Recurrence at secondary locations, often years after removal of the primary tumor, accounts for most of the mortality associated with solid tumors. Metastasis, resistance to chemo- and radiotherapy, and eventual relapse have been attributed to a distinct tumor subpopulation known as cancer stem cells (CSCs). In this review, we consider the properties of CSCs that lead to these outcomes, in particular the relation between epithelial-to-mesenchymal transition, stemness, and tumor initiation. We compare recent clinical and laboratory studies of breast cancer, glioblastoma, and melanoma that illustrate how most current anticancer regimens select for cells with mesenchymal and CSC properties and therefore sow the seeds of relapse. Finally, we discuss the emerging paradigm of combined therapy that targets both CSC and non-CSC tumor components.

CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized human mammary epithelial cells (HMEC) caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The primary sequence of the CLCA2 TMS was found to be conserved in CLCA2 orthologs throughout mammals, suggesting that its interaction with EVA1 co-evolved with the mammary gland. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines.

The Chloride Channel Accessory (CLCA) protein family was first characterized as regulators of calcium-activated chloride channel (CaCC) currents (ICaCC), but the mechanism has not been fully established. We hypothesized that CLCAs might regulate ICaCC by modulating intracellular calcium levels. In cells stably expressing human CLCA2 or vector, we found by calcium imaging that CLCA2 moderately enhanced intracellular-store release but dramatically increased store-operated entry of calcium upon cytosolic depletion. Moreover, another family member, CLCA1, produced similar effects on intracellular calcium mobilization. Co-immunoprecipitation revealed that CLCA2 interacted with the plasma membrane store-operated calcium channel ORAI-1 and the ER calcium sensor STIM-1. The effect of CLCA2 on ICaCC was tested in HEK293 stably expressing calcium-activated chloride channel TMEM16A. Co-expression of CLCA2 nearly doubled ICaCC in response to a calcium ionophore. These results unveil a new mechanism by which CLCA family members activate ICaCC and suggest a broader role in calcium-dependent processes.

CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized HMEC caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The TMS primary sequence of CLCA2 was found to be conserved throughout mammals. Moreover, competitive inhibition of the interaction by ectopic expression of the TMS caused partial EMT, suggesting that the interaction is important for differentiation. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines. Citation Format: Grace N.T. Ramena, Yufang Yin, Yang Yu, Vijay Walia, Randolph C. Elble. CLCA2 interaction with EVA1 promotes mammary epithelial differentiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1677.

CLCA2 is a p53-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during MET in immortalized HMEC, and its knockdown causes EMT. To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1), confirmed by co-immunoprecipitation and immunolocalization experiments. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53. It mediates homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Like CLCA2, knockdown of EVA1 resulted in rapid EMT in immortalized HMEC and increased invasiveness. The interacting domains were delimited by deletion analysis, revealing that the CLCA2 transmembrane segment (TMS) alone was sufficient for the interaction with EVA1. Surprisingly, EVA1 bound to the TMS only in the context of full length CLCA2 precursor, but not in the context of the C-terminal cleavage product, suggesting that cleavage of CLCA2 causes a conformational shift and release of EVA1. The interaction was specific, as other transmembrane proteins did not interact with CLCA2 or EVA1. To determine whether CLCA2 regulates EVA1 localization to tight junctions or hemophilic ligation, we fused the CLCA2 or EVA1 TMS to a secreted heterologous protein to create competitive inhibitors. These results identify the CLCA2-EVA1 complex as pivotal in maintaining epithelial differentiation and explain the downregulation of both genes during tumor progression. Citation Format: Randolph C. Elble, Grace Ramena, Vijay Walia. CLCA2 interactor EVA1 maintains epithelial differentiation of HMEC. [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 LB-276. doi:10.1158/1538-7445.AM2013-LB-276