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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+].

Recent studies reveal that tumor microenvironment contributes to breast cancer (BRCA) development, progression, and therapeutic response. However, the contribution of the tumor microenvironment-related genes in routine diagnostic testing or therapeutic decision making for BRCA remains elusive. Immune/stromal/ESTIMATE scores calculated by the ESTIMATE algorithm quantify immune and stromal components in a tumor, and thus can reflect tumor microenvironment. To investigate the association of the tumor microenvironment-related genes with invasive BRCA prognosis, here we analyzed the immune/stromal/ESTIMATE scores in combination with The Cancer Genome Atlas (TCGA) database in invasive BRCA. We found that immune/stromal/ESTIMATE scores were significantly correlated with the invasive BRCA clinicopathological factors. Based on the immune/stromal/ESTIMATE scores, we extracted a series of differential expression genes (DEGs) related to the tumor microenvironment. Survival analysis was further performed to identify a list of high-frequency DEGs (HF-DEGs), which exhibited prognostic value in invasive BRCA. Importantly, consistent with the results of bioinformatics analysis, immunohistochemistry results showed that high SASH3 expression was associated with a good prognosis in invasive BRCA patients. Our findings suggest that the tumor microenvironment-related HF-DEGs identified in this study have prognostic values and may serve as potential biomarkers and therapeutic targets for invasive BRCA. [ABSTRACT FROM AUTHOR]

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]

Enhancer of zeste homologue 2 (EZH2), a catalytic subunit of polycomb repressive complex 2, is overexpressed in a number of different tumors including breast cancer, and serves important roles in cell cycle regulation, proliferation, apoptosis, tumorigenesis and drug resistance. However, it remains unclear whether EZH2 contributes to tamoxifen resistance in breast cancer. In the present study, the role of EZH2 in tamoxifen resistance in MCF-7 cells was investigated. EZH2 was overexpressed in MCF-7 tamoxifen-resistant (MCF-7 TamR) cells. EZH2 overexpression decreased the sensitivity of MCF-7 cells to tamoxifen, and EZH2 knockdown improved the sensitivity of MCF-7 TamR cells to tamoxifen. Furthermore, EZH2 knockdown induced cell cycle arrest in MCF-7 TamR cells, accompanied by a decrease in cyclin D1 expression and an increase in p16 expression. EZH2 knockdown reduced p16 gene methylation in MCF-7 TamR cells. These findings suggested that EZH2 overexpression may contribute to tamoxifen resistance in breast cancer, and EZH2 inhibition may reverse tamoxifen resistance in breast cancer by regulating the cell cycle via the demethylation of the p16 gene. Thus, EZH2 inhibitors may be effective for treating tamoxifen resistance in breast cancer. [ABSTRACT FROM AUTHOR]

TMEM16A, also known as anoctamin 1, is a recently identified Ca2+-activated chloride channel and the first member of a 10-member TMEM16 family. TMEM16A dysfunction is implicated in many diseases such as cancer, hypertension, and cystic fibrosis. TMEM16A channels are well known to be dually regulated by voltage and Ca2+. In addition, recent studies have revealed that TMEM16A channels are regulated by many molecules such as calmodulin, protons, cholesterol, and phosphoinositides, and a diverse range of stimuli such as thermal and mechanical stimuli. A better understanding of the regulatory mechanisms of TMEM16A is important to understand its physiological and pathological role. Recently, the crystal structure of a TMEM16 family member from the fungus Nectria haematococcaten (nhTMEM16) is discovered, and provides valuable information for studying the structure and function of TMEM16A. In this review, we discuss the structure and function of TMEM16A channels based on the crystal structure of nhTMEM16A and focus on the regulatory mechanisms of TMEM16A channels. J. Cell. Physiol. 232: 707-716, 2017. © 2016 Wiley Periodicals, Inc. [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]

The calcium-activated chloride channel Ano1 (TMEM16A) is overexpressed in many tumors. Although Ano1 overexpression is found in breast cancer due to 11q13 amplification, it remains unclear whether signaling pathways are involved in Ano1 overexpression during breast cancer tumorigenesis in vivo. Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) have been known to contribute to breast cancer progression. It is unclear whether Ano1 is associated with clinical outcomes in breast cancer patients with different ER, PR and HER2 status. In the present study, we investigated the Ano1 expression in 431 patients with invasive ductal breast carcinoma and 46 patients with fibroadenoma, using immunohistochemistry, and analyzed the association between Ano1 expression and clinical characteristics and outcomes of breast cancer patients with different ER, PR, and HER2 status. Ano1 was overexpressed in breast cancer compared with fibroadenoma. Ano1 was significantly more associated with breast cancer with the lower clinical stage (stage I or II), or triple-negative status. Mostly importantly, Ano1 overexpression was associated with good prognosis in patients with the PR-positive or HER2-negative status, and in patients following tamoxifen treatment. Multivariate Cox regression analysis showed that Ano1 overexpression was a prognostic factor for longer overall survival in PR-positive or HER2-negative patients, and a predictive factor for longer overall survival in patients following tamoxifen treatment. Our findings suggest that Ano1 may be a potential marker for good prognosis in PR-positive or HER2-negative patients following tamoxifen treatment. The PR and HER2 status defines a subtype of breast cancer in which Ano1 overexpression is associated with good prognosis following tamoxifen treatment. [ABSTRACT FROM AUTHOR]

Anoctamin1 (Ano1, or TMEM16A) is a Ca2+-activated chloride channel that is gated by both voltage and Ca2+. We have previously identified that the first intracellular loop that contains a high density of acidic residues mediates voltage- and calcium-dependent gating of Ano1. Mutation of the four consecutive glutamates (444EEEE447) inhibits the voltage-dependent activation of Ano1, whereas deletion of these residues decreases apparent Ca2+ sensitivity. In the present study, we further found that deletion of 444EEEEEAVKD452 produced a more than 40-fold decrease in the apparent Ca2+ sensitivity with altered activation kinetics. We then systematically mutated each acidic residue into alanine, and analyzed the voltage- and calcium dependent activation of each mutation. Activation kinetics of wild type Ano1 consisted of a fast component (τfast) that represented voltage-dependent mode, and a slow component (τslow) that reflected the Ca2+-dependent modal gating. E444A, E445A, E446A, E447A, E448A, and E457A mutations showed a decrease in the τfast, significantly inhibited voltage-dependent activation of Ano1 in the absence of Ca2+, and greatly shifted the G-V curve to the right, suggesting that these glutamates are involved in voltage-gating of Ano1. Furthermore, D452A, E464A, E470A, and E475A mutations that did not alter voltage-dependent activation of the channel, significantly decreased Ca2+ dependence of G-V curve, exhibited an increase in the τslow, and produced a 2–3 fold decrease in the apparent Ca2+ sensitivity, suggesting that these acidic residues are involved in Ca2+-dependent gating of the channel. Our data show that acidic residues in the first intracellular loop are the important structural determinant that couples the voltage and calcium dependent gating of Ano1. [ABSTRACT FROM AUTHOR]

The pTEN/AKT/mTOR signaling pathways play a critical role in balancing cell proliferation, differentiation, and survival. Recent studies researched the associations of core genes in the pTEN/AKT/mTOR pathway polymorphisms with the cancer susceptibility; however, the results are inconclusive. Therefore, a systematically meta-analysis was performed to evaluate the association between the five SNPs (mTOR rs2295080 and rs2536, AKT1 rs2494750 and rs2494752, pTEN rs701848) and cancer risk by systematic review of the literature in 31 eligible studies. The results showed a significant decreased risk between rs2295080 TG, GG genotype, and GG/TG genotypes and overall cancer [TG vs.TT: OR(95% CI) = 0.82(0.76, 0.89), GG/TG vs. TT: OR(95% CI) = 0.82(0.76, 0.88), and GG vs., Tg/tt: OR(95% CI) = 0.67(0.51, 0.88)] and the subgroup of urinary system cancer and digestive system cancer. Moreover, the SNP rs701848 CC, TC genotype showed significantly increased the overall cancer risk both in dominant model [CC/TC vs. TT: OR(95% CI) = 1.25(1.15, 1.36)] and recessive model [CC vs., Tc/tt: OR(95% CI) = 1.20(1.09, 1.32)], and digestive system cancer and urinary system cancer. In addition, AG genotype and GG/AG genotype of rs2494752 was associated with increased risk of cancer. Therefore, this meta-analysis provided genetic risk factors for carcinogenesis and the most valid cancer prevalence estimate for Asian population.

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.

The calcium-activated chloride channel Ano1 (TMEM16A) is overexpressed in many tumors. However, conflicting data exist regarding the role of Ano1 in cell proliferation. Here, we performed immunohistochemistry to investigate the expression of Ano1 and Ki67 in 403 patients with breast cancer, and analyzed the association between the expression of Ano1 and Ki67 in breast cancer subtypes categorized according to estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Ano1 expression was negatively correlated with Ki67 expression. Ano1 overexpression more frequently occurred in ER-positive or HER2-negative patients with the low expression of Ki67. Ano1 overexpression was associated with longer overall survival (OS) in breast cancer with the low expression of Ki67, especially in ER-positive, PR-positive, and HER2-negative breast cancer. Multivariate Cox regression analysis showed that Ano1 overexpression was a prognostic factor for longer overall survival in ER-positive, PR-positive, or HER2-negative patients with the low expression of Ki67. Furthermore, Ano1 promoted cell proliferation in ER-positive, PR-positive, and HER2-negative MCF7 cells, but inhibited cell proliferation in ER-negative, PR-negative, and HER2-negative MDA-MB-435S cells. Our findings suggest that Ano1 may differentially regulate cell proliferation in a subtype of breast cancer defined by ER, PR, and HER2. Combined expression of Ano1 and Ki67 may be used for predicting clinical outcomes of breast cancer patients with different subtypes of ER, PR, and HER2., Competing Interests: CONFLICTS OF INTEREST All authors declare no conflict of interest with regard to the submitted manuscript.

Cancer stem cells (CSCs) have been suggested to serve an important role in tumor recurrence and metastasis in breast cancer. The hedgehog (Hh) signaling pathway is essential for the maintenance of breast CSCs. The present study used immunohistochemistry to investigate the expression of Patched (PTCH) and Gli1, which are the main components of the Hh signaling pathway, as well as the expression of cluster of differentiation (CD)44/CD24, which are markers for breast CSCs, in 266 patients with breast cancer. The combined expression of PTCH and Gli1 was significantly associated with larger tumors (>2.0 cm; P=0.001), lymph node metastasis (P=0.003), invasive lobular carcinoma (P=0.016) and Grade II‑III tumors (P<0.001). In addition, PTCH and Gli1 expression was associated with lymph node metastasis (P=0.005 and P=0.001) and Grade II‑II tumors (P=0.020 and P=0.033) in breast cancer patients with the CD44+/CD24‑ phenotype. The expression of PTCH and Gli1 was also associated with significantly shorter overall survival and disease‑free survival (DFS) in breast cancer patients with the CD44+/CD24‑ phenotype. Multivariate Cox regression analysis demonstrated that PTCH expression and the CD44+/CD24‑ phenotype were independent prognostic factors for decreased DFS in patients with breast cancer. These findings suggest that the Hh signaling pathway in breast CSCs may contribute to the poor outcome of patients with breast cancer.

This study investigated the expression of ALDH1A1 and β-catenin in breast cancer patients, and analyzed the correlation of their combined expression with clinicopathological features, chemotherapeutic responses, and prognosis of breast cancer patients. In total 276 human breast cancer tissues and 80 benign hyperplasia tissues were included. The expression of ALDH1A1 and β-catenin was examined using tissue microarray-based immunohistochemistry. ROC curve analysis was performed to determine an optimal cut-off score for the expression of ALDH1A1 and β-catenin, based on the survival status of breast cancer patients. Survival probabilities were estimated by the Kaplan-Meier method. ALDH1A1 expression was higher, but β-catenin showed no significant difference in breast cancer samples compared to controls. Compared with the membrane expression of β-catenin [β-catenin(m)], the cytoplasmic expression of β-catenin [β-catenin(c)] occurred significantly more frequently in breast cancer with the high expression of ALDH1A1 [ALDH1A1(high)] than in breast cancer with the low expression of ALDH1A1 [ALDH1A1(low)] (P=0.014). The expression level of ALDH1A was significantly higher in β-catenin(c) breast cancer than in β-catenin(m) breast cancer (P=0.020). ALDH1A1(high) expression or β-catenin(c) expression alone was associated with lymph node metastasis, and worse clinical outcome in breast cancer patients, especially in patients receiving cyclophosphamide treatment. Combined expression of ALDH1A1(high) and β-catenin(c) was associated with lymph node metastasis, poor outcome, and resistance to cyclophosphamide treatment. β-catenin may regulate ALDH1A1 expression in a subtype of breast cancer with ALDH1A1(high) and β-catenin(c) expression. ALDH1A1(high) and β-catenin, ((c) expression may be used as a biomarker for predicting poor prognosis in breast cancer following cyclophosphamide treatment.)

BCSCs (breast cancer stem cells) have been shown to be resistant to chemotherapy. However, the mechanisms underlying BCSC-mediated chemoresistance remain poorly understood. The Hh (Hedgehog) pathway is important in the stemness maintenance of CSCs. Nonetheless, it is unknown whether the Hh pathway is involved in BCSC-mediated chemoresistance. In the present study, we cultured breast cancer MCF-7 cells in suspension in serum-free medium to obtain BCSC-enriched MCF-7 MS (MCF-7 mammosphere) cells. We showed that MCF-7 MS cells are sensitive to salinomycin, but not paclitaxel, distinct from parent MCF-7 cells. The expression of the critical components of Hh pathway, i.e., PTCH (Patched), SMO (Smoothened), Gli1 and Gli2, was significantly up-regulated in MCF-7 MS cells; salinomycin, but not paclitaxel, treatment caused a remarkable decrease in expression of those genes in MCF-7 MS cells, but not in MCF-7 cells. Salinomycin, but not paclitaxel, increased apoptosis, decreased the migration capacity of MCF-7 MS cells, accompanied by a decreased expression of c-Myc, Bcl-2 and Snail, the target genes of the Hh pathway. The salinomycin-induced cytotoxic effect could be blocked by Shh (Sonic Hedgehog)-mediated Hh signalling activation. Inhibition of the Hh pathway by cyclopamine could sensitize MCF-7 MS cells to paclitaxel. In addition, salinomycin, but not paclitaxel, significantly reduced the tumour growth, accompanied by decreased expression of PTCH, SMO, Gli1 and Gli2 in xenograft tumours. Furthermore, the expression of SMO and Gli1 was positively correlated with the expression of CD44+ / CD24-, and the expression of SMO and Gli1 in CD44+ / CD24- tissues was associated with a significantly shorter OS (overall survival) and DFS (disease-free survival) in breast cancer patients receiving chemotherapy., (© 2015 Authors; published by Portland Press Limited.)

The chemokine receptor CXCR4 and signal transducer and activator of transcription 3 (STAT3) play an important role in breast cancer malignancy and metastasis. However, it remains unknown whether STAT3 can be activated by CXCR4 in human breast cancer. The expression levels of CXCR4, STAT3 and p-STAT3 in 208 breast cancer tissues and 26 tumor-adjacent tissues were examined by immunohistochemistry. Flow cytometry, western blot analysis and immunoprecipitation were used to study activation of STAT3 by CXCL12-CXCR4 signaling in human breast cancer cell lines. The expression levels of CXCR4, STAT3 and p-STAT3 were higher in the breast cancer samples than these levels in the tumor-adjacent samples. The combined expression of CXCR4 and p-STAT3 was correlated with TNM stage, tumor size, lymph node metastasis and histological grade of breast cancer. In the breast cancer cells, CXCL12 treatment increased the expression of p-STAT3. The CXCR4 antagonist AMD3100 and the Janus kinase 2 (JAK2) antagonist AG490 inhibited the CXCL12-induced increase in the phosphorylation of STAT3. Furthermore, CXCL12 promoted direct binding of JAK2 to CXCR4. Our findings suggest that activation of the JAK2/STAT3 pathway via CXCL12-CXCR4 signaling plays an important role in breast cancer malignancy and metastasis. Targeting the CXCL12-CXCR4/JAK2/STAT3 signaling pathway may be a potential therapeutic strategy for the treatment of breast cancer.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been used to treat colorectal cancer (CRC). However, resistance to EGFR-TKIs presents a great challenge for the treatment of CRC, and the mechanisms of resistance are poorly understood. The adenomatous polyposis coli (APC) protein has been known to contribute to the carcinogenesis of CRC. However, its role in the sensitivity of CRC cells to gefitinib has not been investigated. Human CRC HCT-116 (wild-type APC) and HT-29 (mutant APC) cells were used to investigate the effect of APC on the sensitivity of CRC cells to gefitinib. The MTT assay was used to measure cell viability after exposure to gefitinib. Cell apoptosis, migration and invasion were determined by flow cytometry, wound healing assay and transwell assay, respectively. Knockdown and overexpression of APC were performed, and activation of the EGFR and its downstream pathway was determined. Gefinitib inhibited viability, promoted apoptosis, and reduced the migration of HCT-116 and HT-29 cells. HT-29 cells exhibited increased sensitivity to gefinitib when compared to HCT-116 cells. Knockdown of APC expression increased the sensitivity of HCT-16 cells to gefitinib, accompanied by downregulation of pEGFR, p-AKT and pERK1/2. In contrast, overexpression of APC decreased the sensitivity of HT-29 cells to gefitinib, accompanied by upregulation of pEGFR, p-AKT and pERK1/2. APC plays an important role in the sensitivity of CRC cells to gefitinib. APC may represent a potential therapeutic target for the treatment of CRC.

Until recently, anion (Cl(-)) channels have received considerably less attention than cation channels. One reason for this may be that many Cl(-) channels perform functions that might be considered cell-biological, like fluid secretion and cell volume regulation, whereas cation channels have historically been associated with cellular excitability, which typically happens more rapidly. In this review, we discuss the recent explosion of interest in Cl(-) channels, with special emphasis on new and often surprising developments over the past five years. This is exemplified by the findings that more than half of the ClC family members are antiporters, and not channels, as was previously thought, and that bestrophins, previously prime candidates for Ca(2+)-activated Cl(-) channels, have been supplanted by the newly discovered anoctamins and now hold a tenuous position in the Cl(-) channel world.

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.

This article reviews the current state of knowledge about the bestrophins, a newly identified family of proteins that can function both as Cl(-) channels and as regulators of voltage-gated Ca(2+) channels. The founding member, human bestrophin-1 (hBest1), was identified as the gene responsible for a dominantly inherited, juvenile-onset form of macular degeneration called Best vitelliform macular dystrophy. Mutations in hBest1 have also been associated with a small fraction of adult-onset macular dystrophies. It is proposed that dysfunction of bestrophin results in abnormal fluid and ion transport by the retinal pigment epithelium, resulting in a weakened interface between the retinal pigment epithelium and photoreceptors. There is compelling evidence that bestrophins are Cl(-) channels, but bestrophins remain enigmatic because it is not clear that the Cl(-) channel function can explain Best disease. In addition to functioning as a Cl(-) channel, hBest1 also is able to regulate voltage-gated Ca(2+) channels. Some bestrophins are activated by increases in intracellular Ca(2+) concentration, but whether bestrophins are the molecular counterpart of Ca(2+)-activated Cl(-) channels remains in doubt. Bestrophins are also regulated by cell volume and may be a member of the volume-regulated anion channel family.