MARCKS contributes to stromal cancer-associated fibroblast activation and facilitates ovarian cancer metastasis.
ABSTRACT: The Cancer Genome Atlas network has revealed that the 'mesenchymal' epithelial ovarian cancer (EOC) subtype represents the poorest outcome, indicating a crucial role of stromal cancer-associated fibroblasts (CAFs) in disease progression. The cooperative role of CAFs in EOC metastasis has long been recognized, but the mechanisms of stromal CAFs activation are still obscure. Therefore, we carried out an integrative analysis to identify the regulator genes that are responsible for CAFs activation in microdissected tumor stroma profiles. Here, we determined that myristoylated alanine-rich C-kinase substrate (MARCKS) was highly expressed in ovarian stroma, and was required for the differentiation and tumor promoting function of CAFs. Suppression of MARCKS resulted in the loss of CAF features, and diminished role of CAFs in supporting tumor cell growth in 3D organotypic cultures and in murine xenograft model. Mechanistically, we found that MARCKS maintained CAF activation through suppression of cellular senescence and activation of the AKT/Twist1 signaling. Moreover, high MARCKS expression was associated with poor patient survival in EOC. Collectively, our findings identify the potential of MARCKS inhibition as a novel stroma-oriented therapy in EOC.
Project description:Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Identification of new therapeutic targets is crucial. MARCKS, myristoylated alanine-rich C-kinase substrate, has been implicated in aggressiveness of several cancers and MARCKS inhibitors are in development. Using immunohistochemistry (IHC), we retrospectively assessed MARCKS expression in epithelial and stromal cells of 118 pre-chemotherapy EOC samples and 40 normal ovarian samples from patients treated at Salah Azaiez Institute. We compared MARCKS expression in normal versus cancer samples, and searched for correlations with clinicopathological features, including overall survival (OS). Seventy-five percent of normal samples showed positive epithelial MARCKS staining versus 50% of tumor samples (p = 6.02 × 10-3). By contrast, stromal MARCKS expression was more frequent in tumor samples (77%) than in normal samples (22%; p = 1.41 × 10-9). There was no correlation between epithelial and stromal IHC MARCKS statutes and prognostic clinicopathological features. Stromal MARCKS expression was correlated with shorter poor OS in uni- and multivariate analyses. Stromal MARCKS overexpression in tumors might contribute to cancer-associated fibroblasts activation and to the poor prognosis of EOC, suggesting a potential therapeutic interest of MARCKS inhibition for targeting the cooperative tumor stroma.
Project description:Most solid tumors contain cancer-associated fibroblasts (CAFs) that support tumorigenesis and malignant progression. However, the cellular origins of CAFs in epithelial ovarian cancers (EOCs) remain poorly understood, and their utility as a source of clinical biomarkers for cancer diagnosis has not been explored in great depth. Here, we report establishing in vitro and in vivo models of CAFs in ovarian cancer development. Normal ovarian fibroblasts and mesenchymal stem cells cultured in the presence of EOC cells acquired a CAF-like phenotype, and promoted EOC cell migration in vitro. CAFs also promoted ovarian cancer growth in vivo in both subcutaneous and intraperitoneal murine xenograft assays. Molecular profiling of CAFs identified gene expression signatures that were highly enriched for extracellular and secreted proteins. We identified novel candidate CAF-specific biomarkers for ovarian cancer including NPPB, which was expressed in the stroma of 60% primary ovarian cancer tissues (n?=?145) but not in the stroma of normal ovaries (n?=?4). NPPB is a secreted protein that was also elevated in the blood of 50% of women with ovarian cancer (n?=?8). Taken together, these data suggest that the tumor stroma is a novel source of biomarkers, including NPPB, that may be of clinical utility for detection of EOC.
Project description:Adrenergic signaling is known to promote tumor growth and metastasis, but the effects on tumor stroma are not well understood. An unbiased bioinformatics approach analyzing tumor samples from patients with known biobehavioral profiles identified a prominent stromal signature associated with cancer-associated fibroblasts (CAFs) in those with a high biobehavioral risk profile (high Center for Epidemiologic Studies Depression Scale [CES-D] score and low social support). In several models of epithelial ovarian cancer, daily restraint stress resulted in significantly increased CAF activation and was abrogated by a nonspecific β-blocker. Adrenergic signaling-induced CAFs had significantly higher levels of collagen and extracellular matrix components than control tumors. Using a systems-based approach, we found INHBA production by cancer cells to induce CAFs. Ablating inhibin β A decreased CAF phenotype both in vitro and in vivo. In preclinical models of breast and colon cancers, there were increased CAFs and collagens following daily restraint stress. In an independent data set of renal cell carcinoma patients, there was an association between high depression (CES-D) scores and elevated expression of ACTA2, collagens, and inhibin β A. Collectively, our findings implicate adrenergic influences on tumor stroma as important drivers of CAFs and establish inhibin β A as an important regulator of the CAF phenotype in ovarian cancer.
Project description:The connection between signaling pathways activating cancer-associated fibroblasts (CAFs) remains to be determined. Metabolic alterations linked to autophagy have also been implicated in CAF activation. CSL/RBPJ, a transcriptional repressor that mediates Notch signaling, suppresses the gene expression program(s), leading to stromal senescence and CAF activation. Deregulated GLI signaling can also contribute to CAF conversion. Here, we report that compromised CSL function depends on GLI activation for conversion of human dermal fibroblasts into CAFs, separately from cellular senescence. Decreased CSL upregulates the expression of the ULK3 kinase, which binds and activates GLI2. Increased ULK3 also induces autophagy, which is unlinked from GLI and CAF activation. ULK3 upregulation occurs in the CAFs of several tumor types, and ULK3 silencing suppresses the tumor-enhancing properties of these cells. Thus, ULK3 links two key signaling pathways involved in CAF conversion and is an attractive target for stroma-focused anti-cancer intervention.
Project description:The tumor microenvironment plays an important role in the progression of cancer. This study focused on carcinoma-associated fibroblasts (CAFs) and stromal-epithelial interaction between CAFs and epithelial ovarian carcinoma (EOC) cells. We isolated and established primary cultures of CAFs and co-cultured CAFs and EOC cells in vitro. The co-culture conditioned medium (CC-CM) was harvested and its influence on EOC cells was examined. Cytokine, chemokine, and growth factor levels were screened using a biotin label-based human antibody array system. We found that the stromal-epithelial crosstalk provided a suitable microenvironment for the progression of ovarian cancer cells in vitro.
Project description:Pancreatic tumors are known to harbor an abundant and highly desmoplastic stroma. Among the various cell types that reside within tumor stroma, cancer-associated fibroblasts (CAFs) have gained a lot of attention in the cancer field due to their contributions to carcinogenesis and tumor architecture. These cells are not a homogeneous population, but have been shown to have different origins, phenotypes, and contributions. In pancreatic tumors, CAFs generally emerge through the activation and/or recruitment of various cell types, most notably resident fibroblasts, pancreatic stellate cells (PSCs), and tumor-infiltrating mesenchymal stem cells (MSCs). In recent years, single cell transcriptomic studies allowed the identification of distinct CAF populations in pancreatic tumors. Nonetheless, the exact sources and functions of those different CAF phenotypes remain to be fully understood. Considering the importance of stromal cells in pancreatic cancer, many novel approaches have aimed at targeting the stroma but current stroma-targeting therapies have yielded subpar results, which may be attributed to heterogeneity in the fibroblast population. Thus, fully understanding the roles of different subsets of CAFs within the stroma, and the cellular dynamics at play that contribute to heterogeneity in CAF subsets may be essential for the design of novel therapies and improving clinical outcomes. Fortunately, recent advances in technologies such as microfluidics and bio-printing have made it possible to establish more advanced <i>ex vivo</i> models that will likely prove useful. In this review, we will present the different roles of stromal cells in pancreatic cancer, focusing on CAF origin as a source of heterogeneity, and the role this may play in therapy failure. We will discuss preclinical models that could be of benefit to the field and that may contribute to further clinical development.
Project description:<h4>Background & aims</h4>Cancer-associated fibroblasts (CAFs) from pancreatic adenocarcinoma (PDA) present high protein synthesis rates. CAFs express the G-protein-coupled somatostatin receptor sst1. The sst1 agonist SOM230 blocks CAF protumoral features in vitro and in immunocompromised mice. We have explored here the therapeutic potential of SOM230, and underlying mechanisms, in immunocompetent models of murine PDA mimicking the heavy fibrotic and immunosuppressive stroma observed in patient tumors.<h4>Methods</h4>Large-scale mass spectrometry analyses were performed on media conditioned from 9 patient PDA-derived CAF primary cultures. Spontaneous transgenic and experimental (orthotopic co-graft of tumor cells plus CAFs) PDA-bearing mice were longitudinally ultrasound-monitored for tumor and metastatic progression. Histopathology and flow cytometry analyses were performed on primary tumors and metastases. Stromal signatures were functionally validated through bioinformatics using several published, and 1 original, PDA database.<h4>Results</h4>Proteomics on the CAF secretome showed that SOM230 controls stromal activities including inflammatory responses. Among the identified secreted proteins, we validated that colony-stimulating factor 1 (CSF-1) (a macrophage growth factor) was reduced by SOM230 in the tumor and plasma of PDA-harboring mice, alongside intratumor stromal normalization (reduced CAF and macrophage activities), and dramatic metastasis reduction. In transgenic mice, these SOM230 benefits alleviate the chemotherapy-induced (gemcitabine) immunosuppressive stroma reshaping. Mechanistically, SOM230 acts in vivo on CAFs through sst1 to disrupt prometastatic CAF production of CSF-1 and cross-talk with macrophages. We found that in patients, stromal CSF-1 was associated with aggressive PDA forms.<h4>Conclusions</h4>We propose SOM230 as an antimetastatic therapy in PDA for its capacity to remodel the fibrotic and immunosuppressive myeloid stroma. This pharmacotherapy should benefit PDA patients treated with chemotherapies.
Project description:UNLABELLED:Cancer-associated fibroblasts (CAF) are a major constituent of the tumor stroma, but little is known about how cancer cells transform normal fibroblasts into CAFs. microRNAs (miRNA) are small noncoding RNA molecules that negatively regulate gene expression at a posttranscriptional level. Although it is clearly established that miRNAs are deregulated in human cancers, it is not known whether miRNA expression in resident fibroblasts is affected by their interaction with cancer cells. We found that in ovarian CAFs, miR-31 and miR-214 were downregulated, whereas miR-155 was upregulated when compared with normal or tumor-adjacent fibroblasts. Mimicking this deregulation by transfecting miRNAs and miRNA inhibitors induced a functional conversion of normal fibroblasts into CAFs, and the reverse experiment resulted in the reversion of CAFs into normal fibroblasts. The miRNA-reprogrammed normal fibroblasts and patient-derived CAFs shared a large number of upregulated genes highly enriched in chemokines, which are known to be important for CAF function. The most highly upregulated chemokine, CCL5, (C-C motif ligand 5) was found to be a direct target of miR-214. These results indicate that ovarian cancer cells reprogram fibroblasts to become CAFs through the action of miRNAs. Targeting these miRNAs in stromal cells could have therapeutic benefit. SIGNIFICANCE:The mechanism by which quiescent fibroblasts are converted into CAFs is unclear. The present study identifies a set of 3 miRNAs that reprogram normal fibroblasts to CAFs. These miRNAs may represent novel therapeutic targets in the tumor microenvironment.
Project description:Cancer-associated fibroblasts (CAFs) are the key components of the densely proliferated stroma in pancreatic ductal adenocarcinoma (PDAC) and contribute to tumor progression and drug resistance. CAFs comprise heterogeneous subpopulations playing unique and vital roles. However, the commonly used mouse models have not been able to fully reproduce the histological and functional characteristics of clinical human CAF. Here, we generated a human cell-derived stroma-rich CDX (Sr-CDX) model, to reproduce the clinical tumor microenvironment. By co-transplanting human adipose-derived mesenchymal stem cells (AD-MSCs) and a human PDAC cell line (Capan-1) into mice, the Sr-CDX model recapitulated the characteristics of clinical pancreatic cancer, such as accelerated tumor growth, abundant stromal proliferation, chemoresistance, and dense stroma formed from the heterogeneous CAFs. Global RNA sequencing, single-cell based RNA sequencing, and histological analysis of CAFs in the Sr-CDX model revealed that the CAFs of the Sr-CDX mice were derived from the transplanted AD-MSCs and composed of heterogeneous subpopulations of CAF, including known and unknown subtypes. These lines of evidences suggest that our new tumor-bearing mouse model has the potential to address an open question in CAF research, that is the mechanism of CAF differentiation.
Project description:Sphingosine kinase 1 (SPHK1), the enzyme that produces sphingosine 1 phosphate (S1P), is known to be highly expressed in many cancers. However, the role of SPHK1 in cells of the tumor stroma remains unclear. Here, we show that SPHK1 is highly expressed in the tumor stroma of high-grade serous ovarian cancer (HGSC), and is required for the differentiation and tumor promoting function of cancer-associated fibroblasts (CAFs). Knockout or pharmacological inhibition of SPHK1 in ovarian fibroblasts attenuated TGF-?-induced expression of CAF markers, and reduced their ability to promote ovarian cancer cell migration and invasion in a coculture system. Mechanistically, we determined that SPHK1 mediates TGF-? signaling via the transactivation of S1P receptors (S1PR2 and S1PR3), leading to p38 MAPK phosphorylation. The importance of stromal SPHK1 in tumorigenesis was confirmed in vivo, by demonstrating a significant reduction of tumor growth and metastasis in SPHK1 knockout mice. Collectively, these findings demonstrate the potential of SPHK1 inhibition as a novel stroma-targeted therapy in HGSC.