Disruption of STAT3-DNMT1 interaction by SH-I-14 induces re-expression of tumor suppressor genes and inhibits growth of triple-negative breast tumor.

Epigenetic regulation of gene expression is an emerging target to treat several human diseases including cancers. In cancers, expressions of many tumor suppressor genes are suppressed by hyper-methylation in their regulatory regions. Herein, we describe a novel carbazole SH-I-14 that decreased the level of the acetyl-STAT3 at the K685 residue. Mutation analysis revealed that SH-I-14 disrupted STAT3-DNMT1 interaction by removing acetyl group from K685 of STAT3. Finally, the inhibition of STAT3-DNMT1 interaction by SH-I-14 resulted in re-expression of tumor suppressor genes such as VHL and PDLIM4 through de-methylation of their promoter regions. In addition, SH-I-14 showed anti-proliferative effect in triple-negative breast cancer (TNBC) cell lines in vitro and anti-tumor effect in a mouse xenograft model of MDA-MB-231 tumor. Taken together, our results suggest that targeting acetyl-STAT3 (K685) provides potential therapeutic opportunity to treat a subset of human cancers.

Dual inhibition of EGFR and MET induces synthetic lethality in triple-negative breast cancer cells through downregulation of ribosomal protein S6.

Triple-negative breast cancer (TNBC) exhibits innate resistance to the EGFR inhibition despite high level expression of EGFR. Recently, we found that the proliferation of basal-like (BL) subtype TNBC cells is synergistically inhibited by combination of EGFR and PI3K/AKT inhibitors. On the contrary, TNBC cells of mesenchymal stem-like (MSL) subtype are resistant to these combinations. To identify potential synthetic lethal interaction of compounds for treatment of MSL subtype TNBC cells, we performed MTT screening of MDA-MB­231 cells with a small library of receptor tyrosine kinase inhibitors (RTKIs) in the presence of gefitinib, an EGFR inhibitor. We identified MET inhibitors as potent RTKIs that caused synthetic lethality in combination with gefitinib in MDA-MB­231 cells. We demonstrated that combination of a MET inhibitor SU11274 with various EGFR inhibitors resulted in synergistic suppression of cell viability (in MTT assay) and cell survival (in colony formation assay) of MSL subtype TNBC cells. We further demonstrated that SU11274 alone induced G2 arrest and gefitinib/SU11274 combination sustained the SU11274-induced G2 arrest in these cells. In addition, SU11274/gefitinib combination synergistically reduced the level of ribosomal protein S6 (RPS6) in MSL subtype TNBC cells. In addition, knockdown of RPS6 itself, in both HS578T and MDA-MB­231, markedly reduced the proliferation of these cells. Taken together, our data suggest that dual targeting of EGFR and MET inhibits the proliferation of MSL subtype TNBC cells through downregulation of RPS6.

ß-TrCP1 degradation is a novel action mechanism of PI3K/mTOR inhibitors in triple-negative breast cancer cells.

An F-box protein, ß-TrCP recognizes substrate proteins and destabilizes them through ubiquitin-dependent proteolysis. It regulates the stability of diverse proteins and functions as either a tumor suppressor or an oncogene. Although the regulation by ß-TrCP has been widely studied, the regulation of ß-TrCP itself is not well understood yet. In this study, we found that the level of ß-TrCP1 is downregulated by various protein kinase inhibitors in triple-negative breast cancer (TNBC) cells. A PI3K/mTOR inhibitor PI-103 reduced the level of ß-TrCP1 in a wide range of TNBC cells in a proteasome-dependent manner. Concomitantly, the levels of c-Myc and cyclin E were also downregulated by PI-103. PI-103 reduced the phosphorylation of ß-TrCP1 prior to its degradation. In addition, knockdown of ß-TrCP1 inhibited the proliferation of TNBC cells. We further identified that pharmacological inhibition of mTORC2 was sufficient to reduce the ß-TrCP1 and c-Myc levels. These results suggest that mTORC2 regulates the stability of ß-TrCP1 in TNBC cells and targeting ß-TrCP1 is a potential approach to treat human TNBC.

HER2 confers drug resistance of human breast cancer cells through activation of NRF2 by direct interaction.

Overexpression and/or activation of HER2 confers resistance of cancer cells to chemotherapeutic drugs. NRF2 also gives drug resistance of cancer cells through induction of detoxification and/or drug efflux proteins. Although several upstream effectors of NRF2 overlapped with the downstream molecules of HER2 pathway, no direct link between HER2 and NRF2 has ever been established. Here, we identified that co-expression of a constitutively active HER2 (HER2CA) and NRF2 increased the levels of NRF2 target proteins, HO-1 and MRP5. We also identified HER2CA activated the DNA-binding of NRF2 and the antioxidant response element (ARE)-mediated transcription in an NRF2-dependent manner. In addition, NRF2 and HER2CA cooperatively up-regulated the mRNA expression of various drug-resistant and detoxifying enzymes including GSTA2, GSTP1, CYP3A4, HO-1, MRP1, and MRP5. We also demonstrated that NRF2 binds to HER2 not only in transiently transfected HEK293T cells but also in HER2-amplified breast cancer cells. Functionally, overexpression of HER2CA gave resistance of MCF7 breast cancer cells to either paraquat or doxorubicin. Overexpression of dominant negative NRF2 (DN-NRF2) reduced the HER2CA-induced resistance of MCF7 cells to these agents. Taken together, these results suggest that active HER2 binds and regulates the NRF2-dependent transcriptional activation and induces drug resistance of cancer cells.

Novel carbazole inhibits phospho-STAT3 through induction of protein-tyrosine phosphatase PTPN6.

The aberrant activation of STAT3 occurs in many human cancers and promotes tumor progression. Phosphorylation of a tyrosine at amino acid Y705 is essential for the function of STAT3. Synthesized carbazole derived with fluorophore compound 12 was discovered to target STAT3 phosphorylation. Compound 12 was found to inhibit STAT3-mediated transcription as well as to reduce IL-6 induced STAT3 phosphorylation in cancer cell lines expressing both elevated and low levels of phospho-STAT3 (Y705). Compound 12 potently induced apoptosis in a broad number of TNBC cancer cell lines in vitro and was effective at inhibiting the in vivo growth of human TNBC xenograft tumors (SUM149) without any observed toxicity. Compound 12 also effectively inhibited the growth of human lung tumor xenografts (A549) harboring aberrantly active STAT3. In vitro and in vivo studies showed that the inhibitory effects of 12 on phospho-STAT3 were through up-regulation of the protein-tyrosine phosphatase PTPN6. Our present studies strongly support the continued preclinical evaluation of compound 12 as a potential chemotherapeutic agent for TNBC and cancers with constitutive STAT3 signaling.

BRCA1 and Oxidative Stress.

The breast cancer susceptibility gene 1 (BRCA1) has been well established as a tumor suppressor and functions primarily by maintaining genome integrity. Genome stability is compromised when cells are exposed to oxidative stress. Increasing evidence suggests that BRCA1 regulates oxidative stress and this may be another mechanism in preventing carcinogenesis in normal cells. Oxidative stress caused by reactive oxygen species (ROS) is implicated in carcinogenesis and is used strategically to treat human cancer. Thus, it is essential to understand the function of BRCA1 in oxidative stress regulation. In this review, we briefly summarize BRCA1's many binding partners and mechanisms, and discuss data supporting the function of BRCA1 in oxidative stress regulation. Finally, we consider its significance in prevention and/or treatment of BRCA1-related cancers.

Combination of dasatinib and gemcitabine reduces the ALDH1A1 expression and the proliferation of gemcitabine-resistant pancreatic cancer MIA PaCa-2 cells.

Gemcitabine-based chemotherapy is the standard for treatment of pancreatic cancer; however, intrinsic and acquired resistance to gemcitabine commonly occurs. Aldehyde dehydrogenase 1A1 (ALDH1A1), one of the characteristic features of tumor-initiating and/or cancer stem cell (CSC) properties, is important in both intrinsic and acquired resistance to gemcitabine. In this study, we investigated the effectiveness of dasatinib, an SRC inhibitor, and gemcitabine combination to inhibit the survivals of parental (MIA PaCa-2/P) and gemcitabine-resistant (MIA PaCa-2/GR) cell lines. In MIA PaCa-2/GR cells, the levels of phospho-SRC and ALDH1A1 were increased compared to MIA PaCa-2/P cells. Inhibition of SRC by dasatinib or siRNA synergistically enhanced gemcitabine-induced anti-proliferative effects and induced apoptotic cell death in these cells. Furthermore, combination of SRC inhibition (either by dasatinib or siRNA) and gemcitabine significantly decreased the levels of ALDH1A1 expression. These results suggest that dasatinib and gemcitabine combination may be a potential therapeutic strategy to overcome gemcitabine resistance by decreasing the levels of ALDH1A1 expression.

Inhibition of NRF2 by PIK-75 augments sensitivity of pancreatic cancer cells to gemcitabine.

We describe the potential benefit of PIK-75 in combination of gemcitabine to treat pancreatic cancer in a preclinical mouse model. The effect of PIK-75 on the level and activity of NRF2 was characterized using various assays including reporter gene, quantitative PCR, DNA-binding and western blot analyses. Additionally, the combinatorial effect of PIK-75 and gemcitabine was evaluated in human pancreatic cancer cell lines and a xenograft model. PIK-75 reduced NRF2 protein levels and activity to regulate its target gene expression through proteasome-mediated degradation of NRF2 in human pancreatic cancer cell lines. PIK-75 also reduced the gemcitabine-induced NRF2 levels and the expression of its downstream target MRP5. Co-treatment of PIK-75 augmented the antitumor effect of gemcitabine both in vitro and in vivo. Our present study provides a strong mechanistic rationale to evaluate NRF2 targeting agents in combination with gemcitabine to treat pancreatic cancers.

Targeting mutant p53 by a SIRT1 activator YK-3-237 inhibits the proliferation of triple-negative breast cancer cells.

Many types of mutations in tumor suppressor p53 are oncogenic through gain-of-function. Therefore, targeting mutant p53 (mtp53) is a promising therapeutic approach to fight against many types of cancers. We report here a small molecule compound YK-3-237 that reduces acetylation of mtp53 and exhibits anti-proliferative effects toward triple-negative breast cancer (TNBC) cells carrying mtp53. YK-3-237 activates SIRT1 enzyme activities in vitro and deacetylation of both mtp53 in a SIRT1-dependent manner. Deacetylation of mtp53 resulted in depletion of mtp53 protein level and up-regulated the expression of WTp53-target genes, PUMA and NOXA. YK-3-237 also induces PARP-dependent apoptotic cell death and arrests the cell cycle at G2/M phase of mtp53 TNBC cells. Taken together, our data suggest that targeting acetylation of mtp53 is a potential target to treat human cancers.

Inhibition of checkpoint kinase 2 (CHK2) enhances sensitivity of pancreatic adenocarcinoma cells to gemcitabine.

Checkpoint kinase 2 (CHK2) plays pivotal function as an effector of cell cycle checkpoint arrest following DNA damage. Recently, we found that co-treatment of NSC109555 (a potent and selective CHK2 inhibitor) potentiated the cytotoxic effect of gemcitabine (GEM) in pancreatic cancer MIA PaCa-2 cells. Here, we further examined whether NSC109555 could enhance the antitumour effect of GEM in pancreatic adenocarcinoma cell lines. In this study, the combination treatment of NSC109555 plus GEM demonstrated strong synergistic antitumour effect in four pancreatic cancer cells (MIA PaCa-2, CFPAC-1, Panc-1 and BxPC-3). In addition, the GEM/NSC109555 combination significantly increased the level of intracellular reactive oxygen species (ROS), accompanied by induction of apoptotic cell death. Inhibition of ROS generation by N-acetyl cysteine (NAC) significantly reversed the effect of GEM/NSC109555 in apoptosis and cytotoxicity. Furthermore, genetic knockdown of CHK2 by siRNA enhanced GEM-induced apoptotic cell death. These findings suggest that inhibition of CHK2 would be a beneficial therapeutic approach for pancreatic cancer therapy in clinical treatment.

Correlations between BRCA1 defect and environmental factors in the risk of breast cancer.

The risk factors for breast cancer, the most common female malignant cancer, include environmental factors such as radiation, tobacco, a high-fat diet, and xenoestrogens as well as hormones. In addition, BRCA1 and BRCA2 are the most well-known genetic factors that increase risk for breast cancer. Coincidence of those environmental and genetic factors might augment the risk of tumorigenesis of breast. To verify this hypothesis, we briefly evaluated the carcinogenic potency of various environmental factors in the absence or presence of BRCA1 as a genetic factor in a normal mammary epithelial cell line, MCF10A. Many environmental factors tested increased cellular ROS level in the absence of other insult. In addition, TCDD, DMBA, 3MC, and BPA enhanced the BaP-induced ROS production. BRCA1 knockdown (BRCA1-KD) cells by siRNA significantly induced cellular accumulation of ROS compared to control cells. In this setting, the addition of paraquat, TCDD, DMBA, 2OHE2 or 4OHE2 significantly augmented ROS generation in BRCA1-KD MCF10A cells. Measurements of BaP-DNA adduct formation as a marker of DNA damage also revealed that BRCA1 deficiency leads increased DNA damage. In addition, TCDD and DMBA significantly increased BaP-DNA adduct formation in the absence of BRCA1. These results imply that elevated level of ROS is correlated with increase of DNA damage in BRCA1 defective cells. Taken together, our study suggests that several environmental factors might increase the risk of tumorigenesis in BRCA1 defective breast epithelial cells.

Inhibition of the PI3K/AKT pathway potentiates cytotoxicity of EGFR kinase inhibitors in triple-negative breast cancer cells.

Triple-negative breast cancers (TNBCs) are known to be intrinsically resistant to inhibitors for epidermal growth factor receptor (EGFR). Until now, clinical trials for TNBCs using EGFR inhibitors (EGFRis) as single agents have yielded disappointing results. Here, we report that combinatorial treatment using EGFRis, such as gefitinib or erlotinib, with PI3K/AKT pathway inhibitors (PI3K/AKTis) demonstrated a synergistic, anti-proliferative effect in cell lines of the basal-like (BL) subtype, a subtype of TNBC. Western blot analysis revealed that the gefitinib/PI-103 combination significantly reduced the level of both phospho-AKT and phospho-ERK in two susceptible BL subtype cell lines, SUM149PT and MDA-MB-468, whereas it had little or no effect on the level of phospho-ERK in two non-susceptible cell lines (HS578T and MDA-MB-231) of mesenchymal stem-like (MSL) TNBC subtype. The gefitinib/PI-103 combination also significantly induced caspase-3/7-mediated PARP cleavage and reduced two anti-apoptotic proteins, XIAP and Bcl-2 in the susceptible cell lines. In addition, the level of myeloid cell leukemia 1 (Mcl-1) protein was markedly decreased by gefitinib/PI-103 combination in the BL TNBC cells, but showed no significant change by this combination in MSL subtype cells. These results suggest that pharmacological inhibition of EGFR used in combination of PI3K/AKTis is a potential therapeutic approach to treat a subtype of TNBCs.

The synergistic apoptotic interaction of Indole-3-Carbinol and Genistein with TRAIL on endometrial cancer cells.

Induction of apoptosis in target cells is a key mechanism by which chemotherapy promotes cell killing. The purpose of this study was to determine whether Indole-3-Carbinol (I3C) and Genistein in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induce apoptosis in endometrial cancer cell (Ishikawa) and to assess apoptotic mechanism. The MTT assay and flow cytometry were performed to determine cell viability and cell cycle. The induction of apoptosis was measured by caspase-3 activity test, DNA fragmentation assay, annexin V binding assay and western blot analysis. There was no effect in cell growth inhibition and cell cycle progression alone or in two-combination. However, the treatment of I3C and Genistein followed by TRAIL showed significant cell death and marked increase in sub-G1 arrest. Three-combination treatment revealed elevated expression of DR4, DR5 and cleaved forms of caspase-3, caspase-8, PARP. The Flip was found down regulated. Moreover, increase in caspase-3 activity and DNA fragmentation indicated the induction of apoptosis. The results indicate that I3C and Genistein with TRAIL synergistically induced apoptosis via death receptor dependent pathway. Our findings might provide a new insight into the development of novel combination therapies against endometrial cancer.

Salinomycin inhibits Akt/NF-κB and induces apoptosis in cisplatin resistant ovarian cancer cells.

BACKGROUND: Despite advances in treatment, ovarian cancer is the most lethal gynecologic malignancy. Therefore significant efforts are being made to develop novel strategies for the treatment of ovarian cancer. Salinomycin has been shown to be highly effective in the elimination of cancer stem cells both in vitro and in vivo. The present study focused on investigating important cell signaling molecules such as Akt and NF-κB during salinomycin-induced apoptosis in cisplatin resistant ovarian cancer cells (A2780cis). METHODS: MTT assay was performed to determine cell viability. Flow cytometry and DNA fragmentation assay were performed to analyze the effect on cell cycle and apoptosis. The expression of apoptosis related proteins was evaluated by Western blot analysis. RESULTS: The cell viability was significantly reduced by salinomycin treatment in a dose dependent manner. The flow cytometry result showed an increase in sub-G1 phase. Salinomycin inhibited the nuclear transportation of NF-κB, and downregulated Akt expression. Declined Bcl-2, activation of caspase-3 and increased PARP cleavage triggered apoptosis. Moreover, DNA fragmentation assay also revealed apoptotic induction. CONCLUSION: The result suggested that salinomycin-induced apoptosis in A2780cis was associated with inhibition of Akt/NF-κB. It may become a potential chemotherapeutic agent for the cisplatin resistant ovarian cancer therapy.

Regulatory role of osteopontin in malignant transformation of endometrial cancer.

Osteopontin (OPN) involves in the tumor-promoting or metastasis in human endometrial cancer. Depletion of OPN gene expression in endometrial cancer cells was significantly decreased in cell viability and the cells undergo apoptotic cell death. The status of OPN in THESC, RL95, Hec1A and Ishikawa cell lines were analyzed by RT-PCR and western blot. After OPN-siRNA transfection, mRNA and protein expression levels of OPN were determined in Hec1A and Ishikawa cells. Cell proliferation and cell cycle distribution were observed by MTT and flow cytometry analysis. DNA fragmentation assay was used to measure cell apoptosis. Cell migration was assessed by wound healing assay. Depletion of OPN gene expression in endometrial cancer cell lines (Hec1A and Ishikawa cells) reproducibly changed their ability of proliferation. Concomitant changes were seen in the expression of OPN binding cell surface receptors, cell cycle-regulatory genes, cell invasion and colony formation nature of the tumor cells. Decreased colonizing potential in the absence of OPN was reversed in the presence of recombinant OPN. Inhibition of anchorage-independent growth was observed in the presence of metabolic inhibitors of the PI3K, Src and integrin signaling cascades, which was ameliorated in the presence of exogenously added OPN. Our result showed the role of OPN in endometrial cancer, in particular on the malignancy-promoting aspects of OPN that may pave way for new approaches to the clinical management of endometrial cancer.

Inhibition of constitutively activated phosphoinositide 3-kinase/AKT pathway enhances antitumor activity of chemotherapeutic agents in breast cancer susceptibility gene 1-defective breast cancer cells.

Loss or decrease of wild type BRCA1 function, by either mutation or reduced expression, has a role in hereditary and sporadic human breast and ovarian cancers. We report here that the PI3K/AKT pathway is constitutively active in BRCA1-defective human breast cancer cells. Levels of phospho-AKT are sustained even after serum starvation in breast cancer cells carrying deleterious BRCA1 mutations. Knockdown of BRCA1 in MCF7 cells increases the amount of phospho-AKT and sensitizes cells to small molecule protein kinase inhibitors (PKIs) targeting the PI3K/AKT pathway. Restoration of wild type BRCA1 inhibits the activated PI3K/AKT pathway and de-sensitizes cells to PKIs targeting this pathway in BRCA1 mutant breast cancer cells, regardless of PTEN mutations. In addition, clinical PI3K/mTOR inhibitors, PI-103, and BEZ235, showed anti-proliferative effects on BRCA1 mutant breast cancer cell lines and synergism in combination with chemotherapeutic drugs, cisplatin, doxorubicin, topotecan, and gemcitabine. BEZ235 synergizes with the anti-proliferative effects of gemcitabine by enhancing caspase-3/7 activity. Our results suggest that the PI3K/AKT pathway can be an important signaling pathway for the survival of BRCA1-defective breast cancer cells and pharmacological inhibition of this pathway is a plausible treatment for a subset of breast cancers.

BML-275, an AMPK inhibitor, induces DNA damage, G2/M arrest and apoptosis in human pancreatic cancer cells.

Adenosine monophosphate-activated protein kinase (AMPK) is a principal intracellular energy sensor which regulates energy producing pathways and energy requiring pathways when the cellular AMP/ATP ratio is altered. BML-275 (compound C), a well-known inhibitor of AMPK, has been found to induce apoptosis in myeloma, glioma and prostate cancer cells. However, the mechanisms responsible for the selective apoptotic effect(s) by BML-275 in cancer cells remain unknown. In the present study, BML-275 was investigated for its antitumor effect(s) in human pancreatic cancer cell lines. BML-275 inhibited the cell proliferation of 4 human pancreatic cancer cell lines (MIA PaCa-2, Panc-1, Colo-357 and AsPC-1). In addition, BML-275 significantly increased the generation of intracellular reactive oxygen species (ROS), followed by induction of DNA damage signaling and apoptosis. Furthermore, BML-275 induced cell cycle arrest in the G2/M phase. The inhibition of ROS generation by N-acetyl cysteine (NAC) significantly prevented the induction of DNA damage and apoptosis, but failed to prevent the induction of G2/M arrest by BML-275. Small interfering RNA (siRNA)-mediated knockdown of AMPKα increased the generation of intracellular ROS, DNA damage signaling and apoptosis without cell cycle arrest at the G2/M phase. These findings suggest that BML-275 exerts its antitumor effects by inducing ROS generation, DNA damage and apoptosis via inhibition of the AMPK pathway and by inducing G2/M arrest via a pathway independent of AMPK, implicating its potential application as an antitumor agent for pancreatic cancer.

Aldehyde dehydrogenase 1A1 confers intrinsic and acquired resistance to gemcitabine in human pancreatic adenocarcinoma MIA PaCa-2 cells.

Gemcitabine (GEM) is the front-line standard chemotherapy used for the treatment of pancreatic cancer; however, chemoresistance to GEM remains the major obstacle to the successful control of this disease. Both the expression levels and activity of aldehyde dehydrogenase 1A1 (ALDH1A1) are important features of tumor-initiating and/or cancer stem cell properties in multiple types of human cancer. As one of the intrinsic properties of cancer stem cells is drug resistance, in this study, we examined the correlation between the level and activity of endogenous ALDH1A1 and GEM resistance in the MIA PaCa-2 cell line that contains high expression levels and activity of ALDH1A1. We used small interfering RNAs (siRNAs) to deplete ALDH1A1 and investigate its potential role in conferring GEM resistance. The ALDH1A1 knockdown markedly reduced ALDH1A1 expression and activity and inhibited cell proliferation. Moreover, the combination of ALDH1A1-siRNA and GEM significantly decreased cell viability, increased apoptotic cell death and increased the accumulation of cells at the S-phase compared to the controls. Our data also demonstrated that ALDH1A1 expression and activity were significantly higher in the GEM-resistant MIA PaCa-2 cell line (MIA PaCa-2/GR), compared to the parental MIA PaCa-2 cell line (MIA PaCa-2/P). In the MIA PaCa-2/GR cells, the combination of ALDH1A1-siRNA and GEM also showed a significant decrease in cell viability and an increase in apoptotic cell death, emphasizing the importance of ALDH1A1 in both intrinsic and acquired GEM resistance. This potentially powerful combination treatment of ALDH1A1-siRNA and GEM warrants further investigation as an effective therapeutic regimen to overcome the resistance of pancreatic cancer to GEM.