Regulation of PTEN expression by the SWI/SNF chromatin-remodelling protein BRG1 in human colorectal carcinoma cells

Background:

Aberrant expression of Brahma-related gene-1 (BRG1), a core component of the SWI/SNF chromatin-remodelling complex, has been implicated in cancer development; however, the biological significance of BRG1 in colorectal carcinoma (CRC) remains unknown.

Methods:

In CRC tissues, expression of BRG1 and Brahma (BRM) was investigated immunohistochemically. Colorectal carcinoma-derived DLD-1 cells were used for knockdown of BRG1 and PTEN with small interfering RNA (siRNA) and transduction of Akt. Complementary DNA (cDNA) microarray analysis was performed to explore the genes affected by BRG1.

Results:

Expression of BRG1, but not BRM, was frequently elevated in CRC specimens, and knockdown of BRG1 suppressed cell proliferation of DLD-1 cells. By cDNA microarray, we determined that PTEN expression was negatively regulated by BRG1 in DLD-1 cells, which subsequently influenced the cyclin D1 levels via the phosphoinositide 3-OH kinase (PI3K)–Akt signalling pathway. The interplay of BRG1 on cyclin D1 expression was confirmed by the introduction of Akt and knockdown of PTEN in the BRG1 siRNA-transduced DLD-1 cells. Interestingly, this positive correlation between BRG1 and cyclin D1 expression was also observed in CRC specimens.

Conclusion:

Brahma-related gene-1 has an important role in the process of CRC development by activating the PI3K–Akt signalling pathway and resultant upregulation of cyclin D1 levels.     

Loss of GPER identifies new targets for therapy among a subgroup of ERα-positive endometrial cancer patients with poor outcome

Background:

The G protein-coupled oestrogen receptor, GPER, has been suggested as an alternative oestrogen receptor. Our purpose was to investigate the potential of GPER as a prognostic and predictive marker in endometrial carcinoma and to search for new drug candidates to improve treatment of aggressive disease.

Materials and method:

A total of 767 primary endometrial carcinomas derived from three patient series, including an external dataset, were studied for protein and mRNA expression levels to investigate and validate if GPER loss identifies poor prognosis and new targets for therapy in endometrial carcinoma. Gene expression levels, according to ERα/GPER status, were used to search the connectivity map database for small molecular inhibitors with potential for treatment of metastatic disease for receptor status subgroups.

Results:

Loss of GPER protein is significantly correlated with low GPER mRNA, high FIGO stage, non-endometrioid histology, high grade, aneuploidy and ERα loss (all P-values ⩽0.05). Loss of GPER among ERα-positive patients identifies a subgroup with poor prognosis that until now has been unrecognised, with reduced 5-year survival from 93% to 76% (P=0.003). Additional loss of GPER from primary to metastatic lesion counterparts further supports that loss of GPER is associated with disease progression.

Conclusion:

These results support that GPER status adds clinically relevant information to ERα status in endometrial carcinoma and suggest a potential for new inhibitors in the treatment of metastatic endometrial cancers with ERα expression and GPER loss.     

A leukotriene B4 receptor-2 is associated with paclitaxel resistance in MCF-7/DOX breast cancer cells

Background:

Breast cancer is the most common malignancy in women. Although chemotherapeutic agents, such as paclitaxel, are effective treatments for the majority of breast cancer patients, recurrence is frequent and often leads to death. Thus, there is an urgent need to identify novel therapeutic targets that sensitise tumour cells to existing chemotherapy agents.

Methods:

The levels of leukotriene B₄ receptor-2 (BLT2) in multidrug-resistant MCF-7/DOX cells were determined using quantitative PCR and FACS analysis. The potential role of BLT2 in the paclitaxel resistance of MCF-7/DOX cells was assessed using a pharmacological inhibitor and small interfering RNA knockdown, and the BLT2-associated resistance mechanism was assessed.

Results:

The expression levels of BLT2 were markedly upregulated in MCF-7/DOX cells. The inhibition of BLT2 by pre-treatment with {"type":"entrez-nucleotide","attrs":{"text":"LY255283","term_id":"1257961172","term_text":"LY255283"}}LY255283 or siBLT2 knockdown significantly sensitised MCF-7/DOX cells to paclitaxel and induced significant levels of apoptotic death, suggesting that BLT2 mediates paclitaxel resistance. We also demonstrated that BLT2-induced paclitaxel resistance was associated with the upregulation of P-glycoprotein. Finally, co-treatment with a BLT2 inhibitor and paclitaxel markedly reduced tumour growth in an MCF-7/DOX in vivo model.

Conclusion:

Together, our results demonstrate that BLT2 is a novel therapeutic target that sensitises drug-resistant breast cancer cells to paclitaxel.     

Specific activity of cyclin-dependent kinase I is a new potential predictor of tumour recurrence in stage II colon cancer

Background:

There are no established biomarkers to identify tumour recurrence in stage II colon cancer. As shown previously, the enzymatic activity of the cyclin-dependent kinases 1 and 2 (CDK1 and CDK2) predicts outcome in breast cancer. Therefore, we investigated whether CDK activity identifies tumour recurrence in colon cancer.

Methods:

In all, 254 patients with completely resected (R0) UICC stage II colon cancer were analysed retrospectively from two independent cohorts from Munich (Germany) and Leiden (Netherlands). None of the patients received adjuvant treatment. Development of distant metastasis was observed in 27 patients (median follow-up: 86 months). Protein expression and activity of CDKs were measured on fresh-frozen tumour samples.

Results:

Specific activity (SA) of CDK1 (CDK1SA), but not CDK2, significantly predicted distant metastasis (concordance index=0.69, 95% confidence interval (CI): 0.55–0.79, P=0.036). Cutoff derivation by maximum log-rank statistics yielded a threshold of CDK1SA at 11 (SA units, P=0.029). Accordingly, 59% of patients were classified as high-risk (CDK1SA ⩾11). Cox proportional hazard analysis revealed CDK1SA as independent prognostic variable (hazard ratio=6.2, 95% CI: 1.44–26.9, P=0.012). Moreover, CKD1SA was significantly elevated in microsatellite-stable tumours.

Conclusion:

Specific activity of CDK1 is a promising biomarker for metastasis risk in stage II colon cancer.     

miR-320c regulates gemcitabine-resistance in pancreatic cancer via SMARCC1

Background:

Gemcitabine-based chemotherapy is the standard treatment for pancreatic cancer. However, the issue of resistance remains unresolved. The aim of this study was to identify microRNAs (miRNAs) that govern the resistance to gemcitabine in pancreatic cancer.

Methods:

miRNA microarray analysis using gemcitabine-resistant clones of MiaPaCa2 (MiaPaCa2-RGs), PSN1 (PSN1-RGs), and their parental cells (MiaPaCa2-P, PSN1-P) was conducted. Changes in the anti-cancer effects of gemcitabine were studied after gain/loss-of-function analysis of the candidate miRNA. Further assessment of the putative target gene was performed in vitro and in 66 pancreatic cancer clinical samples.

Results:

miR-320c expression was significantly higher in MiaPaCa2-RGs and PSN1-RGs than in their parental cells. miR-320c induced resistance to gemcitabine in MiaPaCa2. Further experiments showed that miR-320c-related resistance to gemcitabine was mediated through SMARCC1, a core subunit of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. In addition, clinical examination revealed that only SMARCC1-positive patients benefited from gemcitabine therapy with regard to survival after recurrence (P=0.0463).

Conclusion:

The results indicate that miR-320c regulates the resistance of pancreatic cancer cells to gemcitabine through SMARCC1, suggesting that miR-320c/SMARCC1 could be suitable for prediction of the clinical response and potential therapeutic target in pancreatic cancer patients on gemcitabine-based therapy.     

G-protein Coupled Estrogen Receptor 1 Expression in Primary Breast Cancers and Its Correlation with Clinicopathological Variables

Purpose

G-protein coupled estrogen receptor 1 (GPER) probably play important roles in the progression of breast cancer including endocrine therapeutic resistance. We evaluated GPER in primary breast cancers.

Methods

Immunohistochemistry was used to detect GPER in paraffin-embedded tissues of primary breast cancers from 423 patients and GPER expression was correlated with clinicopathological factors.

Results

GPER was expressed in 63.8% of specimens, coexpressed with estrogen receptor alpha (ERα) in 36.6% of tumors and was positive in 62.5% of the ERα-negative tumors. The expression of GPER had no relationship with the status of ERα, progesterone receptor and HER2. Although the expression of GPER was significantly inversely related with nodal status (p=0.045), no correlation between GPER expression and other clinicopathological variables (age, menstruation status, tumor size, stage, histologic grade, Nottingham Prognostic Index or pathological type) was found.

Conclusion

GPER and ERα exhibited independent expression pattern of distribution in primary breast cancers. A long-term follow-up and a more definite molecular phenotype for ER are necessary in confirming studies.     

PSMB7 is associated with anthracycline resistance and is a prognostic biomarker in breast cancer

Background:

To date individual markers have failed to correctly predict resistance against anticancer agents in breast cancer. We used gene expression patterns attributable to chemotherapy-resistant cells to detect potential new biomarkers related to anthracycline resistance. One of the genes, PSMB7, was selected for further functional studies and clinical validation.

Methods:

We contrasted the expression profiles of four pairs of different human tumour cell lines and of their counterparts resistant to doxorubicin. Observed overexpression of PSMB7 in resistant cell lines was validated by immunohistochemistry. To examine its function in chemoresistance, we silenced the gene by RNA interference (RNAi) in doxorubicin-resistant MCF-7 breast cancer cells, then cell vitality was measured after doxorubicin treatment. Microarray gene expression from GEO raw microarray samples with available progression-free survival data was downloaded, and expression of PSMB7 was used for grouping samples.

Results:

After doxorubicin treatment, 79.8±13.3% of resistant cells survived. Silencing of PSMB7 in resistant cells decreased survival to 31.8±6.4% (P>0.001). A similar effect was observed after paclitaxel treatment. In 1592 microarray samples, the patients with high PSMB7 expression had a significantly shorter survival than the patients with low expression (P<0.001).

Conclusion:

Our findings suggest that high PSMB7 expression is an unfavourable prognostic marker in breast cancer.     

Effective treatment of chemoresistant breast cancer in vitro and in vivo by a factor VII-targeted photodynamic therapy

Background:

The purpose of this study was to test a novel, dual tumour vascular endothelial cell (VEC)- and tumour cell-targeting factor VII-targeted Sn(IV) chlorin e6 photodynamic therapy (fVII-tPDT) by targeting a receptor tissue factor (TF) as an alternative treatment for chemoresistant breast cancer using a multidrug resistant (MDR) breast cancer line MCF-7/MDR.

Methods:

The TF expression by the MCF-7/MDR breast cancer cells and tumour VECs in MCF-7/MDR tumours from mice was determined separately by flow cytometry and immunohistochemistry using anti-human or anti-murine TF antibodies. The efficacy of fVII-tPDT was tested in vitro and in vivo and was compared with non-targeted PDT for treatment of chemoresistant breast cancer. The in vitro efficacy was determined by a non-clonogenic assay using crystal violet staining for monolayers, and apoptosis and necrosis were assayed to elucidate the underlying mechanisms. The in vivo efficacy of fVII-tPDT was determined in a nude mouse model of subcutaneous MCF-7/MDR tumour xenograft by measuring tumour volume.

Results:

To our knowledge, this is the first presentation showing that TF was expressed on tumour VECs in chemoresistant breast tumours from mice. The in vitro efficacy of fVII-tPDT was 12-fold stronger than that of ntPDT for MCF-7/MDR cancer cells, and the mechanism of action involved induction of apoptosis and necrosis. Moreover, fVII-tPDT was effective and safe for the treatment of chemoresistant breast tumours in the nude mouse model.

Conclusions:

We conclude that fVII-tPDT is effective and safe for the treatment of chemoresistant breast cancer, presumably by simultaneously targeting both the tumour neovasculature and chemoresistant cancer cells. Thus, this dual-targeting fVII-tPDT could also have therapeutic potential for the treatment of other chemoresistant cancers.     

Prognostic importance of CDK4/6-specific activity as a predictive marker for recurrence in patients with endometrial cancer,with or without adjuvant chemotherapy

Background:

Pathologically low-risk endometrial cancer patients do not receive postoperative treatment; however, 10–15% of these patients show recurrence with poor prognosis. We evaluated the clinical importance of cyclin-dependent kinase 4/6 (CDK4/6) activity, and its significance as a novel biomarker for the prognosis and chemo-sensitivity of endometrioid endometrial carcinoma (EEC).

Methods:

Cyclin-dependent kinase 4/6 expression and enzyme activity in 109 tumour samples from patients with EEC were examined with a cell-cycle profiling (C2P) assay. CDK4/6-specific activity (CDK4/6SA) was determined, and its relationship with clinicopathological factors and expression of Ki-67 was analysed.

Results:

CDK4/6-specific activity was significantly correlated with Ki-67 (P=0.035), but not with any other clinicopathological characteristics. CDK4/6SA was significantly higher (P=0.002) in pathologically low-risk patients (not receiving adjuvant chemotherapy, n=74) than in intermediate- or high-risk patients (receiving adjuvant chemotherapy, n=35). In addition, patients with high CDK4/6SA (>3.0) showed significantly (P=0.024) shorter progression-free survival (PFS) than those with low CDK4/6SA (<3.0). Although Ki-67 expression itself was not a marker for prognosis, the combination of high CDK4/6SA and high Ki-67 expression (>15%) was robustly associated with shorter PFS (P=0.015), and this combination was an independent poor prognostic factor in the low-risk group. Inversely, in the intermediate-/high-risk group, patients with high CDK4/6SA had a tendency of a more favourable prognosis compared with patients with low CDK4/6SA (P=0.063).

Conclusions:

CDK4/6-specific activity can be used as a biomarker to predict prognosis and, possibly, chemo-sensitivity. The combination of Ki-67 expression might strengthen the clinical usefulness of CDK4/6SA as a biomarker.     

In vivo and in vitro properties of STX2484: a novel non-steroidal anti-cancer compound active in taxane-resistant breast cancer

Background:

STX2484 is a novel non-steroidal compound with potent anti-proliferative activity. These studies aimed to identify STX2484''s mechanism of action, in vivo efficacy and activity in taxane-resistant breast cancer models.

Methods:

Effects of STX2484 and paclitaxel on proliferation, cell cycle and apoptosis were assessed in vitro in drug-resistant (MCF-7_DOX) and non-resistant cells (MCF-7_WT). STX2484 efficacy in βIII tubulin overexpression in MCF-7 cells was also determined. Anti-angiogenic activity was quantified in vitro by a co-culture model and in vivo using a Matrigel plug assay. An MDA-MB-231 xenograft model was used to determine STX2484 efficacy in vivo.

Results:

STX2484 is a tubulin disruptor, which induces p53 expression, Bcl2 phosphorylation, caspase-3 cleavage, cell cycle arrest and apoptosis. In addition, STX2484 is a potent anti-angiogenic agent in vitro and in vivo. In breast cancer xenografts, STX2484 (20 mg kg⁻¹ p.o.) suppressed tumour growth by 84% after 35 days of daily dosing, with limited toxicity. In contrast to paclitaxel, STX2484 efficacy was unchanged in two clinically relevant drug-resistant models.

Conclusions:

STX2484 is an orally bioavailable microtubule-disrupting agent with in vivo anti-angiogenic activity and excellent in vivo efficacy with no apparent toxicity. Crucially, STX2484 has superior efficacy to paclitaxel in models of clinical drug resistance.     

Tumour hypoxia determines the potential of combining mTOR and autophagy inhibitors to treat mammary tumours

Background:

Hypoxia can activate autophagy, a self-digest adaptive process that maintains cell turnover. Mammalian target of rapamycin (mTOR) inhibitors are used to treat cancer but also stimulate autophagy.

Methods:

Human mammary cancer cells and derived xenografts were used to examine whether hypoxia could exacerbate autophagy-mediated resistance to the mTOR inhibitor rapamycin.

Results:

Rapamycin exerted potent antitumour effects in MCF-7 and MDA-MB-231 mammary tumours through a marked inhibition of angiogenesis, but the autophagy inhibitor chloroquine (CQ) failed to further sensitise tumours to mTOR inhibition. Rapamycin treatment actually led to tumour reoxygenation, thereby preventing the development of autophagy. Chloroquine alone, however, blocked the growth of MCF-7 tumours and in vitro blunted the hypoxia-induced component of autophagy in these cells. Finally, when initiating CQ treatment in large, hypoxic tumours, a robust antitumour effect could be observed, which also further increased the antiproliferative effects of rapamycin.

Conclusion:

The mTOR inhibitor rapamycin significantly contributes to tumour growth inhibition and normalisation of the tumour vasculature through potent antiangiogenic effects. The resulting reduction in hypoxia accounts for a lack of sensitisation by the autophagy inhibitor CQ, except if the tumours are already at an advanced stage, and thus largely hypoxic at the initiation of the combination of rapamycin and CQ treatment.     

MicroRNA-1246 expression associated with CCNG2-mediated chemoresistance and stemness in pancreatic cancer

Background:

Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance.

Methods:

We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining.

Results:

The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients.

Conclusions:

miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.     

Exogenous administration of protease-resistant,non-matrix-binding IGFBP-2 inhibits tumour growth in a murine model of breast cancer

Background:

Insulin-like growth factors (IGF-I and IGF-II) signal via the type 1 IGF receptor (IGF-1R) and IGF-II also activates the insulin receptor isoform A (IR-A). Signalling via both receptors promotes tumour growth, survival and metastasis. In some instances IGF-II action via the IR-A also promotes resistance to anti-IGF-1R inhibitors. This study assessed the efficacy of two novel modified IGF-binding protein-2 (IGFBP-2) proteins that were designed to sequester both IGFs. The two modified IGFBP-2 proteins were either protease resistant alone or also lacked the ability to bind extracellular matrix (ECM).

Methods:

The modified IGFBP-2 proteins were tested in vitro for their abilities to inhibit cancer cell proliferation and in vivo to inhibit MCF-7 breast tumour xenograft growth.

Results:

Both mutants retained low nanomolar affinity for IGF-I and IGF-II (0.8–2.1-fold lower than IGFBP-2) and inhibited cancer cell proliferation in vitro. However, the combined protease resistant, non-matrix-binding mutant was more effective in inhibiting MCF-7 tumour xenograft growth and led to inhibition of angiogenesis.

Conclusions:

By removing protease cleavage and matrix-binding sites, modified IGFBP-2 was effective in inhibiting tumour growth and reducing tumour angiogenesis.     

An RNAi-based screen reveals PLK1, CDK1 and NDC80 as potential therapeutic targets in malignant pleural mesothelioma

Background:

Malignant pleural mesothelioma (MPM) is an aggressive tumour originating in the thoracic mesothelium. Prognosis remains poor with 9- to 12-month median survival, and new targets for treatments are desperately needed.

Methods:

Utilising an RNA interference (RNAi)-based screen of 40 genes overexpressed in tumours, including genes involved in the control of cell cycle, DNA replication and repair, we investigated potential therapeutic targets for MPM. Following in vitro characterisation of the effects of target silencing on MPM cells, candidates were assessed in tumour samples from 154 patients.

Results:

Gene knockdown in MPM cell lines identified growth inhibition following knockdown of NDC80, CDK1 and PLK1. Target knockdown induced cell-cycle arrest and increased apoptosis. Using small-molecule inhibitors specific for these three proteins also led to growth inhibition of MPM cell lines, and Roscovitine (inhibitor of CDK1) sensitised cells to cisplatin. Protein expression was also measured in tumour samples, with markedly variable levels of CDK1 and PLK1 noted. PLK1 expression in over 10% of cells correlated significantly with a poor prognosis.

Conclusion:

These results suggest that RNAi-based screening has utility in identifying new targets for MPM, and that inhibition of NDC80, CDK1 and PLK1 may hold promise for treatment of this disease.     

Cell cycle genes and ovarian cancer susceptibility: a tagSNP analysis

Background:

Dysregulation of the cell cycle is a hallmark of many cancers including ovarian cancer, a leading cause of gynaecologic cancer mortality worldwide.

Methods:

We examined single nucleotide polymorphisms (SNPs) (n=288) from 39 cell cycle regulation genes, including cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors, in a two-stage study. White, non-Hispanic cases (n=829) and ovarian cancer-free controls (n=941) were genotyped using an Illumina assay.

Results:

Eleven variants in nine genes (ABL1, CCNB2, CDKN1A, CCND3, E2F2, CDK2, E2F3, CDC2, and CDK7) were associated with risk of ovarian cancer in at least one genetic model. Seven SNPs were then assessed in four additional studies with 1689 cases and 3398 controls. Association between risk of ovarian cancer and ABL1 rs2855192 found in the original population [odds ratio, OR_{BB vs AA} 2.81 (1.29–6.09), P=0.01] was also observed in a replication population, and the association remained suggestive in the combined analysis [OR_{BB vs AA} 1.59 (1.08–2.34), P=0.02]. No other SNP associations remained suggestive in the replication populations.

Conclusion:

ABL1 has been implicated in multiple processes including cell division, cell adhesion and cellular stress response. These results suggest that characterization of the function of genetic variation in this gene in other ovarian cancer populations is warranted.     

Mutation analysis of components of the Mediator kinase module in MED12 mutation-negative uterine leiomyomas

Background:

Kinase module of Mediator complex (‘CDK8 submodule'') consists of four subunits: CDK8, Cyclin C, MED12, and MED13. Recently, we reported recurrent MED12 mutations in 70% of uterine leiomyomas. The aim of this study was to analyse whether mutations in other components of the module contribute to the development of these lesions.

Methods:

Mutation screening of altogether 70 MED12 mutation-negative uterine leiomyomas was carried out by direct sequencing.

Results:

None of the tumours displayed somatic mutations in the coding regions of CDK8/CDK19, CCNC, or MED13.

Conclusions:

Mutations in CDK8/CDK19, CCNC, and MED13 do not frequently contribute to genesis of uterine leiomyomas.