Calcium influx into cells via plasma membrane protein channels is tightly regulated to maintain cellular homeostasis. Calcium channel proteins in the plasma membrane and endoplasmic reticulum have been linked to cancer, specifically during the epithelial-mesenchymal transition (EMT), a cell state transition process implicated in both cancer cell migration and drug resistance. The transcription factor SNAI1 (SNAIL) is upregulated during EMT and is responsible for gene expression changes associated with EMT, but the calcium channels required for Snai1 expression remain unknown. In this study, we show that blocking store-operated calcium entry (SOCE) with 2-aminoethoxydiphenylborane (2APB) reduces cell migration but, paradoxically, increases the level of TGF-β dependent Snai1 gene activation. We determined that this increased Snai1 transcription involves signaling through the AKT pathway and subsequent binding of NF-κB (p65) at the Snai1 promoter in response to TGF-β. We also demonstrated that the calcium channel protein ORAI3 and the stromal interaction molecule 1 (STIM1) are required for TGF-β dependent Snai1 transcription. These results suggest that calcium channels differentially regulate cell migration and Snai1 transcription, indicating that each of these steps could be targeted to ensure complete blockade of cancer progression.

Poly- adenosine diphosphate (ADP)-ribose (PAR) is a polymer synthesized as a posttranslational modification by some poly (ADP-ribose) polymerases (PARPs), namely PARP-1, PARP-2, tankyrase-1, and tankyrase-2 (TNKS-1/2). PARP-1 is nuclear and has also been detected in extracellular vesicles. PARP-2 and TNKS-1/2 are distributed in nuclei and cytoplasm. PARP or PAR alterations have been described in tumors, and in particular by influencing the Epithelial- Mesenchymal Transition (EMT), which influences cell migration and drug resistance in cancer cells. Pro-EMT and anti-EMT effects of PARP-1 have been reported while whether PAR changes occur specifically during EMT is currently unknown. The PARP-1/2 inhibitor Olaparib (OLA) is approved by FDA to treat certain patients harboring cancers with impaired homologous recombination. Here, we studied PAR changes and OLA effects on EMT. Total and nuclear PAR increased in EMT while PAR belts were disassembled. OLA prevented EMT, according to: (i) molecular markers evaluated by immuno-cytofluorescence/image quantification, Western blots, and RNA quantitation, (ii) morphological changes expressed as anisotropy, and (iii) migration capacity in the scratch assay. OLA also partially reversed EMT. OLA might work through unconventional mechanisms of action (different from synthetic lethality), even in non-BRCA (breast cancer 1 gene) mutated cancers.

Breast cancer is the most common cancer among women worldwide, with many breast cancer‐associated deaths being attributed to metastasis of the primary tumor. In a process known as Epithelial to Mesenchymal transition (EMT), normal epithelial cells lose their cell‐to‐cell adhesions, become elongated and migratory, resulting in the mesenchymal phenotype. The SNAIL protein is known to be a master regulator of EMT, serving to repress expression of genes associated with cellular adhesion and activate genes involved in chemoresistance. In vivo and in vitro studies have both indicated that high levels of SNAIL correlate with poor prognosis, increased chemoresistance, and greater probability of recurrence of the tumor.Calcium signaling in the cell is important for several biological functions, and was implicated in EMT and breast cancer. To determine which calcium channels were involved in SNAIL upregulation during TGF‐β induced EMT, we blocked store‐operated calcium entry (SOCE) with 2‐aminoethoxydiphenylborane (2APB). This reduced cell migration but, paradoxically, increased the level of TGF‐β dependent SNAIL gene activation. We determined that this increased SNAIL transcription involved signaling through the AKT pathway and subsequent binding of NF‐κB (p65) at the SNAIL promoter in response to TGF‐β. We also demonstrated that the calcium channel protein ORAI3 and the stromal interaction molecule 1 (STIM1) are required for TGF‐β dependent SNAIL transcription. These results suggest that calcium channels differentially regulate cell migration and SNAIL transcription, indicating that each of these steps could be targeted to ensure complete blockade of cancer progression. We are currently testing whether this increase in SNAIL contributes to chemoresistance, and if blockade of ORAI3 can inhibit chemoresistance.Support or Funding InformationThis work was funded by grant support from the National Institutes of Health grants P20‐GM104360 to Archana Dhasarathy and UND School of Medicine and Health Sciences.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.