Chemotherapy enriches for pro-inflammatory macrophage phenotypes that support cancer stem-like cells and disease progression in ovarian cancer.

High-grade serous ovarian cancer (HGSOC) remains a poorly understood disease with a high mortality rate. While most patients respond to cytotoxic therapies, a majority will experience recurrence. This may be due to a minority of drug resistant cancer stem-like cells (CSCs) that survive chemotherapy and are capable of repopulating heterogenous tumors. It remains unclear how CSCs are supported in the tumor microenvironment (TME) particularly during chemotherapy exposure. Tumor associated macrophages (TAMs) make up half of the immune population of the ovarian TME and are known to support CSCs and contribute to cancer progression. TAMs are plastic cells that alter their phenotype in response to environmental stimuli and thus may influence CSC maintenance during chemotherapy. Given the plasticity of TAMs we studied the effects of carboplatin on macrophage phenotypes using both THP-1- and peripheral blood mononuclear cell (PBMC)- derived macrophages and whether this supports CSCs and ovarian cancer progression following treatment. We found that carboplatin exposure induces an M1-like pro-inflammatory phenotype that promotes SOX2 expression, spheroid formation, and CD117+ ovarian CSCs, and that macrophage-secreted CCL2/MCP-1 is at least partially responsible for this effect. Depletion of TAMs during carboplatin exposure results in fewer CSCs and prolonged survival in a xenograft model of ovarian cancer. This study supports a role for platinum-based chemotherapies in promoting a transient pro-inflammatory M1-like TAM that enriches for CSCs during treatment. Improving our understanding of TME responses to cytotoxic drugs and identifying novel mechanisms of CSC maintenance will enable the development of better therapeutic strategies for HGSOC.

Catalytically distinct IDH1 mutants tune phenotype severity in tumor models.

Mutations in isocitrate dehydrogenase 1 (IDH1) impart a neomorphic reaction that produces the oncometabolite D-2-hydroxyglutarate (D2HG), which can inhibit DNA and histone demethylases to drive tumorigenesis via epigenetic changes. Though heterozygous point mutations in patients primarily affect residue R132, there are myriad D2HG-producing mutants that display unique catalytic efficiency of D2HG production. Here, we show that catalytic efficiency of D2HG production is greater in IDH1 R132Q than R132H mutants, and expression of IDH1 R132Q in cellular and mouse xenograft models leads to higher D2HG concentrations in cells, tumors, and sera compared to R132H-expressing models. Reduced representation bisulfite sequencing (RRBS) analysis of xenograft tumors shows expression of IDH1 R132Q relative to R132H leads to hypermethylation patterns in pathways associated with DNA damage. Transcriptome analysis indicates that the IDH1 R132Q mutation has a more aggressive pro-tumor phenotype, with members of EGFR, Wnt, and PI3K signaling pathways differentially expressed, perhaps through non-epigenetic routes. Together, these data suggest that the catalytic efficiency of IDH1 mutants modulate D2HG levels in cellular and in vivo models, resulting in unique epigenetic and transcriptomic consequences where higher D2HG levels appear to be associated with more aggressive tumors.

Omental Preadipocytes Stimulate Matrix Remodeling and IGF Signaling to Support Ovarian Cancer Metastasis.

Ovarian cancer can metastasize to the omentum, which is associated with a complex tumor microenvironment. Omental stromal cells facilitate ovarian cancer colonization by secreting cytokines and growth factors. An improved understanding of the tumor-supportive functions of specific cell populations in the omentum could identify strategies to prevent and treat ovarian cancer metastasis. Here, we showed that omental preadipocytes enhance the tumor initiation capacity of ovarian cancer cells. Secreted factors from preadipocytes supported cancer cell viability during nutrient and isolation stress and enabled prolonged proliferation. Coculturing with preadipocytes led to the upregulation of genes involved in extracellular matrix (ECM) organization, cellular response to stress, and regulation of insulin-like growth factor (IGF) signaling in ovarian cancer cells. IGF1 induced ECM genes and increased alternative NF-κB signaling by activating RelB. Inhibiting the IGF1 receptor initially increased tumor omental adhesion but decreased the growth of established preadipocyte-induced subcutaneous tumors as well as established intraperitoneal tumors. Together, this study shows that omental preadipocytes support ovarian cancer progression, which has implications for targeting metastasis. Significance: Omental preadipocyte-mediated IGF1 signaling promotes ovarian cancer tumorigenesis and metastasis via extracellular matrix remodeling, revealing a role for preadipocytes in regulating ovarian cancer progression and highlighting potential therapeutic targets for metastatic disease.

TWEAK-Fn14-RelB Signaling Cascade Promotes Stem Cell-like Features that Contribute to Post-Chemotherapy Ovarian Cancer Relapse.

Disease recurrence in high-grade serous ovarian cancer may be due to cancer stem-like cells (CSC) that are resistant to chemotherapy and capable of reestablishing heterogeneous tumors. The alternative NF-κB signaling pathway is implicated in this process; however, the mechanism is unknown. Here we show that TNF-like weak inducer of apoptosis (TWEAK) and its receptor, Fn14, are strong inducers of alternative NF-κB signaling and are enriched in ovarian tumors following chemotherapy treatment. We further show that TWEAK enhances spheroid formation ability, asymmetric division capacity, and expression of SOX2 and epithelial-to-mesenchymal transition genes VIM and ZEB1 in ovarian cancer cells, phenotypes that are enhanced when TWEAK is combined with carboplatin. Moreover, TWEAK in combination with chemotherapy induces expression of the CSC marker CD117 in CD117- cells. Blocking the TWEAK-Fn14-RelB signaling cascade with a small-molecule inhibitor of Fn14 prolongs survival following carboplatin chemotherapy in a mouse model of ovarian cancer. These data provide new insights into ovarian cancer CSC biology and highlight a signaling axis that should be explored for therapeutic development. IMPLICATIONS: This study identifies a unique mechanism for the induction of ovarian cancer stem cells that may serve as a novel therapeutic target for preventing relapse.

Insulin-like growth factor binding protein 5: Diverse roles in cancer.

Insulin-like growth factor binding proteins (IGFBPs) and the associated signaling components in the insulin-like growth factor (IGF) pathway regulate cell differentiation, proliferation, apoptosis, and adhesion. Of the IGFBPs, insulin-like growth factor binding protein 5 (IGFBP5) is the most evolutionarily conserved with a dynamic range of IGF-dependent and -independent functions, and studies on the actions of IGFBP5 in cancer have been somewhat paradoxical. In cancer, the IGFBPs respond to external stimuli to modulate disease progression and therapeutic responsiveness in a context specific manner. This review discusses the different roles of IGF signaling and IGFBP5 in disease with an emphasis on discoveries within the last twenty years, which underscore a need to clarify the IGF-independent actions of IGFBP5, the impact of its subcellular localization, the differential activities of each of the subdomains, and the response to elements of the tumor microenvironment (TME). Additionally, recent advances addressing the role of IGFBP5 in resistance to cancer therapeutics will be discussed. A better understanding of the contexts in which IGFBP5 functions will facilitate the discovery of new mechanisms of cancer progression that may lead to novel therapeutic opportunities.

Characterization of SOX2, OCT4 and NANOG in Ovarian Cancer Tumor-Initiating Cells.

The identification of tumor-initiating cells (TICs) has traditionally relied on surface markers including CD133, CD44, CD117, and the aldehyde dehydrogenase (ALDH) enzyme, which have diverse expression across samples. A more reliable indication of TICs may include the expression of embryonic transcription factors that support long-term self-renewal, multipotency, and quiescence. We hypothesize that SOX2, OCT4, and NANOG will be enriched in ovarian TICs and may indicate TICs with high relapse potential. We evaluated a panel of eight ovarian cancer cell lines grown in standard 2-D culture or in spheroid-enriching 3-D culture, and correlated expression with growth characteristics, TIC marker expression, and chemotherapy resistance. RNA-sequencing showed that cell cycle regulation pathways involving SOX2 were elevated in 3-D conditions. HGSOC lines had longer doubling-times, greater chemoresistance, and significantly increased expression of SOX2, OCT4, and NANOG in 3-D conditions. CD117+ or ALDH+/CD133+ cells had increased SOX2, OCT4, and NANOG expression. Limiting dilution in in vivo experiments implicated SOX2, but not OCT4 or NANOG, with early tumor-initiation. An analysis of patient data suggested a stronger role for SOX2, relative to OCT4 or NANOG, for tumor relapse potential. Overall, our findings suggest that SOX2 may be a more consistent indicator of ovarian TICs that contribute to tumor repopulation following chemotherapy. Future studies evaluating SOX2 in TIC biology will increase our understanding of the mechanisms that drive ovarian cancer relapse.

Small intestinal submucosa-derived extracellular matrix bioscaffold significantly enhances angiogenic factor secretion from human mesenchymal stromal cells.

INTRODUCTION: The in vivo therapeutic effect of mesenchymal stromal cells (MSCs) is currently believed to be tightly linked to their paracrine secretion ability. However, insufficient or imprecise cell delivery, low cell survival and retention post-transplant, along with harsh donor site microenvironments, are major barriers to the clinical success of MSC therapies. Here we tested a small intestinal submucosa (SIS)-derived extracellular matrix (ECM) bioscaffold augmented with MSCs, with the hypothesis that they will facilitate the precise delivery of increased numbers of MSCs therefore improving cell viability and retention. METHODS: In this study, we evaluated the secretion of angiogenic factors from three human MSC lines cultured on SIS ECM. We used human antibody array and enzyme-linked immunosorbent assay to measure the level of angiogenic factors released from MSCs when cultured on SIS ECM or regular tissue culture plastic. We tested MSCs cultured for three different time points. RESULTS: We found that the SIS ECM culture environment can significantly enhance the release of several angiogenic factors when compared to MSCs cultured on standard tissue culture plastic. Specifically, vascular endothelial growth factor and interleukin-8 secretion was significantly increased at 24, 48 and 72 hours postseeding onto SIS ECM whereas vascular endothelial growth factor release for cells cultured on plastic surface remained the same during these time points. We also observed significant donor to donor variation in cytokine production. CONCLUSIONS: This study demonstrates that MSCs transplanted onto a SIS ECM may greatly increase their therapeutic potential through an increase in pro-angiogenic cytokine release.

Kinase suppressor of Ras (KSR1) modulates multiple kit-ligand-dependent mast cell functions.

The intricately regulated Ras pathway coordinates multiple kit-ligand-induced mast cell functions, including chemotaxis, proliferation, and degranulation. However, the intracellular proteins that modulate the intensity and duration of stem cell factor-induced signals and the consequent cellular response are incompletely understood. Scaffolding proteins coordinate the spatial organization of mitogen-activated protein kinase proteins that may potentiate and/or inhibit cell functions. The kinase suppressor of Ras (KSR1) protein is known to function as a molecular scaffold and coordinates the organization of Raf/Mek/Erk in response to receptor tyrosine kinases. However, the impact of KSR1 in myeloid mast cell functions and in response to stem cell factor remains unknown. In the present study, we investigated the role of KSR1 in regulating cellular functions of bone marrow-derived mast cells of KSR1-deficient ((-/-)) mice. Genetic disruption of KSR1 resulted in both striking reductions in kit-ligand-mediated proliferation and degranulation, which are commonly attributed to mitogen-activated protein kinase signals. Surprisingly, disruption of the KSR1 scaffold also resulted in a decline in migration that is generally not linked to Raf-Erk signals. We found that loss of KSR1 does impact the biochemical activation of p21-activated kinase, a kinase that is known to modulate Raf-Erk signals and also F-actin polymerization key to mast cell migration. Collectively, these studies demonstrate that the scaffolding protein KSR1 has an important role in multiple kit-ligand-mediated mast cell functions. This study elucidates varied mast cell physiological functions for KSR1, including those related to cytoskeletal organization, and it suggests a novel molecular target for attenuating mast cell-mediated inflammation.