Expression and function of ABCG2 and XIAP in glioblastomas.

Despite multimodal treatment that includes surgery, radiation and chemotherapy, virtually all glioblastomas (GBM) recur, indicating that these interventions are insufficient to eradicate all malignant cells. To identify potential new therapeutic targets in GBMs, we examined the expression and function of proteins that are associated with therapy resistance and cancer cell survival. We measured the expression of eight such proteins in 50 GBM samples by immunohistochemistry and analyzed patient survival. We report that GBM patients with high expression of ABCG2 (also called BCRP) or XIAP at the protein level had worse survival than those with low expression. The adjusted hazard ratio for ABCG2 was 2.35 and for XIAP was 2.65. Since glioma stem cells (GSCs) have been shown to be more resistant than bulk tumor cells to anti-cancer therapies and to express high levels of these proteins, we also sought to determine if ABCG2 and XIAP have functional roles in GSCs. We used small molecule inhibitors to treat patient-derived GBM tumorspheres in vitro and observed that inhibitors of ABCG2, Ko143 and fumitremorgin, significantly reduced self-renewal. These results suggest that ABCG2 and XIAP proteins may be useful indicators of patient survival and that inhibition of ABCG2 may be a promising therapeutic strategy in GBMs.

Use and Yield of Baseline Imaging and Laboratory Testing in Stage II Breast Cancer.

BACKGROUND: Despite guideline recommendations, baseline laboratory testing and advanced imaging are widely ordered in clinical practice to stage asymptomatic patients with clinical stage II breast cancer (BC). MATERIALS AND METHODS: A retrospective study at two academic centers in Boston, Massachusetts, between 2006 and 2007 explored the use, results, and implications of laboratory tests, tumor markers, and imaging in patients with clinical stage II BC. RESULTS: Among 411 patients, 233 (57%) had liver function testing, 134 (33%) had tumor marker tests, and 237 (58%) had computed tomography (CT) as part of their initial diagnostic workup. Median age was 52 (range, 23-90 years). On multivariable analysis, young age, more advanced stage, and tumor subtype (human epidermal growth receptor-positive [HER2+] and triple-negative breast cancer [TNBC]) were significantly associated with baseline CT. The rate of detection of true metastatic disease with use of baseline staging imaging was 2.1% (95% confidence interval, 0.7%-5%). It was 2.2% (3 of 135) for estrogen receptor/progesterone receptor-positive disease, 1.9% (1 of 54) for HER2+ disease, and 2.1% (1 of 48) for TNBC. At 5 years of follow-up, 46 of 406 patients were diagnosed with metastatic breast cancer. Thirty-four of 46 (73.9%) who developed recurrent disease had imaging at their initial diagnosis, and of these, five had abnormalities on their initial imaging that was correlated with where they developed metastatic disease. CONCLUSION: In this cohort of women with stage II BC, staging imaging at diagnosis had a low yield in detecting distant metastases (2.1%). The detection rate was not higher with HER2+ disease or TNBC, despite the trend that patients with these subtypes were more likely to undergo imaging. IMPLICATIONS FOR PRACTICE: Despite guideline recommendations, asymptomatic patients with stage II breast cancer (BC) often undergo staging imaging with computed tomography, bone scanning, or positron emission tomography. Physicians have often reported that they order imaging despite recommendations because they believe that younger patients or patients with more aggressive BC phenotypes, such as human epidermal receptor 2-positive BC or triple-negative BC, benefit from staging imaging. In this cohort of women younger than those in prior studies, the yield of detecting distant metastatic disease in patients with clinical stage II BC was very low and the detection rate was not higher in the presence of HER2-positive or triple-negative BC.

Frequency of mutations in individuals with breast cancer referred for BRCA1 and BRCA2 testing using next-generation sequencing with a 25-gene panel.

BACKGROUND: Next-generation sequencing (NGS) allows for simultaneous sequencing of multiple cancer susceptibility genes and, for an individual, may be more efficient and less expensive than sequential testing. The authors assessed the frequency of deleterious germline mutations among individuals with breast cancer who were referred for BRCA1 and BRCA2 (BRCA1/2) gene testing using a panel of 25 genes associated with inherited cancer predisposition. METHODS: This was a cross-sectional study using NGS in 2158 individuals, including 1781 who were referred for commercial BRCA1/2 gene testing (cohort 1) and 377 who had detailed personal and family history and had previously tested negative for BRCA1/2 mutations (cohort 2). RESULTS: Mutations were identified in 16 genes, most frequently in BRCA1, BRCA2, CHEK2, ATM, and PALB2. Among the participants in cohort 1, 9.3% carried a BRCA1/2 mutation, 3.9% carried a mutation in another breast/ovarian cancer susceptibility gene, and 0.3% carried an incidental mutation in another cancer susceptibility gene unrelated to breast or ovarian cancer. In cohort 2, the frequency of mutations in breast/ovarian-associated genes other than BRCA1/2 was 2.9%, and an additional 0.8% had an incidental mutation. In cohort 1, Lynch syndrome-related mutations were identified in 7 individuals. In contrast to BRCA1/2 mutations, neither age at breast cancer diagnosis nor family history of ovarian or young breast cancer predicted for other mutations. The frequency of mutations in genes other than BRCA1/2 was lower in Ashkenazi Jews compared with non-Ashkenazi individuals (P=.026). CONCLUSIONS: Using an NGS 25-gene panel, the frequency of mutations in genes other than BRCA1/2 was 4.3%, and most mutations (3.9%) were identified in genes associated with breast/ovarian cancer.

Safety and outcome of patients treated with a modified outpatient intraperitoneal regimen for epithelial ovarian, primary peritoneal or fallopian tube cancer.

BACKGROUND: Despite the survival benefit of intraperitoneal (IP) chemotherapy observed in GOG172, significant toxicity and poor treatment completion rates have prevented the widespread acceptance of this regimen. Here, we report our experience with a modified outpatient GOG172 regimen. METHODS: Eligible patients had stage III, optimally debulked epithelial ovarian, fallopian tube or primary peritoneal cancer that underwent IP port placement for administration of a modified GOG172 regimen consisting of: (i) intravenous paclitaxel 135 mg/m² on day 1 over 3 h; (ii) intraperitoneal cisplatin 75 mg/m² on day 2, and (iii) intraperitoneal paclitaxel 60 mg/m² on day 8. Day 8 IP paclitaxel was omitted until tolerance of the first cycle of IP cisplatin had been established. RESULTS: Four or more cycles of IP chemotherapy were completed by 72.5% (29) of 40 eligible patients; 20% of patients exhibited catheter-related complications requiring port removal and discontinuation of IP chemotherapy. Grade 3-4 hematologic, metabolic and gastrointestinal toxicities occurred in 36, 8 and 21% of the patients, respectively. With a median follow-up of 47.7 months, progression-free and overall survival was comparable to GOG172. CONCLUSIONS: This modified outpatient GOG172 regimen is associated with less toxicity and improved completion rates compared to the original GOG172 regimen.

mTOR inhibitors in renal cell carcinoma.

The mammalian target of rapamycin (mTOR) is a downstream effector of the PI3-K/Akt/mTOR pathway. Allosteric inhibitors of mTOR, everolimus and temsirolimus, have shown promising clinical activity in advanced renal cell carcinoma but their effect is far from durable and only a subset of patients experience substantial benefit from these agents. The PI3-K/Akt/mTOR pathway represents an intricate network of fine regulation and feedback loops, and resistance to allosteric mTOR inhibitors may be embedded within this complexity. In this article we highlight the molecular elements of the PI3-K/Akt/mTOR pathway, the clinical experience with everolimus and temsirolimus in advanced renal cell carcinoma, and the future directions in terms of sequential therapy, combinational therapy and development of novel therapeutic agents.

Response to gefitinib and erlotinib in Non-small cell lung cancer: a restrospective study.

BACKGROUND: In Non-small cell lung cancer (NSCLC), an overactive epidermal growth factor receptor (EGFR) pathway is a component of the malignant phenotype. Two tyrosine kinase inhibitors (TKIs) of EGFR, gefinitib and erlotinib, have been used with variable benefit. METHODS: We have analyzed outcome data of a population of NSCLC patients that received these TKIs to determine the benefit derived and to define the clinical and molecular parameters that correlate with response. Tumor tissue from a subgroup of these patients was analyzed by immunohistochemistry to measure the expression level of EGFR and four activated (phosphorylated) members of the pathway, pEGFR, pERK, pAKT, and pSTAT3. RESULTS: Erlotinib was slightly superior to gefitinib in all measures of response, although the differences were not statistically significant. The most robust clinical predictors of time to progression (TTP) were best response and rash (p < 0.0001). A higher level of pEGFR was associated with longer TTP, while the total EGFR level was not associated with response. Higher levels of pAKT and pSTAT3 were also associated with longer TTP. In contrast, a higher level of pERK1/2 was associated with shorter TTP. CONCLUSION: These observations suggest the hypothesis that tumor cells that have activated EGFR pathways, presumably being utilized for survival, are clinically relevant targets for pathway inhibition. An accurate molecular predictive model of TKI response should include activated members of the EGFR pathway. TKIs may be best reserved for tumors expressing pEGFR and pAKT or pSTAT, and little pERK. In the absence of molecular predictors of response, the appearance of a rash and a positive first scan are good clinical indicators of response.

NRAGE mediates p38 activation and neural progenitor apoptosis via the bone morphogenetic protein signaling cascade.

Understanding the molecular events that govern neural progenitor lineage commitment, mitotic arrest, and differentiation into functional progeny are germane to our understanding of neocortical development. Members of the family of bone morphogenetic proteins (BMPs) play pivotal roles in regulating neural differentiation and apoptosis during neurogenesis through combined actions involving Smad and TAK1 activation. We demonstrate that BMP signaling is required for the induction of apoptosis of neural progenitors and that NRAGE is a mandatory component of the signaling cascade. NRAGE possesses the ability to bind and function with the TAK1-TAB1-XIAP complex facilitating the activation of p38. Disruption of NRAGE or any other member of the noncanonical signaling cascaded is sufficient to block p38 activation and thus the proapoptotic signals generated through BMP exposure. The function of NRAGE is independent of Smad signaling, but the introduction of a dominant-negative Smad5 also rescues neural progenitor apoptosis, suggesting that both canonical and noncanonical pathways can converge and regulate BMP-mediated apoptosis. Collectively, these results establish NRAGE as an integral component in BMP signaling and clarify its role during neural progenitor development.

Targeted BMI1 inhibition impairs tumor growth in lung adenocarcinomas with low CEBPα expression.

Lung cancer is the most common cause of cancer deaths. The expression of the transcription factor C/EBPα (CCAAT/enhancer binding protein α) is frequently lost in non-small cell lung cancer, but the mechanisms by which C/EBPα suppresses tumor formation are not fully understood. In addition, no pharmacological therapy is available to specifically target C/EBPα expression. We discovered a subset of pulmonary adenocarcinoma patients in whom negative/low C/EBPα expression and positive expression of the oncogenic protein BMI1 (B lymphoma Mo-MLV insertion region 1 homolog) have prognostic value. We also generated a lung-specific mouse model of C/EBPα deletion that develops lung adenocarcinomas, which are prevented by Bmi1 haploinsufficiency. BMI1 activity is required for both tumor initiation and maintenance in the C/EBPα-null background, and pharmacological inhibition of BMI1 exhibits antitumor effects in both murine and human adenocarcinoma lines. Overall, we show that C/EBPα is a tumor suppressor in lung cancer and that BMI1 is required for the oncogenic process downstream of C/EBPα loss. Therefore, anti-BMI1 pharmacological inhibition may offer a therapeutic benefit for lung cancer patients with low expression of C/EBPα and high BMI1.

Expression of vascular endothelial growth factor, mitogen-activated protein kinase and p53 in human colorectal cancer.

BACKGROUND: VEGF is a growth factor involved in the regulation of angiogenesis, a process that plays a central role in tumor growth. It has been suggested that mutations of p53 and activation of the Ras/MAPK pathway may contribute to the up-regulation of VEGF expression and induction of angiogenesis. PATIENTS AND METHODS: We explored the expression of p53 and VEGF and p44MAPK phosphorylation in 43 human colorectal carcinomas, as well as in peritumoral mucosas, and in normal mucosas in order to establish a correlation between VEGF expression and either mutations of p53 or phosphorylation of p44MAPK. Overexpression of p53 in tumor tissues was interpreted as evidence of mutations. RESULTS: p53 was overexpressed in 22 out of 43 tumors; MAPK was phosphorylated in 25 out of 43 cases whereas only 4 out of 22 peritumoral mucosas showed a moderate phosphorylation of p44MAPK VEGF was up-regulated in 22 out of 43 tumors, moderately expressed in 4 out of 22 peritumoral mucosas and not detectable in normal mucosa. Immunohistochemical analysis showed the presence of a phosphorylated form of p44 MAPK only in neoplastic cells. Statistical analysis demonstrated a significant correlation between p53 and VEGF expression (p<0.03) as well as between VEGF expression and p44 MAPK phosphorylation (p<0.002). CONCLUSION: These data suggest that mutations of p53 and activation of the Ras/MAPK pathway may play a role in the induction of VEGF expression in human colorectal cancer.

Sublethal concentrations of 17-AAG suppress homologous recombination DNA repair and enhance sensitivity to carboplatin and olaparib in HR proficient ovarian cancer cells.

The promise of PARP-inhibitors(PARPis) in the management of epithelial ovarian cancer(EOC) is tempered by the fact that approximately 50% of patients with homologous recombination (HR)-proficient tumors do not respond well to these agents. Combination of PARPis with agents that inhibit HR may represent an effective strategy to enhance their activity in HR-proficient tumors. Using a bioinformatics approach, we identified that heat shock protein 90 inhibitors(HSP90i) may suppress HR and thus revert HR-proficient to HR-deficient tumors. Analysis of publicly available gene expression data showed that exposure of HR-proficient breast cancer cell lines to HSP90i 17-AAG(17-allylamino-17-demethoxygeldanamycin) downregulated HR, ATM and Fanconi Anemia pathways. In HR-proficient EOC cells, 17-AAG suppressed HR as assessed using the RAD51 foci formation assay and this was further confirmed using the Direct Repeat-GFP reporter assay. Furthermore, 17-AAG downregulated BRCA1 and/or RAD51 protein levels, and induced significantly more γH2AX activation in combination with olaparib compared to olaparib alone. Finally, sublethal concentrations of 17-AAG sensitized HR-proficient EOC lines to olaparib and carboplatin but did not affect sensitivity of the HR-deficient OVCAR8 line arguing that the 17-AAG mediated sensitization is dependent on suppression of HR. These results provide a preclinical rationale for using a combination of olaparib/17-AAG in HR-proficient EOC.

The RNA-binding protein Musashi-1 regulates neural development through the translational repression of p21WAF-1.

RNA-binding proteins regulate cell fate decisions during nervous system development. The Msi family of RNA-binding proteins is expressed in multipotential neural progenitors, and is required for maintaining cells in a proliferative state. We demonstrate that Msi-1's ability to regulate progenitor maintenance is through the translational inhibition of the cyclin-dependent kinase inhibitor p21WAF-1. Msi-1 ectopic expression increases the proliferation rate and the capacity to regulate p21WAF-1 protein expression, independent of p53. The 3' untranslated region (UTR) of the native p21(WAF-1) mRNA contains a Msi-1 consensus-binding site that permits Msi-1 to directly repress the translation of p21WAF-1 protein. Reduction of Msi-1 through antisense leads to aberrant p21WAF-1 expression, which significantly impairs neural differentiation. A double knockdown for p21WAF-1 and Msi-1 rescues the production of mature MAP+ neurons. Our results further elucidate the symbiotic relationship between cell cycle withdrawal and the onset of differentiation in the developing nervous system, as well as increasing the understanding of the influence that RNA-binding proteins serve in regulating these processes.

Notch activation suppresses fibroblast growth factor-dependent cellular transformation.

Aberrant activations of the Notch and fibroblast growth factor receptor (FGFR) signaling pathways have been correlated with neoplastic growth in humans and other mammals. Here we report that the suppression of Notch signaling in NIH 3T3 cells by the expression of either the extracellular domain of the Notch ligand Jagged1 or dominant-negative forms of Notch1 and Notch2 results in the appearance of an exaggerated fibroblast growth factor (FGF)-dependent transformed phenotype characterized by anchorage-independent growth in soft agar. Anchorage-independent growth exhibited by Notch-repressed NIH 3T3 cells may result from prolonged FGFR stimulation caused by both an increase in the expression of prototypic and oncogenic FGF gene family members and the nonclassical export of FGF1 into the extracellular compartment. Interestingly, FGF exerts a negative effect on Notch by suppressing CSL (CBF-1/RBP-Jk/KBF2 in mammals, Su(H) in Drosophila and Xenopus, and Lag-2 in Caenorhabditis elegans)-dependent transcription, and the ectopic expression of constitutively active forms of Notch1 or Notch2 abrogates FGF1 release and the phenotypic effects of FGFR stimulation. These data suggest that communication between the Notch and FGFR pathways may represent an important reciprocal autoregulatory mechanism for the regulation of normal cell growth.