Y. wu, Sunila Pradeep, Hee Dong Han, Behrouz Zand, Heather Dalton, Rebecca Previs, Morgan Taylor, Justin Bottsford-Miller, Lingegowda S. Mangala, Cristian Rodriguez-Aguayo, Mariela De Biasi, Gabriel Lopez-Berestein, Steve Cole, Susan K. Lutgendorf, Anil K. Sood. Sustained adrenergic signaling promotes intratumoral innervation through BDNF induction. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1062. doi:10.1158/1538-7445.AM2014-1062