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Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of [C1.sup.-1] ion channels that are activated by intracellular [Ca.sup.2+]. At present, the structures and mechanisms responsible for [Ca.sup.2+] sensing remain unknown. Here, we have used a combination of molecular modeling, density functional--binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in [Ca.sup.2+] sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential tot [Ca.sup.2+] sensing by hBest1. The cluster of five amino acids (293-308) is crucial for normal channel gating by [Ca.sup.2+] because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by [Ca.sup.2+]. Using homology, models built on the crystal structure of calmodulin (CAM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind [Ca.sup.2+] with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative [Ca.sup.2+]-binding loop (312-323) reduced the apparent [Ca.sup.2+] affinity by 20-fold. In addition, the D312G and D323N mutations abolished [Ca.sup.2+]-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350-390) that is required for [Ca.sup.2+] activation and plays a role in current rundown. These experiments identity a region of hBest1 (312-323) that is involved in the gating of hBest1 by [Ca.sup.2+] and suggest a model in which [Ca.sup.2+] binding to EF1 activates the channel in a process that requires the acidic domain (293-308) and another regulatory domain (350-390). Many of the ~100 disease-causing mutations in hBest1 are located in this region that we have implicated in [Ca.sup.2+] sensing, suggesting that these mutations disrupt hBestl channel gating by [Ca.sup.2+].

Background and Purpose: Ca2+‐activated Cl− channels (Ano1 channels) contribute to the pathogenesis of colorectal cancer. Honokiol is known to inhibit cell proliferation and tumour growth in colorectal cancer. However, the molecular target of honokiol remains unclear. This study aimed to investigate whether honokiol inhibited cell proliferation of colorectal cancer by targeting Ano1 channels. Experimental Approach Patch‐clamp techniques were performed to study the effect of honokiol on Ca2+‐activated Cl− currents in HEK293 cells overexpressing Ano1‐ or Ano2‐containing plasmids or in human colorectal carcinoma SW620 cells. Site‐directed mutagenesis was used to identify the critical residues for honokiol‐induced Ano1 inhibition. Proliferation of SW620 cells or human intestinal epithelial NCM460 cells by CCK‐8 assays. Key Results: Honokiol blocked Ano1 currents in Ano1‐overexpressing HEK293 cells and SW620 cells. Honokiol more potently inhibited Ano1 currents than Ano2 currents. Three amino acids (R429, K430 and N435) were critical for honokiol‐induced Ano1 inhibition. The R429A/K430L/N435G mutation reduced the sensitivity of Ano1 to honokiol. Honokiol inhibited SW620 cell proliferation, and this effect was reduced by Ano1‐shRNAs. Furthermore, Ano1 overexpression promoted proliferation in NCM460 cells with low Ano1 endogenous expression and resulted in an increased sensitivity to honokiol. Overexpression of the R429A/K430L/N435G mutation reduced WT Ano1‐induced increase in the sensitivity of NCM460 cells to honokiol. Conclusion and Implications: We identified a new anticancer mechanism of honokiol, through the inhibition of cell proliferation, by targeting Ano1 Ca2+‐activated Cl− channels. [ABSTRACT FROM AUTHOR]

Double strand breaks induced by genotoxic agents, if inappropriately repaired, will cause cell death or induce cancer. Poly(ADP-ribose) polymerase-3 (PARP-3) serves a role in double strand break repair, and may be involved in tumorigenesis. To the best of our knowledge, the role of PARP-3 in breast cancer has not yet been examined. In the present study, the expression of PARP-3 was investigated in 493 breast cancer samples and 54 tumor-adjacent control samples using tissue-microarray-based immunohistochemistry. PARP-3 expression was higher in breast cancer samples compared with control samples. PARP-3 overexpression was significantly associated with histological grade II–III (P=0.012). In addition, PARP-3 overexpression was significantly associated with shorter disease-free survival (DFS; P=0.027) time and exhibited a tendency toward shorter overall survival (OS; P=0.183) time in patients with breast cancer compared with patients with lower PARP-3 expression, particularly in BRCA1-positive patients (P=0.004 for disease-free survival and P=0.095 for OS). Multivariate Cox regression analysis indicated that PARP-3 was an independent prognostic factor in patients with breast cancer. Furthermore, it was revealed that PARP-3 overexpression was associated with shorter survival time in patients with cyclophosphamide/doxorubicin or epirubicin/5-fluorouracil (CAF/CEF) chemotherapy compared with low PARP-3 expression, but not in patients with CAF/CEF + docetaxel chemotherapy. The present study suggested that PARP-3 may be used as a biomarker for predicting the clinical outcome of patients receiving chemotherapy, and targeting PARP-3 may be a potential therapeutic strategy for the treatment of breast cancer. [ABSTRACT FROM AUTHOR]

The genetic variants of the ATP-binding cassette, subfamily G, member 2 (ABCG2) are known to be involved in developing cancer risk and interindividual differences in chemotherapeutic response. The polymorphisms in ABCG2 gene were genotyped by using PCR-RFLP assays. We found that ABCG2 G34A GA/AA genotype, C421A AA genotype, and haplotypes 34A-421C and 34G-421A were significantly associated with increased risk for developing breast carcinoma. Furthermore, ABCG2 C421A AA homozygote had a significant enhanced therapeutic response in patients with neoadjuvant anthracycline-based chemotherapy. Moreover, ABCG2 G34A AA genotype carriers displayed a longer OS in ER positive patients or PR positive patients after postoperative anthracycline-based chemotherapy. These results suggested that the ABCG2 polymorphisms might be a candidate pharmacogenomic factor to assess susceptibility and prognosis for breast carcinoma patients. [ABSTRACT FROM AUTHOR]

TMEM16A (known as anoctamin 1) Ca 2+ -activated chloride channel is overexpressed in many tumors. TMEM16A overexpression can be caused by gene amplification in many tumors harboring 11q13 amplification. TMEM16A expression is also controlled in many cancer cells via transcriptional regulation, epigenetic regulation and microRNAs. In addition, TMEM16A activates different signaling pathways in different cancers, e.g. the EGFR and CAMKII signaling in breast cancer, the p38 and ERK1/2 signaling in hepatoma, the Ras-Raf-MEK-ERK1/2 signaling in head and neck squamous cell carcinoma and bladder cancer, and the NFκB signaling in glioma. Furthermore, TMEM16A overexpression has been reported to promote, inhibit, or produce no effects on cell proliferation and migration in different cancer cells. Since TMEM16A exerts different roles in different cancer cells via activation of distinct signaling pathways, we try to develop the idea that TMEM16A regulates cancer cell proliferation and migration in a cell-dependent mechanism. The cell-specific role of TMEM16A may depend on the cellular environment that is predetermined by TMEM16A overexpression mechanisms specific for a particular cancer type. TMEM16A may exert its cell-specific role via its associated protein networks, phosphorylation by different kinases, and involvement of different signaling pathways. In addition, we discuss the role of TMEM16A channel activity in cancer, and its clinical use as a prognostic and predictive marker in different cancers. This review highlights the cell-type specific mechanisms of TMEM16A in cancer, and envisions the promising use of TMEM16A inhibitors as a potential treatment for TMEM16A-overexpressing cancers.

Mutations in the bestrophin-1 (Best1) gene are linked to several kinds of macular degeneration in both humans and dogs. Although bestrophins have been shown clearly to be Cl(-) ion channels, it is controversial whether Cl(-) channel dysfunction can explain the diseases. It has been suggested that bestrophins are multifunctional proteins: they may regulate voltage-gated Ca(2+) channels in addition to functioning as Cl(-) channels. Here, we show that human Best1 gene (hBest1) differentially modulates Ca(V)1.3 (L-type) voltage-gated Ca(2+) channels through association with the Ca(V)beta subunit. In transfected human embryonic kidney 293 cells, hBest1 inhibited Ca(V)1.3. Inhibition of Ca(V)1.3 was not observed in the absence of the beta subunit. Also, the hBest1 C terminus binds to Ca(V)beta subunits, suggesting that the effect of hBest1 was mediated by the Ca(V)beta subunit. The region of hBest1 responsible for the effect was localized to a region (amino acids 330-370) in the cytoplasmic C terminus that contains a predicted src-homology-binding domain that is not present in other bestrophin subtypes. Mutation of Pro(330) and Pro(334) abolished the effects of hBest1 on Ca(V)1.3. The effect was specific to hBest1; it was not observed with mouse Best1 (mBest1), mBest2, or mBest3. Wild-type hBest1 and the disease-causing mutants R92S, G299R, and D312N inhibited Ca(V) currents the same amount, whereas the A146K and G222E mutants were less effective. We propose that hBest1 regulates Ca(V) channels by interacting with the Ca(V)beta subunit and altering channel availability. Our findings reveal a novel function of bestrophin in regulation of Ca(V) channels and suggest a possible mechanism for the role of hBest1 in macular degeneration.