sFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance.

Cancer is a disease of ageing. Clinically, aged cancer patients tend to have a poorer prognosis than young. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumour progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression, we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. Here we find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signalling cascade in melanoma cells that results in a decrease in ß-catenin and microphthalmia-associated transcription factor (MITF), and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to DNA damage induced by reactive oxygen species, rendering the cells more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumour progression, offering new possibilities for the design of therapy for the elderly.

Modeling the two-way feedback between contractility and matrix realignment reveals a nonlinear mode of cancer cell invasion.

Cancer cell invasion from primary tumors is mediated by a complex interplay between cellular adhesions, actomyosin-driven contractility, and the physical characteristics of the extracellular matrix (ECM). Here, we incorporate a mechanochemical free-energy-based approach to elucidate how the two-way feedback loop between cell contractility (induced by the activity of chemomechanical interactions such as Ca2+ and Rho signaling pathways) and matrix fiber realignment and strain stiffening enables the cells to polarize and develop contractile forces to break free from the tumor spheroids and invade into the ECM. Interestingly, through this computational model, we are able to identify a critical stiffness that is required by the matrix to break intercellular adhesions and initiate cell invasion. Also, by considering the kinetics of the cell movement, our model predicts a biphasic invasiveness with respect to the stiffness of the matrix. These predictions are validated by analyzing the invasion of melanoma cells in collagen matrices of varying concentration. Our model also predicts a positive correlation between the elongated morphology of the invading cells and the alignment of fibers in the matrix, suggesting that cell polarization is directly proportional to the stiffness and alignment of the matrix. In contrast, cells in nonfibrous matrices are found to be rounded and not polarized, underscoring the key role played by the nonlinear mechanics of fibrous matrices. Importantly, our model shows that mechanical principles mediated by the contractility of the cells and the nonlinearity of the ECM behavior play a crucial role in determining the phenotype of the cell invasion.

Age-related increases in fibroblast-secreted IGFBP2 increase melanoma cell invasion and lipid synthesis.

Aged melanoma patients (>65 years old) have more aggressive disease relative to young patients (<55 years old) for reasons that are not completely understood. Analysis of the young and aged secretome from human dermal fibroblasts identified >5-fold levels of insulin-like growth factor binding protein 2 (IGFBP2) in the aged fibroblast secretome. IGFBP2 functionally triggers upregulation of the PI3K-dependent fatty acid biosynthesis program in melanoma cells. Melanoma cells co-cultured with aged dermal fibroblasts have higher levels of lipids relative to co-cultured with young dermal fibroblasts, which can be lowered by silencing IGFBP2 expression in fibroblasts, prior to treating with conditioned media. Conversely, ectopically treating melanoma cells with recombinant IGFBP2 in the presence of conditioned media from young fibroblasts, or overexpressing IGFBP2 in melanoma cells promoted lipid synthesis and accumulation in the melanoma cells. Treatment of young mice with rIGFBP2 increases tumor growth. Neutralizing IGFBP2 in vitro reduces migration and invasion in melanoma cells, and in vivo studies demonstrate that neutralizing IGFBP2 in syngeneic aged mice reduces tumor growth amd metastasis. Our results suggest that aged dermal fibroblasts increase melanoma cell aggressiveness through increased secretion of IGFBP2, stressing the importance of considering age when designing studies and treatment.

Myeloid-Derived Suppressor Cells Are a Major Source of Wnt5A in the Melanoma Microenvironment and Depend on Wnt5A for Full Suppressive Activity.

Metastatic dissemination remains a significant barrier to successful therapy for melanoma. Wnt5A is a potent driver of invasion in melanoma and is believed to be secreted from the tumor microenvironment (TME). Our data suggest that myeloid-derived suppressor cells (MDSC) in the TME are a major source of Wnt5A and are reliant upon Wnt5A for multiple actions. Knockdown of Wnt5A specifically in the myeloid cells demonstrated a clear decrease in Wnt5A expression within the TME in vivo as well as a decrease in intratumoral MDSC and regulatory T cell (Treg). Wnt5A knockdown also decreased the immunosuppressive nature of MDSC and decreased expression of TGFß1 and arginase 1. In the presence of Wnt5A-depleted MDSC, tumor-infiltrating lymphocytes expressed decreased PD-1 and LAG3, suggesting a less exhausted phenotype. Myeloid-specific Wnt5A knockdown also led to decreased lung metastasis. Tumor-infiltrating MDSC from control animals showed a strong positive correlation with Treg, which was completely ablated in animals with Wnt5A-negative MDSC. Overall, our data suggest that while MDSC contribute to an immunosuppressive and less immunogenic environment, they exhibit an additional function as the major source of Wnt5A in the TME. SIGNIFICANCE: These findings demonstrate that myeloid cells provide a major source of Wnt5A to facilitate metastatic potential in melanoma cells and rely on Wnt5A for their immunosuppressive function.

Activation of JAK2/STAT3 signaling by osteopontin promotes tumor growth in human breast cancer cells.

Deregulation of signal transducer and activator of transcription (STAT)-3 signaling plays crucial role in oncogenesis of various cancers. However, the molecular mechanism by which osteopontin (OPN), a chemokine-like extracellular matrix-associated protein, regulates STAT3 activation that leads to tumor progression and inhibits apoptosis in breast cancer cells is not well understood. In this study, we for the first time report that OPN upregulates alphavbeta3 integrin-mediated Janus kinase 2 (JAK2) phosphorylation and STAT3 activation in breast cancer (MDA-MB-468 and MCF-7) cells. Pretreatment of cells with JAK2 inhibitor (AG 490) suppresses OPN-induced STAT3 phosphorylation, its nuclear localization and DNA binding indicating that JAK2 is involved in this process. Transfection of cells with wild-type (wt) STAT3 enhanced whereas mutant STAT3 (STAT3 Y705F) suppressed OPN-induced breast tumor cell migration. Treatment of cells with OPN followed by staurosporine (STS) showed that OPN protects the cells from STS-induced apoptosis. Moreover, transfection of cells with wt STAT3 upregulates whereas STAT3 Y705F downregulates Bcl2 and cyclin D1 expressions in response to OPN. Interestingly, STAT3-overexpressing cells when injected to non-obese diabetic/severe combined immunodeficiency mice followed by OPN treatment, the mice developed enhanced tumor growth as compared with STAT3 Y705F-injected mice or mice injected with OPN alone. The levels of Bcl2 and cyclin D1 in wt STAT3 tumors were significantly higher than controls. Clinical specimen analysis revealed that increased OPN and pSTAT3 expressions correlate with enhanced breast tumor progression. Thus, targeting OPN and its regulated STAT3 signaling could be a potent therapeutic approach and understanding these mechanisms may form the basis of new therapeutic regimen for the management of breast cancer.

Changes in Aged Fibroblast Lipid Metabolism Induce Age-Dependent Melanoma Cell Resistance to Targeted Therapy via the Fatty Acid Transporter FATP2.

Older patients with melanoma (>50 years old) have poorer prognoses and response rates to targeted therapy compared with young patients (<50 years old), which can be driven, in part, by the aged microenvironment. Here, we show that aged dermal fibroblasts increase the secretion of neutral lipids, especially ceramides. When melanoma cells are exposed to the aged fibroblast lipid secretome, or cocultured with aged fibroblasts, they increase the uptake of lipids via the fatty acid transporter FATP2, which is upregulated in melanoma cells in the aged microenvironment and known to play roles in lipid synthesis and accumulation. We show that blocking FATP2 in melanoma cells in an aged microenvironment inhibits their accumulation of lipids and disrupts their mitochondrial metabolism. Inhibiting FATP2 overcomes age-related resistance to BRAF/MEK inhibition in animal models, ablates tumor relapse, and significantly extends survival time in older animals. SIGNIFICANCE: These data show that melanoma cells take up lipids from aged fibroblasts, via FATP2, and use them to resist targeted therapy. The response to targeted therapy is altered in aged individuals because of the influences of the aged microenvironment, and these data suggest FATP2 as a target to overcome resistance.See related commentary by Montal and White, p. 1255.This article is highlighted in the In This Issue feature, p. 1241.

Age Correlates with Response to Anti-PD1, Reflecting Age-Related Differences in Intratumoral Effector and Regulatory T-Cell Populations.

Purpose: We have shown that the aged microenvironment increases melanoma metastasis, and decreases response to targeted therapy, and here we queried response to anti-PD1.Experimental Design: We analyzed the relationship between age, response to anti-PD1, and prior therapy in 538 patients. We used mouse models of melanoma, to analyze the intratumoral immune microenvironment in young versus aged mice and confirmed our findings in human melanoma biopsies.Results: Patients over the age of 60 responded more efficiently to anti-PD-1, and likelihood of response to anti-PD-1 increased with age, even when we controlled for prior MAPKi therapy. Placing genetically identical tumors in aged mice (52 weeks) significantly increased their response to anti-PD1 as compared with the same tumors in young mice (8 weeks). These data suggest that this increased response in aged patients occurs even in the absence of a more complex mutational landscape. Next, we found that young mice had a significantly higher population of regulatory T cells (Tregs), skewing the CD8+:Treg ratio. FOXP3 staining of human melanoma biopsies revealed similar increases in Tregs in young patients. Depletion of Tregs using anti-CD25 increased the response to anti-PD1 in young mice.Conclusions: While there are obvious limitations to our study, including our inability to conduct a meta-analysis due to a lack of available data, and our inability to control for mutational burden, there is a remarkable consistency in these data from over 500 patients across 8 different institutes worldwide. These results stress the importance of considering age as a factor for immunotherapy response. Clin Cancer Res; 24(21); 5347-56. ©2018 AACR See related commentary by Pawelec, p. 5193.

The multifaceted roles of osteopontin in cell signaling, tumor progression and angiogenesis.

Osteopontin (OPN) is a chemokine like phosphorylated glycoprotein that plays important role in cancer progression. Extensive research from various laboratories has demonstrated the likely role of OPN in regulating the cell signaling that ultimately controls tumor growth and metastasis. Several earlier reports indicated that OPN is associated with various cancers; but its functional role in carcinogenesis is still not well defined. Besides the role of OPN in tumor biology, several studies have demonstrated the pathophysiological role of OPN in diverse biological events. This review will focus on recent advances in understanding the molecular mechanism by which OPN regulates a series of signaling cascades through activation of various kinases and transcription factors that ultimately control the expression of downstream effector genes, which contribute to tumor progression and angiogenesis in vitro and animal models. We will also provide evidences that suggest the enhanced expression of OPN is not only associated with several tumor types, but its level of expression is directly correlated to various stages of the clinical specimens of breast and prostate cancers. These studies may be useful for identifying novel OPN-based therapeutic approach for the treatment of cancer.

Inhibition of Age-Related Therapy Resistance in Melanoma by Rosiglitazone-Mediated Induction of Klotho.

Purpose: Aging is a poor prognostic factor for melanoma. We have shown that melanoma cells in an aged microenvironment are more resistant to targeted therapy than identical cells in a young microenvironment. This is dependent on age-related secreted factors. Klotho is an age-related protein whose serum levels decrease dramatically by age 40. Most studies on klotho in cancer have focused on the expression of klotho in the tumor cell. We have shown that exogenous klotho inhibits internalization and signaling of Wnt5A, which drives melanoma metastasis and resistance to targeted therapy. We investigate here whether increasing klotho in the aged microenvironment could be an effective strategy for the treatment of melanoma.Experimental Design: PPARγ increases klotho levels and is increased by glitazones. Using rosiglitazone, we queried the effects of rosiglitazone on Klotho/Wnt5A cross-talk, in vitro and in vivo, and the implications of that for targeted therapy in young versus aged animals.Results: We show that rosiglitazone increases klotho and decreases Wnt5A in tumor cells, reducing the burden of both BRAF inhibitor-sensitive and BRAF inhibitor-resistant tumors in aged, but not young mice. However, when used in combination with PLX4720, tumor burden was reduced in both young and aged mice, even in resistant tumors.Conclusions: Using glitazones as adjuvant therapy for melanoma may provide a new treatment strategy for older melanoma patients who have developed resistance to vemurafenib. As klotho has been shown to play a role in other cancers too, our results may have wide relevance for multiple tumor types. Clin Cancer Res; 23(12); 3181-90. ©2017 AACR.

Cancer-Associated Fibroblasts Neutralize the Anti-tumor Effect of CSF1 Receptor Blockade by Inducing PMN-MDSC Infiltration of Tumors.

Tumor-associated macrophages (TAM) contribute to all aspects of tumor progression. Use of CSF1R inhibitors to target TAM is therapeutically appealing, but has had very limited anti-tumor effects. Here, we have identified the mechanism that limited the effect of CSF1R targeted therapy. We demonstrated that carcinoma-associated fibroblasts (CAF) are major sources of chemokines that recruit granulocytes to tumors. CSF1 produced by tumor cells caused HDAC2-mediated downregulation of granulocyte-specific chemokine expression in CAF, which limited migration of these cells to tumors. Treatment with CSF1R inhibitors disrupted this crosstalk and triggered a profound increase in granulocyte recruitment to tumors. Combining CSF1R inhibitor with a CXCR2 antagonist blocked granulocyte infiltration of tumors and showed strong anti-tumor effects.

ATG5 Mediates a Positive Feedback Loop between Wnt Signaling and Autophagy in Melanoma.

Autophagy mediates resistance to various anticancer agents. In melanoma, resistance to targeted therapy has been linked to expression of Wnt5A, an intrinsic inhibitor of ß-catenin, which also promotes invasion. In this study, we assessed the interplay between Wnt5A and autophagy by combining expression studies in human clinical biopsies with functional analyses in cell lines and mouse models. Melanoma cells with high Wnt5A and low ß-catenin displayed increased basal autophagy. Genetic blockade of autophagy revealed an unexpected feedback loop whereby knocking down the autophagy factor ATG5 in Wnt5Ahigh cells decreased Wnt5A and increased ß-catenin. To define the physiologic relevance of this loop, melanoma cells with different Wnt status were treated in vitro and in vivo with the potent lysosomotropic compound Lys05. Wnt5Ahigh cells were less sensitive to Lys05 and could be reverted by inducing ß-catenin activity. Our results suggest the efficacy of autophagy inhibitors might be improved by taking the Wnt signature of melanoma cells into account. Cancer Res; 77(21); 5873-85. ©2017 AACR.

Association of osteopontin and cyclooxygenase-2 expression with breast cancer subtypes and their use as potential biomarkers.

Breast cancer is one of the most common malignant tumors among females worldwide and remains a leading cause of cancer-related mortality. Due to the heterogeneous clinical nature of breast cancer, it is necessary to identify new biomarkers that are associated with tumor growth, angiogenesis and metastasis. Osteopontin (OPN) and cyclooxygenase-2 (COX-2) are known to be overexpressed in invasive breast cancer and their overexpression is associated with aggressive histological and clinical features. The present study assessed OPN and COX-2 expression in various subtypes of breast cancer. The expression of OPN and COX-2 was analyzed using immunohistochemistry (IHC) in a cohort of 67 invasive ductal breast carcinoma patients. The statistical analysis was performed using standard statistical software SPSS version 18.0. The associations between OPN and COX-2 and the human epidermal growth factor receptor type 2 (HER2)-overexpressing and non-HER2-overexpressing subtypes were evaluated using the Mann-Whitney U test. The mean OPN level was significantly higher in the HER2-overexpressing subtype compared with the non-HER2-overexpressing subtype. Furthermore, the mean COX-2 expression levels were higher in the HER2-overexpressing subtype compared with the luminal A, luminal B or triple-negative groups. It is well known that carcinomas overexpressing HER2/neu have a worse prognosis than luminal tumors. Hence, it may be hypothesized that an elevated expression of OPN and COX-2 in a HER2-overexpressing subtype may contribute to a more aggressive behavior and be used as diagnostic and prognostic markers in breast cancer.

Osteopontin: a potentially important therapeutic target in cancer.

INTRODUCTION: Cancer is an extremely complex disease and most cancer treatments are limited to chemotherapy, radiation and surgery. The progression of tumours towards malignancy requires the interaction of various cytokines, growth factors, transcription factors and effector molecules. Osteopontin is a cytokine-like, calcium-binding, extracelular-matrix- associated member of the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family of proteins. It plays an important role in determining the oncogenic potential of various cancers. The role of osteopontin in various pathophysiological conditions suggests that the alteration in post-translational modification result in different functional forms that might change its normal physiological functions. AREAS COVERED: Osteopontin -based anticancer therapy, which may provide a new insight for the effective management of cancer. EXPERT OPINION: A better understanding of the signalling mechanism by which osteopontin promotes tumourigenesis may be useful in crafting novel osteopontin -based anticancer therapy. The role of osteopontin in promoting cancer progression is the subject of in depth investigation and thus targeting osteopontin might be a suitable therapeutic approach for the treatment of cancer.

p38 kinase is crucial for osteopontin-induced furin expression that supports cervical cancer progression.

p38 kinases activated by growth factors, hormones, and environmental stresses exert diverse functions in regulating normal and malignant cell pathophysiology. Enhanced levels of activated p38 isoforms have been linked with poor prognosis in breast cancer, although the mechanistic basis for this association is poorly understood. In this study, we report that p38 activation in cervical cancer cells is driven by osteopontin (OPN), an extracellular matrix-associated cytokine that drives invasive progression. OPN regulates CD44-mediated p38 phosphorylation that induces NF-κB activation and NF-κB-dependent expression of furin, an extracellular protease implicated in human papilloma virus (HPV) processing that enhances cervical cancer cell motility. OPN induces CD44-mediated MKK3/6 phosphorylation which in turn phosphorylates p38 in these cells. OPN-induced furin expression and cell motility was impeded by blockades to MKK3/6, p38α/ß or NF-κB signaling. In a mouse xenograft model of human cervical cancer, tumor growth was enhanced by OPN overexpression and blocked by short hairpin RNA (shRNA)-mediated OPN silencing. Furin overexpression similarly augmented tumor growth in the model, whereas blocking MKK3/6, p38, or furin reduced OPN-induced cervical tumor growth. Analysis of clinical specimens revealed that enhanced expression of OPN, phosphorylated NF-κB, p65, and furin correlated with cervical cancer progression, further strengthening the in vitro and in vivo results. In summary, our findings offer a proof of concept for targeting OPN and its downstream p38 signaling as a novel therapeutic strategy to manage cervical cancer.