In Ah Kim

microRNA-7 increases radiosensitivity of human cancer cells with activated EGFR-associated signaling.

Many microRNAs (miRNAs) play crucial roles in regulating expression of oncogenes or tumor suppressor genes. The epidermal growth factor receptor (EGFR) is frequently overexpressed in a wide range of solid tumors and is an important therapeutic target; however, the therapeutic outcome of currently available anti-EGFR agents is often limited due to diverse molecular resistance mechanisms. In this study, we evaluated the potential of targeting miRNA-7 for overcoming radio-resistance of cancer cells with activated EGFR-associated signaling. A panel of human cancer cell lines with increased EGFR-PI3K-Akt signaling was transfected with pre-miR-7 or control miRNA. Ectopic overexpression of miR-7 attenuated EGFR and Akt expression and radiosensitized SQ20B squamous cell carcinoma of the larynx, MDA-MB-468 breast cancer cells, A549 lung carcinoma cells, and U251 and U87 malignant glioma cells. In contrast, antisense-mediated inhibition of mature miR-7 expression led up-regulation of EGFR and its downstream effectors, and increased radio-resistance of U251 glioma cells. Overexpression of miR-7 prolonged radiation-induced γH2AX foci formation and downregulation of DNA-dependent protein kinases (DNA-PKcs). miR-7 may be a useful therapeutic target for overcoming the radio-resistance of human cancers with activated EGFR-PI3K-AKT signaling.

High EGFR gene copy number predicts poor outcome in triple-negative breast cancer

Epidermal growth factor receptor (EGFR) is frequently overexpressed in triple-negative breast cancer and is emerging as a therapeutic target. EGFR gene copy number alteration and mutation are highly variable and scientists have been challenged to define their prognostic significance in triple-negative breast cancer. We examined EGFR protein expression, EGFR gene copy number alteration and mutation of exon 18 to 21 in 151 cases of triple-negative breast cancer and correlated these findings with clinical outcomes. In addition, intratumoral agreement of EGFR protein overexpression and gene copy number alteration was evaluated. EGFR overexpression was found in 97 of 151 cases (64%) and high EGFR gene copy number was detected in 50 cases (33%), including 3 gene amplification (2%) and 47 high polysomy (31%). Five EGFR mutations were detected in 4 of 151 cases (3%) and included G719A in exon 18 (n=1), V786M in exon 20 (n=1), and L858R in exon 21 (n=3). One case had two mutations (G719A and L858R). High EGFR copy number, but not EGFR mutation, correlated with EGFR protein overexpression. Intratumoral heterogeneity of EGFR protein overexpression and EGFR copy number alteration was not significant. In survival analyses, high EGFR copy number was found to be an independent prognostic factor for poor disease-free survival in patients with triple-negative breast cancer. Our findings showed that EGFR mutation was a rare event, but high EGFR copy number was relatively frequent and correlated with EGFR overexpression in triple-negative breast cancer. Moreover, high EGFR copy number was associated with poor clinical outcome in triple-negative breast cancer, suggesting that evaluation of EGFR copy number may be useful for predicting outcomes in patients with triple-negative breast cancer and for selecting patients for anti-EGFR-targeted therapy.

Histone Deacetylase Inhibitor–Mediated Radiosensitization of Human Cancer Cells: Class Differences and the Potential Influence of p53

Histone deacetylase inhibitors (HDI) are emerging as potentially useful components of the anticancer armamentarium and as useful tools to dissect mechanistic pathways. HDIs that globally inhibit histone deacetylases (HDAC) have radiosensitizing effects, but the relative contribution of specific HDAC classes remains unclear. Newly characterized HDIs are now available that preferentially inhibit specific HDAC classes, including SK7041 (inhibits class I HDACs) and splitomicin (inhibits class III HDACs). We investigated in human cancer cells the relative radiosensitizations that result from blocking specific HDAC classes. We found that trichostatin A (TSA; inhibitor of both class I and II HDACs) was the most effective radiosensitizer, followed by the class I inhibitor SK7041, whereas splitomicin (inhibitor of class III) had least effect. Interestingly, radiosensitization by TSA in cell lines expressing p53 was more pronounced than in isogenic lines lacking p53. Radiosensitization of cells expressing p53 by TSA was reduced by pifithrin-α, a small-molecule inhibitor of p53. In contrast, the radiosensitization by TSA of cells expressing low levels of p53 was enhanced by transfection of wild-type p53–expressing vector or pretreatment with leptomycin B, an inhibitor of nuclear export that increased intracellular levels of p53. These effects on radiosensitization were respectively muted or not seen in cells treated with SK7041 or splitomicin. To our knowledge, this may be among the first systematic investigations of the comparative anticancer effects of inhibiting specific classes of HDACs, with results suggesting differences in the degrees of radiosensitization, which in some cell lines may be influenced by p53 expression.

FGFR1 is amplified during the progression of in situ to invasive breast carcinoma

IntroductionGene amplification is an important mechanism for activating oncogenes in malignant tumors. Although amplification of HER2, C-MYC, CCND1 and FGFR1 has been reported in breast cancers, their role in the progression of in situ to invasive breast carcinoma is unclear. To investigate this question we compared the amplification frequencies of these genes in pure ductal carcinoma in situ (DCIS), DCIS associated with invasive carcinoma, and invasive carcinoma.MethodsWe performed fluorescence in situ hybridization of the selected genes on tissue microarrays composed of 179 pure DCIS and 438 invasive carcinomas. Two hundred and sixteen of the latter had DCIS components, and in those cases we compared gene amplification in the intraductal and invasive components of each carcinoma.ResultsThe rate of amplification of FGFR1 was higher in invasive carcinomas than in the pure DCIS, but the opposite was true for HER2 amplification. These findings applied consistently to high-grade tumors, but not to low/intermediate-grade tumors. The amplification status of HER2, C-MYC, CCND1 and FGFR1 was generally similar in the matched invasive and DCIS components of the same tumors. However, FGFR1 amplification was more common in the invasive components than in the DCIS components. In survival analyses, FGFR1 amplification was found to be an independent prognostic factor for poor disease-free survival for all patients with invasive carcinoma and for the hormone receptor-positive subgroup.ConclusionAmplification of HER2, C-MYC and CCND1 seems to play a role in the early development of breast cancer, but not in its progression. However, the increased frequency of FGFR1 amplification in invasive carcinomas compared with pure DCIS and in the invasive components of individual tumors, and its association with decreased disease-free survival, suggests a role for FGFR1 amplification in the progression of breast cancer including in situ-to-invasive transition, as well as initiation.

Targeting epidermal growth factor receptor-associated signaling pathways in non-small cell lung cancer cells: implication in radiation response.

Several studies have shown solid evidence for the potential value of targeting epidermal growth factor receptor (EGFR) signaling to enhance the antitumor activity of radiation. However, therapeutic resistance has emerged as an important clinical issue. Here, we investigated whether strategies for targeting EGFR-associated downstream signaling would radiosensitize a panel of non–small cell lung cancer cell lines. Inhibition of K-RAS using RNA interference attenuated downstream signaling and increased radiosensitivity of A549 and H460 cells, whereas inhibition of EGFR did not. A549 cells harboring a K-RAS mutation at codon V12 were radiosensitized by small interfering RNA (siRNA) targeting this codon. H460 cells having mutation at codon V61 was radiosensitized by siRNA targeting of this mutation. K-RAS siRNA did not radiosensitize H1299 cells possessing wild-type K-RAS. Inhibition of the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin pathway led to significant radiosensitization of the two cell lines, whereas selective inhibition of extracellular signal-regulated kinase signaling did not. Inhibitors targeting the PI3K-AKT-mTOR pathway also abrogated G2 arrest following irradiation and induced γH2AX foci formation. A dual inhibitor of class I PI3K and mammalian target of rapamycin effectively increased the radiosensitivity of A549 and H460 cells. Inhibition of PI3K-AKT signaling was associated with the downregulation of DNA-PKs. Although apoptosis was the primary mode of cell death when cells were pretreated with LY294002 or AKT inhibitor VIII, cells pretreated with rapamycin or PI-103 showed mixed modes of cell death, including apoptosis and autophagy. Our results suggest possible mechanisms for counteracting EGFR prosurvival signaling implicated in radioresistance and offer an alternative strategy for overcoming resistance to EGFR inhibitors used in combination with irradiation. Mol Cancer Res; 8(7); 1027–36. ©2010 AACR.

Enhanced cytotoxic effect of radiation and temozolomide in malignant glioma cells: targeting PI3K-AKT-mTOR signaling, HSP90 and histone deacetylases

BackgroundDespite aggressive treatment with radiation therapy and concurrent adjuvant temozolomide (TMZ), glioblastoma multiform (GBM) still has a dismal prognosis. We aimed to identify strategies to improve the therapeutic outcome of combined radiotherapy and TMZ in GBM by targeting pro-survival signaling from the epidermal growth factor receptor (EGFR).MethodsGlioma cell lines U251, T98G were used. Colony formation, DNA damage repair, mode of cell death, invasion, migration and vasculogenic mimicry as well as protein expression were determined.ResultsU251 cells showing a low level of methyl guanine transferase (MGMT) were highly responsive to the radiosensitizing effect of TMZ compared to T98G cells having a high level of MGMT. Treatment with a dual inhibitor of Class I PI3K/mTOR, PI103; a HSP90 inhibitor, 17-DMAG; or a HDAC inhibitor, LBH589, further increased the cytotoxic effect of radiation therapy plus TMZ in U251 cells than in T98G cells. However, treatment with a mTOR inhibitor, rapamycin, did not discernibly potentiate the radiosensitizing effect of TMZ in either cell line. The mechanism of enhanced radiosensitizing effects of TMZ was multifactorial, involving impaired DNA damage repair, induction of autophagy or apoptosis, and reversion of EMT (epithelial-mesenchymal transition).ConclusionsOur results suggest possible strategies for counteracting the pro-survival signaling from EGFR to improve the therapeutic outcome of combined radiotherapy and TMZ for high-grade gliomas.

The role of carcinoembryonic antigen after neoadjuvant chemoradiotherapy in patients with rectal cancer.

PURPOSE: The purpose of this study was to evaluate the role of CEA after neoadjuvant chemoradiotherapy in patients with rectal cancer. METHODS: This study involved 109 patients with rectal cancer who were treated with preoperative chemoradiotherapy and curative resection. Preoperative serum CEA levels were measured twice, before chemoradiotherapy administration and 4 weeks after chemoradiotherapy. Surgery was performed 6 to 9 weeks after neoadjuvant chemoradiotherapy. RESULTS: The 3-year disease-free survival rate was 85.0%. On univariate analysis, prechemoradiotherapy CEA level, postchemoradiotherapy CEA level, tumor regression grade, ypT, ypN, circumferential resection margin, lymphatic invasion, venous invasion, and perineural invasion were associated with disease-free survival. Based on univariate analysis (group 1: prechemoradiotherapy CEA ≤3.5; group 2: prechemoradiotherapy CEA >3.5, postchemoradiotherapy CEA ≤2.7; group 3: prechemoradiotherapy CEA >3.5, postchemoradiotherapy CEA >2.7), we categorized patients into 3 groups according to their pre- and postchemoradiotherapy CEA levels (ng/mL). The 3-year disease-free survival rate was significantly better in groups 1 and 2 (94.7% and 88.0%) than in group 3 (52.6%, P < .001). On multivariate analysis, tumor regression grade (P = .036), ypN (P = .036), and CEA groups (P = .022) were identified as independent prognostic factors for disease-free survival. Furthermore, postchemoradiotherapy CEA ≤2.7 ng/mL was an independent predictor of good tumor regression (P = .001), ypT0 to 2 (P = .002), and ypN0 (P = .001). CONCLUSIONS: Combined pre- and postchemoradiotherapy CEA levels could be useful as a prognostic factor for disease-free survival in patients with rectal cancer who undergo treatment with neoadjuvant chemoradiotherapy and curative resection. Postchemoradiotherapy CEA may be helpful in a selection of patients who want more conservative surgery after chemoradiotherapy.

Stronger proteasomal inhibition and higher CHOP induction are responsible for more effective induction of paraptosis by dimethoxycurcumin than curcumin

Although curcumin suppresses the growth of a variety of cancer cells, its poor absorption and low systemic bioavailability have limited its translation into clinics as an anticancer agent. In this study, we show that dimethoxycurcumin (DMC), a methylated, more stable analog of curcumin, is significantly more potent than curcumin in inducing cell death and reducing the clonogenicity of malignant breast cancer cells. Furthermore, DMC reduces the tumor growth of xenografted MDA-MB 435S cells more strongly than curcumin. We found that DMC induces paraptosis accompanied by excessive dilation of mitochondria and the endoplasmic reticulum (ER); this is similar to curcumin, but a much lower concentration of DMC is required to induce this process. DMC inhibits the proteasomal activity more strongly than curcumin, possibly causing severe ER stress and contributing to the observed dilation. DMC treatment upregulates the protein levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and Noxa, and the small interfering RNA-mediated suppression of CHOP, but not Noxa, markedly attenuates DMC-induced ER dilation and cell death. Interestingly, DMC does not affect the viability, proteasomal activity or CHOP protein levels of human mammary epithelial cells, suggesting that DMC effectively induces paraptosis selectively in breast cancer cells, while sparing normal cells. Taken together, these results suggest that DMC triggers a stronger proteasome inhibition and higher induction of CHOP compared with curcumin, giving it more potent anticancer effects on malignant breast cancer cells.

Distinct patterns of promoter CpG island methylation of breast cancer subtypes are associated with stem cell phenotypes

Although DNA methylation profiles in breast cancer have been connected to breast cancer molecular subtype, there have been no studies of the association of DNA methylation with stem cell phenotype. This study was designed to evaluate the promoter CpG island methylation of 15 genes in relation to breast cancer subtype, and to investigate whether the patterns of CpG island methylation in each subtype are associated with their cancer stem cell phenotype represented by CD44+/CD24− and ALDH1 expression. We performed MethyLight analysis of the methylation status of 15 promoter CpG island loci involved in breast cancer progression (APC, DLEC1, GRIN2B, GSTP1, HOXA1, HOXA10, IGF2, MT1G, RARB, RASSF1A, RUNX3, SCGB3A1, SFRP1, SFRP4, and TMEFF2) and determined cancer stem cell phenotype by CD44/CD24 and ALDH1 immunohistochemistry in 36 luminal A, 33 luminal B, 30 luminal–HER2, 40 HER2 enriched, and 40 basal-like subtypes of breast cancer. The number of CpG island loci methylated differed significantly between subtypes, and was highest in the luminal–HER2 subtype and lowest in the basal-like subtype. Methylation frequencies and levels in 12 of the 15 genes differed significantly between subtypes, and the basal-like subtype had significantly lower methylation frequencies and levels in nine of the genes than the other subtypes. CD44+/CD24− and ALDH1+ putative stem cell populations were most enriched in the basal-like subtype. Methylation of promoter CpG islands was significantly lower in CD44+/CD24-cell (+) tumors than in CD44+/CD24-cell (−) tumors, even within the basal-like subtype. ALDH1 (+) tumors were also less methylated than ALDH1 (−) tumors. Our findings showed that promoter CpG island methylation was different in relation to breast cancer subtype and stem cell phenotype of tumor, suggesting that breast cancers have distinct patterns of CpG island methylation according to molecular subtypes and these are associated with different stem cell phenotypes of the tumor.

Epigenetic modulation of radiation response in human cancer cells with activated EGFR or HER-2 signaling: Potential role of histone deacetylase 6

BACKGROUND AND PURPOSE Histone deacetylase inhibitors (HDIs) are prototypes of agents targeting epigenetic modifications and have received considerable attention for their promise as targeted anticancer drugs. We examined the effects and potential mechanism(s) of combining LBH589 and irradiation in human cancer cells having activated EGFR or HER-2 signaling, focusing on the role of HDAC6. METHODS AND MATERIALS We evaluated whether the HDI, LBH589, would radiosensitize a panel of human tumor cell lines having activated EGFR or HER-2 signaling. A mechanistic role for the HDAC6 isotype was investigated using RNA interference and ectopic overexpression HDAC6. RESULTS The HDI, LBH589, enhanced the radiosensitivity of the human carcinoma cell lines we tested. Radiosensitization was accompanied by abrogation of radiation-induced G2/M arrest and was associated with aberrant mitotic features and prolonged gammaH2AX foci. Radiation-induced apoptosis was also increased. LBH589 radiosensitized cells with activated EGFR or HER-2 signaling to a greater degree than the HDIs SK7041 or TSA. However radiosensitization by the three HDI was equivalent in cells without activation of this signaling. LBH589 led acetylation of histone H3 and HSP90. This was associated with down-regulation of the client oncoproteins EGFR, HER-2, and decreased phosphorylation of Akt and ERK. Specific inhibition of HDAC6 by RNAi increased radiosensitivity as well as increasing acetylation of HSP90 and reducing the association of HSP90 with its client proteins. Conversely, ectopic overexpression of HDAC6 isotype increased the levels of p-EGFR and p-AKT expression, and reduced LBH589-mediated radiosensitization. CONCLUSIONS These findings define a unique mechanism for counteracting pro-survival signaling from EGFR or HER-2 that is present in many tumor cells.