Cancer Connectors: Connexins, Gap Junctions, and Communication

Despite concerted clinical and research efforts, cancer is a leading cause of death worldwide. Surgery, radiation, and chemotherapy have remained the most common standard-of-care strategies against cancer for decades. However, the side effects of these therapies demonstrate the need to investigate adjuvant novel treatment modalities that minimize the harm caused to healthy cells and tissues. Normal and cancerous cells require communication amongst themselves and with their surroundings to proliferate and drive tumor growth. It is vital to understand how intercellular and external communication impacts tumor cell malignancy. To survive and grow, tumor cells, and their normal counterparts utilize cell junction molecules including gap junctions (GJs), tight junctions, and adherens junctions to provide contact points between neighboring cells and the extracellular matrix. GJs are specialized structures composed of a family of connexin proteins that allow the free diffusion of small molecules and ions directly from the cytoplasm of adjacent cells, without encountering the extracellular milieu, which enables rapid, and coordinated cellular responses to internal and external stimuli. Importantly, connexins perform three main cellular functions. They enable direct gap junction intercellular communication (GJIC) between cells, form hemichannels to allow cell communication with the extracellular environment, and serve as a site for protein-protein interactions to regulate signaling pathways. Connexins themselves have been found to promote tumor cell growth and invasiveness, contributing to the overall tumorigenicity and have emerged as attractive anti-tumor targets due to their functional diversity. However, connexins can also serve as tumor suppressors, and therefore, a complete understanding of the roles of the connexins and GJs in physiological and pathophysiological conditions is needed before connexin targeting strategies are applied. Here, we discuss how the three aspects of connexin function, namely GJIC, hemichannel formation, and connexin-protein interactions, function in normal cells, and contribute to tumor cell growth, proliferation, and death. Finally, we discuss the current state of anti-connexin therapies and speculate which role may be most amenable for the development of targeting strategies

SerpinB3 Drives Cancer Stem Cell Survival in Glioblastoma

Despite therapeutic interventions for glioblastoma (GBM), cancer stem cells (CSCs) drive recurrence. The precise mechanisms underlying CSC resistance, namely inhibition of cell death, are unclear. We built on previous observations that the high cell surface expression of junctional adhesion molecule-A drives CSC maintenance and identified downstream signaling networks, including the cysteine protease inhibitor SerpinB3. Using genetic depletion approaches, we found that SerpinB3 is necessary for CSC maintenance, survival, and tumor growth, as well as CSC pathway activation. Knockdown of SerpinB3 also increased apoptosis and susceptibility to radiation therapy. SerpinB3 was essential to buffer cathepsin L-mediated cell death, which was enhanced with radiation. Finally, we found that SerpinB3 knockdown increased the efficacy of radiation in pre-clinical models. Taken together, our findings identify a GBM CSC-specific survival mechanism involving a cysteine protease inhibitor, SerpinB3, and provide a potential target to improve the efficacy of GBM therapies against therapeutically resistant CSCs

The "intelligent adaptor" Dab2 regulates clathrin-mediated endocytosis

Thesis (Ph.D.)--University of Washington, 2012Clathrin-mediated endocytosis (CME) is an essential process by which cells internalize plasma membrane proteins. The master regulator of the process is thought to be the adaptor protein AP2, which organizes receptors, clathrin, and other necessary factors at the plasma membrane. Disabled-2 (Dab2) is an adaptor protein that allows AP2 to internalize receptors to which it cannot directly bind, such as the low-density lipoprotein receptor (LDLR) and integrin β1. Dab2 has been shown to function independently of AP2 under some conditions. I hypothesized that this AP2-independent function of Dab2 might require Dab2 to interact directly with various endocytic accessory proteins normally recruited by AP2. Here I report the direct interaction of Dab2 with three endocytic accessory proteins, the F-BAR protein FCHO2 and the EH domain proteins Eps15 and Intersectin (ITSN). Interestingly, FCHO2 and the EH domain proteins are both important for the organization and structure of clathrin-coated pits (CCPs). FCHO2 and EH domain proteins are required for CME and their interaction with Dab2 is required for Dab2-mediated endocytosis. The mere presence of EH domain proteins in CCPs is not sufficient for internalization of Dab2 cargoes; Eps15 and ITSN must directly bind to Dab2 to permit receptor endocytosis. This suggests that a cargo-adaptor-EH domain protein complex is required for CME. I also give evidence that Dab2 exists in both "open" and "closed" states and that the change between the two may be regulated by cargo binding. While common perception holds that adaptor proteins simply link two incompatible proteins together, I propose that Dab2 is an "intelligent adaptor" which senses the presence of one molecule and undergoes a conformational change to permit the binding of others

Connexins in Cancer: Jekyll or Hyde?

The expression, localization, and function of connexins, the protein subunits that comprise gap junctions, are often altered in cancer. In addition to cell–cell coupling through gap junction channels, connexins also form hemichannels that allow communication between the cell and the extracellular space and perform non-junctional intracellular activities. Historically, connexins have been considered tumor suppressors; however, they can also serve tumor-promoting functions in some contexts. Here, we review the literature surrounding connexins in cancer cells in terms of specific connexin functions and propose that connexins function upstream of most, if not all, of the hallmarks of cancer. The development of advanced connexin targeting approaches remains an opportunity for the field to further interrogate the role of connexins in cancer phenotypes, particularly through the use of in vivo models. More specific modulators of connexin function will both help elucidate the functions of connexins in cancer and advance connexin-specific therapies in the clinic

Global immune fingerprinting in glioblastoma patient peripheral blood reveals immune-suppression signatures associated with prognosis.

Glioblastoma (GBM) remains uniformly lethal, and despite a large accumulation of immune cells in the microenvironment, there is limited antitumor immune response. To overcome these challenges, a comprehensive understanding of GBM systemic immune response during disease progression is required. Here, we integrated multiparameter flow cytometry and mass cytometry TOF (CyTOF) analysis of patient blood to determine changes in the immune system among tumor types and over disease progression. Utilizing flow cytometry analysis in a cohort of 259 patients ranging from benign to malignant primary and metastatic brain tumors, we found that GBM patients had a significant elevation in myeloid-derived suppressor cells (MDSCs) in peripheral blood but not immunosuppressive Tregs. In GBM patient tissue, we found that increased MDSC levels in recurrent GBM portended poor prognosis. CyTOF analysis of peripheral blood from newly diagnosed GBM patients revealed that reduced MDSCs over time were accompanied by a concomitant increase in DCs. GBM patients with extended survival also had reduced MDSCs, similar to the levels of low-grade glioma (LGG) patients. Our findings provide a rationale for developing strategies to target MDSCs, which are elevated in GBM patients and predict poor prognosis

Global immune fingerprinting in glioblastoma patient peripheral blood reveals immune-suppression signatures associated with prognosis

Glioblastoma (GBM) remains uniformly lethal, and despite a large accumulation of immune cells in the microenvironment, there is limited antitumor immune response. To overcome these challenges, a comprehensive understanding of GBM systemic immune response during disease progression is required. Here, we integrated multiparameter flow cytometry and mass cytometry TOF (CyTOF) analysis of patient blood to determine changes in the immune system among tumor types and over disease progression. Utilizing flow cytometry analysis in a cohort of 259 patients ranging from benign to malignant primary and metastatic brain tumors, we found that GBM patients had a significant elevation in myeloid-derived suppressor cells (MDSCs) in peripheral blood but not immunosuppressive Tregs. In GBM patient tissue, we found that increased MDSC levels in recurrent GBM portended poor prognosis. CyTOF analysis of peripheral blood from newly diagnosed GBM patients revealed that reduced MDSCs over time were accompanied by a concomitant increase in DCs. GBM patients with extended survival also had reduced MDSCs, similar to the levels of low-grade glioma (LGG) patients. Our findings provide a rationale for developing strategies to target MDSCs, which are elevated in GBM patients and predict poor prognosis