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

Context dependent role of the CD36--thrombospondin--histidine-rich glycoprotein axis in tumor angiogenesis and growth

<div><p>The angiogenic switch is a promising therapeutic target in cancer. Work by our laboratory and others has described an important endogenous anti-angiogenic pathway mediated by interactions of CD36, a receptor on microvascular endothelial cells, with proteins containing thrombospondin (TSP) type I repeat domains (TSR). Recent studies revealed that circulating Histidine Rich Glycoprotein (HRG) inhibits the anti-angiogenic potential of the CD36-TSR pathway by functioning as a decoy receptor that binds and sequesters TSR proteins. As tumors of different origin display variable expression profiles of numerous targets, we hypothesized that the TSP-CD36-HRG axis regulates vascularization and growth in the tumor microenvironment in a context, or tumor type, dependent manner. Growth of Lewis Lung Carcinoma (LL2) and B16F1 Melanoma tumor cell implants in syngeneic wild type (WT), <em>hrg</em>, or <em>cd36</em> null mice were used as a model to interrogate this signaling axis. LL2 tumor volumes were greater in <em>cd36</em> null mice and smaller in <em>hrg</em> null mice compared to WT. Immunofluorescent staining showed increased vascularity in <em>cd36</em> null vs. WT and WT vs. <em>hrg</em> null mice. No differences in tumor growth or vascularity were observed with B16F1 implants, consistent with lack of expression of TSP-1 in B16F1 cells. When TSR expression was induced in B16F1 cells by cDNA transfection, tumor growth and vascularity were similar to that seen with LL2 cells. These data show a role for CD36-mediated anti-angiogenic activity in the tumor microenvironment when TSR proteins are available and demonstrate that HRG modulates this activity. Further, they suggest a mechanism by which tumor microenvironments may regulate sensitivity to TSR containing proteins.</p> </div

Thrombospondin-1 expression in LL2 and B16F1 melanoma cells and tumors.

<p>(<b>A</b>) Lewis Lung (LL2) or B16F melanoma cells were cultured in serum free media for 24 hours (1d) at which point proteins in post culture media (CM) were precipitated by TCA, separated under reducing conditions by SDS/PAGE and analyzed by immunoblot using anti-TSP-1 antibody. TSP-1 monomers were detected at 170 kDa in the media conditioned by LL2 cells, but not B16F1 cells. Purified human HRG and TSP were used as controls. (<b>B</b>) B16F1 melanoma tumor tissue was analyzed by western blot analysis for TSP expression. Intact TSP was not observed at 150 kDa, however possible degredation products were observed around 55 kDa. (<b>C</b>) B16F1 and LL2 tumor tissue was analyzed by RT-PCR for expression of TSP. TSP was detected in both tumor types, approximately 7 fold higher in LL2. (<b>D</b>) Conditioned media was collected from 4 different antibiotic resistant clones of TSR transfected B16F melanoma cells and analyzed by immunoblot as in panel A. Clone 11 expressed abundant anti-TSP reactive material at the appropriate molecular weight of recombinant TSR and was utilized for subsequent tumor studies. (<b>E</b>) TSP knockdown efficiency was analyzed by RT-PCR with statistical significance as indicated; **P<0.05; *P = 0.06. In both instances of TSP knockdown 1 (K1 and K2), reductions in TSP message levels were detected as compared with nontargeted control (NT) cells.</p

TSR transfected B16F1 melanoma cells show enhanced tumor growth in <i>cd36</i> null mice and suppressed tumor growth in <i>hrgp</i> null mice.

<p>50,000 cells from a stably transfected B16f1 melanoma cell line (Clone 11) were injected in the backs of <i>cd36</i> null (A) or <i>hrgp</i> null (B) mice. C57BL/6 mice were used as controls. Tumor volumes were assessed at timed points as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040033#pone-0040033-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040033#pone-0040033-g003" target="_blank">3</a>. *P<0.05; **P = 0.08; ***P = 0.06. LL2 cells stabily transfected with nontargeted (NT) or TSP targeted constructs, K1 and K2, constructs were similarly injected subcutaneously onto the backs of wildtype (WT) and cd36 null mice (Null). (<b>C</b>) WT-NT tumors grew smaller than WT-K1 and WT-K2 tumors, with statistically significant differences seen at days 7 and 15 for both NT vs K1 and NT vs K2. (<b>D</b>) No differences were observed between NT, K1 and K1 in cd36 null mice.</p

<i>Hrg</i> deletion in mice suppresses syngeneic tumor growth.

<p>Lewis Lung carcinoma cells (<b>A</b>) or B16F1 melanoma cells (<b>B</b>) were injected in the backs of <i>hrg</i> null or wild type C57BL/6 mice (50,000 cells/mouse). Tumor volumes were assessed over 16 days following implantation. *P<0.05.</p

<i>Hrg</i> deletion in mice suppresses Lewis Lung tumor vascularity.

<p>(<b>A</b>) Lewis Lung tumors as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040033#pone-0040033-g003" target="_blank">Figure 3</a> were dissected, sectioned and examined by immunofluorescence microscopy using anti-VEGF receptor antibody (green) to detect blood vessels. DAPI stained nuclei are blue. Magnification bars represent 100 µm. IgG control is shown in bottom panel as negative control. (<b>B</b>) Vessel densities measured as vessels per mm2. Median vessel density: wt 12.33, cd36 null 6.99.</p

<i>Cd36</i> deletion in mice enhances syngeneic tumor growth.

<p>Lewis Lung carcinoma cells (<b>A</b>) or B16F1 melanoma cells (<b>B</b>) were injected in the backs of <i>cd36</i> null or wild type C57BL/6 mice (50,000 cells/mouse). Tumor volumes were assessed over 17 days following implantation. *P<0.05.</p