Casey Trimmer

Cancer cells metabolically "fertilize" the tumor microenvironment with hydrogen peroxide, driving the Warburg effect: implications for PET imaging of human tumors.

Previously, we proposed that cancer cells behave as metabolic parasites, as they use targeted oxidative stress as a “weapon” to extract recycled nutrients from adjacent stromal cells. Oxidative stress in cancer-associated fibroblasts triggers autophagy and mitophagy, resulting in compartmentalized cellular catabolism, loss of mitochondrial function, and the onset of aerobic glycolysis, in the tumor stroma. As such, cancer-associated fibroblasts produce high-energy nutrients (such as lactate and ketones) that fuel mitochondrial biogenesis, and oxidative metabolism in cancer cells. We have termed this new energy-transfer mechanism the “reverse Warburg effect.” To further test the validity of this hypothesis, here we used an in vitro MCF7-fibroblast co-culture system, and quantitatively measured a variety of metabolic parameters by FACS analysis (analogous to laser-capture micro-dissection). Mitochondrial activity, glucose uptake, and ROS production were measured with highly-sensitive fluorescent probes (MitoTracker, NBD-2-deoxy-glucose, and DCF-DA). Interestingly, using this approach, we directly show that cancer cells initially secrete hydrogen peroxide that then triggers oxidative stress in neighboring fibroblasts. Thus, oxidative stress is contagious (spreads like a virus) and is propagated laterally and vectorially from cancer cells to adjacent fibroblasts. Experimentally, we show that oxidative stress in cancer-associated fibroblasts quantitatively reduces mitochondrial activity, and increases glucose uptake, as the fibroblasts become more dependent on aerobic glycolysis. Conversely, co-cultured cancer cells show significant increases in mitochondrial activity, and corresponding reductions in both glucose uptake and GLUT1 expression. Pre-treatment of co-cultures with extracellular catalase (an anti-oxidant enzyme that detoxifies hydrogen peroxide) blocks the onset of oxidative stress, and potently induces the death of cancer cells, likely via starvation. Given that cancer-associated fibroblasts show the largest increases in glucose uptake, we suggest that PET imaging of human tumors, with Fluoro-2-deoxy-D-glucose (F-2-DG), may be specifically detecting the tumor stroma, rather than epithelial cancer cells.

CAV1 Inhibits Metastatic Potential in Melanomas through Suppression of the Integrin/Src/FAK Signaling Pathway

Caveolin-1 (CAV1) is the main structural component of caveolae, which are plasma membrane invaginations that participate in vesicular trafficking and signal transduction events. Although evidence describing the function of CAV1 in several cancer types has recently accumulated, its role in melanoma tumor formation and progression remains poorly explored. Here, by using B16F10 melanoma cells as an experimental system, we directly explore the function of CAV1 in melanoma tumor growth and metastasis. We first show that CAV1 expression promotes proliferation, whereas it suppresses migration and invasion of B16F10 cells in vitro. When orthotopically implanted in the skin of mice, B16F10 cells expressing CAV1 form tumors that are similar in size to their control counterparts. An experimental metastasis assay shows that CAV1 expression suppresses the ability of B16F10 cells to form lung metastases in C57Bl/6 syngeneic mice. Additionally, CAV1 protein and mRNA levels are found to be significantly reduced in human metastatic melanoma cell lines and human tissue from metastatic lesions. Finally, we show that following integrin activation, B16F10 cells expressing CAV1 display reduced expression levels and activity of FAK and Src proteins. Furthermore, CAV1 expression markedly reduces the expression of integrin β(3) in B16F10 melanoma cells. In summary, our findings provide experimental evidence that CAV1 may function as an antimetastatic gene in malignant melanoma.

Genetic Ablation of Cav1 Differentially Affects Melanoma Tumor Growth and Metastasis in Mice: Role of Cav1 in Shh Heterotypic Signaling and Transendothelial Migration

Both cell-autonomous and non-cell-autonomous factors contribute to tumor growth and metastasis of melanoma. The function of caveolin-1 (Cav1), a multifunctional scaffold protein known to modulate several biologic processes in both normal tissue and cancer, has been recently investigated in melanoma cancer cells, but its role in the melanoma microenvironment remains largely unexplored. Here, we show that orthotopic implantation of B16F10 melanoma cells in the skin of Cav1KO mice increases tumor growth, and co-injection of Cav1-deficient dermal fibroblasts with melanoma cells is sufficient to recapitulate the tumor phenotype observed in Cav1KO mice. Using indirect coculture experiments with fibroblasts and melanoma cells combined with cytokine analysis, we found that Cav1-deficient fibroblasts promoted the growth of melanoma cells via enhanced paracrine cytokine signaling. Specifically, Cav1-deficient fibroblasts displayed increased ShhN expression, which heterotypically enhanced the Shh signaling pathway in melanoma cells. In contrast to primary tumor growth, the ability of B16F10 melanoma cells to form lung metastases was significantly reduced in Cav1KO mice. This phenotype was associated mechanistically with the inability of melanoma cells to adhere to and to transmigrate through a monolayer of endothelial cells lacking Cav1. Together, our findings show that Cav1 may regulate different mechanisms during primary melanoma tumor growth and metastatic dissemination.

Cav1 Suppresses Tumor Growth and Metastasis in a Murine Model of Cutaneous SCC through Modulation of MAPK/AP-1 Activation.

Caveolin-1 (Cav1) is a scaffolding protein that serves to regulate the activity of several signaling molecules. Its loss has been implicated in the pathogenesis of several types of cancer, but its role in the development and progression of cutaneous squamous cell carcinoma (cSCC) remains largely unexplored. Herein, we use the keratinocyte cell line PAM212, a murine model of cSCC, to determine the function of Cav1 in skin tumor biology. We first show that Cav1 overexpression decreases cell and tumor growth, whereas Cav1 knockdown increases these attributes in PAM212 cells. In addition, Cav1 knockdown increases the invasive ability and incidence of spontaneous lymph node metastasis. Finally, we demonstrate that Cav1 knockdown increases extracellular signaling-related kinase 1/2 mitogen-activated protein kinase/activator protein-1 pathway activation. We attribute the growth and invasive advantage conferred by Cav1 knockdown to increased expression of activator protein-1 transcriptional targets, including cyclin D1 and keratin 18, which show inverse expression in PAM212 based on the expression level of Cav1. In summary, we demonstrate that loss of Cav1 affects several characteristics associated with aggressive human skin tumors and that this protein may be an important modulator of tumor growth and invasion in cSCC.

Abstract 1631: Cav1 is a key mediator of tumor-stromal interactions in melanoma.

Several lines of experimental evidence have demonstrated the importance of the tumor microenvironment in controlling melanoma tumor growth and melanoma metastasis. Caveolin1 (Cav1), the main structural component of the plasma membrane microdomains termed Caveolae, is emerging as an important signaling molecule in the stroma of several tumor types. However, Cav1’s function in the melanoma microenvironment of primary tumors and of metastasis remains largely unexplored. Here, we devise various experimental approaches to elucidate the function of stromal Cav1 in the development of melanoma in mice. We show that loss of Cav1 (but not Cav2) in mice promotes the growth of orthotopically implanted melanoma cells. We use cocultures of fibroblasts and melanoma cells coupled with cytokine analysis to identify various Cav1 regulated factors that function in a paracrine fashion in melanoma. Cav1KO fibroblasts secrete increased amount of ShhN, bFGF, and MMP2/3, cytokines known to promote proliferation of melanoma cells and remodeling of the tumor stroma during melanomagenesis. Intradermal xenografts of fibroblasts and melanoma cells (5:1 ratios) confirmed the tumor promoting role of fibroblasts lacking Cav1. Interestingly, constitutive inhibition of the Shh pathway in melanoma cells reverses the growth and tumor-promoting effects of Cav1KO fibroblasts in co-culture or co-injection experiments suggesting a Cav1 mediated Shh heterotypic interaction between fibroblasts and melanoma cells. In summary, these studies reveal previously undefined functions for Cav1 in the melanoma microenvironment that could potentially be targeted for therapy. Citation Format: Casey Trimmer, Franco Capozza. Cav1 is a key mediator of tumor-stromal interactions in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1631. doi:10.1158/1538-7445.AM2013-1631 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 1083: Caveolin-1 in cutaneous squamous cell carcinoma development

Caveolin-1 (Cav1) is the main protein component of a specialized form of lipid raft termed caveolae and serves to compartmentalize and negatively regulate the activity of signaling molecules involved in cell proliferation and survival. Cav1 has been shown to act as a tumor suppressor in several types of cancer, including breast cancer and melanoma, but its role in the pathogenesis of non-melanoma skin cancer remains largely unexplored. Previous work has indicated that Cav1 is expressed in the basal cell layer of both murine and human epidermis; additionally, loss of Cav1 protein has been shown by our lab to render murine skin more susceptible to papilloma development following treatment with a carcinogenic compound. The purpose of the current study was to further examine the role of Cav1 in non-melanoma skin cancer development using various experimental approaches. Given the previous research, our hypothesis is that Cav1 acts as a suppressor of tumor growth and development in the skin. In order to explore this hypothesis, we have used several experimental systems, including human tissue arrays and loss of function and gain of function studies in animal models of cutaneous squamous cell carcinoma. Manipulation of Cav1 expression in (1) a keratinocyte cell line followed by xenotransplantation and (2) an induced model of skin cancer allowed us to assess the effect of this protein on tumor development, growth, and progression. Using these approaches, we were able to show that Cav1 ablation dramatically increases not only in vitro cell growth and invasion, but also in vivo tumorigenesis, tumor growth, and invasion. In conclusion, these studies provide further evidence in support of the hypothesis that Cav1 acts as a tumor suppressor protein in the skin and serve to identify new potential therapeutic targets in the treatment of cutaneous malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1083. doi:10.1158/1538-7445.AM2011-1083