Dileucine-like motifs in the C-terminal tail of connexin32 control its endocytosis and assembly into gap junctions.

Defects in assembly of gap junction-forming proteins, called connexins (Cxs), are observed in a variety of cancers. Connexin32 (Cx32; also known as GJB1) is expressed by the polarized cells in epithelia. We discovered two dileucine-based motifs, which govern the intracellular sorting and endocytosis of transmembrane proteins, in the C-terminal tail of Cx32 and explored their role in regulating its endocytosis and gap junction-forming abilities in pancreatic and prostate cancer cells. One motif, designated as LI, was located near the juxtamembrane domain, whereas the other, designated as LL, was located distally. We also discovered a non-canonical motif, designated as LR, in the C-terminal tail. Our results showed that rendering these motifs non-functional had no effect on the intracellular sorting of Cx32. However, rendering the LL or LR motif nonfunctional enhanced the formation of gap junctions by inhibiting Cx32 endocytosis by the clathrin-mediated pathway. Rendering the LI motif nonfunctional inhibited gap junction formation by augmenting the endocytosis of Cx32 via the LL and LR motifs. Our studies have defined distinct roles of these motifs in regulating the endocytosis of Cx32 and its gap junction-forming ability.This article has an associated First Person interview with the first author of the paper.

An update on minding the gap in cancer.

This article is a report of the "International Colloquium on Gap junctions: 50Years of Impact on Cancer" that was held 8-9 September 2016, at the Amphitheater "Pôle Biologie Santé" of the University of Poitiers (Poitiers, France). The colloquium was organized by M Mesnil (Université de Poitiers, Poitiers, France) and C Naus (University of British Columbia, Vancouver, Canada) to celebrate the 50th anniversary of the seminal work published in 1966 by Loewenstein and Kanno [Intercellular communication and the control of tissue growth: lack of communication between cancer cells, Nature, 116 (1966) 1248-1249] which initiated studies on the involvement of gap junctions in carcinogenesis. During the colloquium, 15 participants presented reviews or research updates in the field which are summarized below.

The carboxyl tail of connexin32 regulates gap junction assembly in human prostate and pancreatic cancer cells.

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size.

Functions of normal and malignant prostatic stem/progenitor cells in tissue regeneration and cancer progression and novel targeting therapies.

This review summarizes the recent advancements that have improved our understanding of the functions of prostatic stem/progenitor cells in maintaining homeostasis of the prostate gland. We also describe the oncogenic events that may contribute to their malignant transformation into prostatic cancer stem/progenitor cells during cancer initiation and progression to metastatic disease stages. The molecular mechanisms that may contribute to the intrinsic or the acquisition of a resistant phenotype by the prostatic cancer stem/progenitor cells and their differentiated progenies with a luminal phenotype to the current therapies and disease relapse are also reviewed. The emphasis is on the critical functions of distinct tumorigenic signaling cascades induced through the epidermal growth factor system, hedgehog, Wnt/beta-catenin, and/or stromal cell-derived factor-1/CXC chemokine receptor-4 pathways as well as the deregulated apoptotic signaling elements and ATP-binding cassette multidrug transporter. Of particular therapeutic interest, we also discuss the potential beneficial effects associated with the targeting of these signaling elements to overcome the resistance to current treatments and prostate cancer recurrence. The combined targeted strategies toward distinct oncogenic signaling cascades in prostatic cancer stem/progenitor cells and their progenies as well as their local microenvironment, which could improve the efficacy of current clinical chemotherapeutic treatments against incurable, androgen-independent, and metastatic prostate cancers, are also described.

E-cadherin differentially regulates the assembly of Connexin43 and Connexin32 into gap junctions in human squamous carcinoma cells.

It is as yet unknown how the assembly of connexins (Cx) into gap junctions (GJ) is initiated upon cell-cell contact. We investigated whether the trafficking and assembly of Cx43 and Cx32 into GJs were contingent upon cell-cell adhesion mediated by E-cadherin. We also examined the role of the carboxyl termini of these Cxs in initiating the formation of GJs. Using cadherin and Cx-null cells, and by introducing Cx43 and Cx32, either alone or in combination with E-cadherin, our studies demonstrated that E-cadherin-mediated cell-cell adhesion was neither essential nor sufficient to initiate GJ assembly de novo in A431D human squamous carcinoma cells. However, E-cadherin facilitated the growth and assembly of preformed GJs composed of Cx43, although the growth of cells on Transwell filters was required to initiate the assembly of Cx32. Our results also documented that the carboxyl termini of both Cxs were required in this cell type to initiate the formation of GJs de novo. Our findings also showed that GJ puncta composed of Cx43 co-localized extensively with ZO-1 and actin fibers at cell peripheries and that ZO-1 knockdown attenuated Cx43 assembly. These findings suggest that the assembly of Cx43 and Cx32 into GJs is differentially modulated by E-cadherin-mediated cell-cell adhesion and that direct or indirect cross-talk between carboxyl tails of Cxs and actin cytoskeleton via ZO-1 may regulate GJ assembly and growth.

Cysteine residues in the C-terminal tail of connexin32 regulate its trafficking.

Gap junctions (GJs) are formed by the assembly of constituent transmembrane proteins called connexins (Cxs). Aberrations in this assembly of Cxs are observed in several genetic diseases as well as in cancers. Hence it becomes imperative to understand the molecular mechanisms underlying such assembly defect. The polarized cells in the epithelia express Connexin32 (Cx32). The C-terminal tail (CT) of Cx32 orchestrates several aspects of GJ dynamics, function and growth. The study here was aimed at determining if post-translational modifications, specifically, palmitoylation of cysteine residues, present in the CT of Cx32, has any effect on GJ assembly. The CT of Cx32 was found to harbor three cysteine residues, which are likely to be modified by palmitoylation. The study here has revealed for the first time that Cx32 is palmitoylated at cysteine 217 (C217) in cell line derived from prostate tumors. However, it was found that mutating C217 to alanine affected neither the trafficking nor the ability of Cx32 to assemble into GJs. Intriguingly, it was discovered that mutating cysteine 280 and 283, only in combination, blocked the trafficking of Cx32 from the trans-Golgi network to the cell surface. The mutants showed reduced stability due to enhanced lysosomal degradation. Overall, the findings reveal the importance of the two C-terminal cysteine residues of Cx32 in regulating its trafficking and stability and hence its ability to assemble into GJs.

Androgen-regulated formation and degradation of gap junctions in androgen-responsive human prostate cancer cells.

The constituent proteins of gap junctions, called connexins (Cxs), have a short half-life. Despite this, the physiological stimuli that control the assembly of Cxs into gap junctions and their degradation have remained poorly understood. We show here that in androgen-responsive human prostate cancer cells, androgens control the expression level of Cx32-and hence the extent of gap junction formation-post-translationally. In the absence of androgens, a major fraction of Cx32 is degraded presumably by endoplasmic reticulum-associated degradation, whereas in their presence, this fraction is rescued from degradation. We also show that Cx32 and Cx43 degrade by a similar mechanism. Thus, androgens regulate the formation and degradation of gap junctions by rerouting the pool of Cxs, which normally would have been degraded from the early secretory compartment, to the cell surface, and enhancing assembly into gap junctions. Androgens had no significant effect on the formation and degradation of adherens and tight junction-associated proteins. The findings that in a cell culture model that mimics the progression of human prostate cancer, degradation of Cxs, as well as formation of gap junctions, are androgen-dependent strongly implicate an important role of junctional communication in the prostate morphogenesis and oncogenesis.

ErbB-2 signaling plays a critical role in regulating androgen-sensitive and castration-resistant androgen receptor-positive prostate cancer cells.

While androgen deprivation therapy (ADT) reduces tumor burden, autocrine growth factor loops such as human epidermal growth factor receptor 2 (HER2/ErbB-2/neu) have been proposed to contribute to prostate cancer (PCa) survival and relapse. However, the role of ErbB-2 in regulating androgen-sensitive (AS) and castration-resistant (CR) cell proliferation remains unclear. Here, we determined the role of ErbB-2 in PCa progression and survival under steroid-reduced conditions using two independent PCa cell progression models. In AR-positive androgen-independent (AI) PCa cells that exhibit the CR phenotype, ErbB-2 was constitutively activated, compared to corresponding AS PCa cells. In AS LNCaP C-33 cells, androgen-induced ErbB-2 activation through ERK1/2 mediates PCa cell proliferation. Further, the ErbB-2-specific but not EGFR-specific inhibitor suppresses basal and androgen-stimulated cell proliferation and also blocks ERK1/2 activation. ErbB-2 ectopic expression and cPAcP siRNA transfection of LNCaP C-33 cells each increases ErbB-2 tyrosine phosphorylation, correlating with increased AI PSA secretion and cell proliferation. Conversely, trapping ErbB-2 by transfected endoplasmic reticulum-targeting ScFv5R expression vector abolished DHT-induced LNCaP C-33 cell growth. Moreover, inhibition of ErbB-2 but not EGFR in AI LNCaP C-81 and MDA PCa2b-AI PCa cells significantly abolished AI cell growth. In contrast to androgens via ErbB-2/ERK1/2 signaling in AS PCa cells, the inhibition of ErbB-2 abrogated AI cell proliferation by inhibiting the cell survival protein Akt in those AI cells. These results suggest that ErbB-2 is a prominent player in mediating the ligand-dependent and -independent activation of AR in AS and AI/CR PCa cells respectively for PCa progression and survival.

Vitamin D3 regulates the formation and degradation of gap junctions in androgen-responsive human prostate cancer cells.

1α-25(OH)2 vitamin D3 (1-25D), an active hormonal form of Vitamin D3, is a well-known chemopreventive and pro-differentiating agent. It has been shown to inhibit the growth of several prostate cancer cell lines. Gap junctions, formed of proteins called connexins (Cx), are ensembles of cell-cell channels, which permit the exchange of small growth regulatory molecules between adjoining cells. Cell-cell communication mediated by gap junctional channels is an important homeostatic control mechanism for regulating cell growth and differentiation. We have investigated the effect of 1-25D on the formation and degradation of gap junctions in an androgen-responsive prostate cancer cell line, LNCaP, which expresses retrovirally-introduced Cx32. Connexin32 is expressed by the luminal and well-differentiated cells of normal prostate and prostate tumors. Our results document that 1-25D enhances the expression of Cx32 and its subsequent assembly into gap junctions. Our results further show that 1-25D prevents androgen-regulated degradation of Cx32, post-translationally, independent of androgen receptor (AR)-mediated signaling. Finally, our findings document that formation of gap junctions sensitizes Cx32-expressing LNCaP cells to the growth inhibitory effects of 1-25D and alters their morphology. These findings suggest that the growth-inhibitory effects of 1-25D in LNCaP cells may be related to its ability to modulate the assembly of Cx32 into gap junctions.

Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells.

The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell-cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif-deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43's endocytosis and assembly into gap junctions.

Retinoids regulate the formation and degradation of gap junctions in androgen-responsive human prostate cancer cells.

The retinoids, the natural or synthetic derivatives of Vitamin A (retinol), are essential for the normal development of prostate and have been shown to modulate prostate cancer progression in vivo as well as to modulate growth of several prostate cancer cell lines. 9-cis-retinoic acid and all-trans-retinoic acid are the two most important metabolites of retinol. Gap junctions, formed of proteins called connexins, are ensembles of intercellular channels that permit the exchange of small growth regulatory molecules between adjoining cells. Gap junctional communication is instrumental in the control of cell growth. We examined the effect of 9-cis-retinoic acid and all-trans retinoic acid on the formation and degradation of gap junctions as well as on junctional communication in an androgen-responsive prostate cancer cell line, LNCaP, which expressed retrovirally introduced connexin32, a connexin expressed by the luminal cells and well-differentiated cells of prostate tumors. Our results showed that 9-cis-retinoic acid and all-trans retinoic acid enhanced the assembly of connexin32 into gap junctions. Our results further showed that 9-cis-retinoic acid and all-trans-retinoic acid prevented androgen-regulated degradation of gap junctions, post-translationally, independent of androgen receptor mediated signaling. Finally, our findings showed that formation of gap junctions sensitized connexin32-expressing LNCaP cells to the growth modifying effects of 9-cis-retinoic acid, all-trans-retinoic acid and androgens. Thus, the effects of retinoids and androgens on growth and the formation and degradation of gap junctions and their function might be related to their ability to modulate prostate growth and cancer.

Decreased nNOS in the PVN leads to increased sympathoexcitation in chronic heart failure: role for CAPON and Ang II.

AIMS: Previously, we showed an enhanced excitatory (N-methyl d-aspartate receptor-NR(1)) and decreased inhibitory neuronal nitric oxide (NO) synthase (nNOS) influence within the paraventricular nucleus (PVN) of rats with chronic heart failure (CHF). Although NR(1) and nNOS are normally linked, they can be disconnected by nNOS sequestering with nNOS-associated protein (CAPON). The aim of this study was to elucidate the underlying mechanism for the disconnection between increased expression of NR(1) and decreased nNOS in the PVN of rats with CHF which leads to enhanced sympathoexcitation. METHODS AND RESULTS: CAPON expression was augmented while nNOS expression was decreased in the PVN of rats with CHF (6-8 weeks after left coronary artery ligation). Angiotensin II (Ang II) type I receptor (AT(1)) antagonist losartan (Los) treatment in rats with CHF reduced renal sympathetic nerve activity with concomitant normalization of protein expression of CAPON and nNOS in the PVN. Los treatment also reversed the blunting of endogenous NO-mediated sympatho-inhibition in rats with CHF. Moreover, Ang II-induced increase in CAPON expression in NG108 neuronal cells was also ameliorated by Los. CONCLUSION: Blocking AT(1) receptors prevents the overexpression of CAPON and concomitant decrease in nNOS in the PVN, resulting in attenuation of sympathoexcitation commonly observed in CHF. Taken together, our data highlight the importance of altered expression and subsequent interaction of nNOS and CAPON within the PVN, leading to increased sympathoexcitation in CHF. Identifying this crucial nNOS/CAPON interaction regulated by AT(1) receptors may provide an important potential therapeutic target in CHF.

Assembly of connexin43 into gap junctions is regulated differentially by E-cadherin and N-cadherin in rat liver epithelial cells.

Cadherins have been thought to facilitate the assembly of connexins (Cxs) into gap junctions (GJs) by enhancing cell-cell contact, however the molecular mechanisms involved in this process have remained unexplored. We examined the assembly of GJs composed of Cx43 in isogenic clones derived from immortalized and nontransformed rat liver epithelial cells that expressed either epithelial cadherin (E-Cad), which curbs the malignant behavior of tumor cells, or neuronal cadherin (N-Cad), which augments the invasive and motile behavior of tumor cells. We found that N-cad expression attenuated the assembly of Cx43 into GJs, whereas E-Cad expression facilitated the assembly. The expression of N-Cad inhibited GJ assembly by causing endocytosis of Cx43 via a nonclathrin-dependent pathway. Knock down of N-Cad by ShRNA restored GJ assembly. When both cadherins were simultaneously expressed in the same cell type, GJ assembly and disassembly occurred concurrently. Our findings demonstrate that E-Cad and N-Cad have opposite effects on the assembly of Cx43 into GJs in rat liver epithelial cells. These findings imply that GJ assembly and disassembly are the down-stream targets of the signaling initiated by E-Cad and N-Cad, respectively, and may provide one possible explanation for the disparate role played by these cadherins in regulating cell motility and invasion during tumor progression and invasion.

MUC4 mucin interacts with and stabilizes the HER2 oncoprotein in human pancreatic cancer cells.

MUC4, a high-molecular weight transmembrane glycoprotein, is overexpressed in pancreatic cancer and is implicated in its pathogenesis. It is a heterodimeric protein containing a large extracellular, heavily glycosylated subunit, MUC4alpha, and a transmembrane growth factor-like subunit, MUC4beta. In the present study, we have shown the interaction of human MUC4 with the receptor tyrosine kinase HER2 in pancreatic adenocarcinoma cells by reciprocal coimmunoprecipitation and cocapping studies. MUC4 colocalized with HER2 at the cell surface and in the cytoplasm. Silencing of MUC4 by transient or stable expression of MUC4-targeted short-interfering RNA led to the down-regulation of HER2 with a concomitant decrease in its phosphorylated form (pY(1248)-HER2). Further analyses revealed that the MUC4-knockdown-mediated decrease in HER2 expression occurred due to the drop in the stability of the receptor. In MUC4-knockdown pancreatic cancer cells, we also observed a reduced phosphorylation of the focal adhesion kinase and p42/44 mitogen-activated protein kinase, which are downstream effector proteins in HER2 signaling. Our findings add a new dimension to MUC4 function as a modulator of cell signaling and provide mechanistic evidence for its role in pancreatic cancer progression.

Improvement of cytotoxic effects induced by mitoxantrone on hormone-refractory metastatic prostate cancer cells by co-targeting epidermal growth factor receptor and hedgehog signaling cascades.

The results of the present study revealed for the first time the possibility to use a combination of mitoxantrone with gefitinib and cyclopamine for inhibiting the growth of epidermal growth factor (EGF), sonic hedgehog- (SHHNp), and serum-stimulated androgen-sensitive LNCaP-C33 and androgen-independent (AI) LNCaP-C81, DU145 and PC3 prostate cancer (PC) cells. The supra-additive anti-proliferative effects of drugs were mediated via a blockade of the PC3 cells in the G(1) and G(2)M phases of the cell cycle. Importantly, the combination of mitoxantrone plus gefitinib and/or cyclopamine also caused a higher rate of apoptotic death of PC cells including enriched fraction of CD44(high) PC3 cell subpopulation as compared to the individual agents or bi-combination of drugs. The cytotoxic effects induced by mitoxantrone, gefitinib and cyclopamine on PC3 cells appear to be at least partly mediated through the depolarization of the mitochondrial membrane, release of cytochrome c into the cytosol, hydrogen peroxide production and activation of caspase cascades. These findings indicate that the simultaneous blockade of EGF-EGFR and sonic hedgehog tumorigenic signaling cascades may represent a promising strategy for improving the efficacy of current mitoxantrone-based therapies against incurable AI and metastatic PCs in the clinics.

Impaired trafficking of connexins in androgen-independent human prostate cancer cell lines and its mitigation by alpha-catenin.

Gap junctions, composed of connexins, provide a pathway of direct intercellular communication for the diffusion of small molecules between cells. Evidence suggests that connexins act as tumor suppressors. We showed previously that expression of connexin-43 and connexin-32 in an indolent prostate cancer cell line, LNCaP, resulted in gap junction formation and growth inhibition. To elucidate the role of connexins in the progression of prostate cancer from a hormone-dependent to -independent state, we introduced connexin-43 and connexin-32 into an invasive, androgen-independent cell line, PC-3. Expression of these proteins in PC-3 cells resulted in intracellular accumulation. Western blot analysis revealed a lack of Triton-insoluble, plaque-assembled connexins. In contrast to LNCaP cells, connexins could not be cell surface-biotinylated and did not reside in the cell surface derived endocytic vesicles, in PC-3 cells, suggesting impaired trafficking to the cell surface. Intracellular accumulation of connexins was observed in several androgen-independent prostate cancer cell lines. Transient expression of alpha-catenin facilitated the trafficking of both connexins to the cell surface and induced gap junction assembly. Our results suggest that impaired trafficking, and not the inability to form gap junctions, is the major cause of communication deficiency in human prostate cancer cell lines.