Citations

Abstract Purpose: Inherited variability in the prognosis of lung cancer patients treated with platinum-based chemotherapy has been widely investigated. However, the overall contribution of genetic variation to platinum response is not well established. To identify novel candidate single nucleotide polymorphisms (SNP)/genes, we carried out a genome-wide association study (GWAS) for cisplatin cytotoxicity by using lymphoblastoid cell lines (LCL), followed by an association study of selected SNPs from the GWAS with overall survival (OS) in lung cancer patients. Experimental Design: A GWAS for cisplatin was conducted with 283 ethnically diverse LCLs. A total of 168 top SNPs were genotyped in 222 small cell lung cancer (SCLC) and 961 non-SCLC (NSCLC) patients treated with platinum-based therapy. Association of the SNPs with OS was determined by using the Cox regression model. Selected candidate genes were functionally validated by siRNA knockdown in human lung cancer cells. Results: Among 157 successfully genotyped SNPs, 9 and 10 SNPs were top SNPs associated with OS for patients with NSCLC and SCLC, respectively, although they were not significant after adjusting for multiple testing. Fifteen genes, including 7 located within 200 kb up or downstream of the 4 top SNPs and 8 genes for which expression was correlated with 3 SNPs in LCLs were selected for siRNA screening. Knockdown of DAPK3 and METTL6, for which expression levels were correlated with the rs11169748 and rs2440915 SNPs, significantly decreased cisplatin sensitivity in lung cancer cells. Conclusions: This series of clinical and complementary laboratory-based functional studies identified several candidate genes/SNPs that might help predict treatment outcomes for platinum-based therapy of lung cancer. Clin Cancer Res; 17(17); 5801–11. ©2011 AACR.

Adenosine monophosphate–activated protein kinase (AMPK) is a major energy sensor that maintains cellular energy homeostasis. Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of CAG repeats in the huntingtin (Htt) gene. In this paper, we report that activation of the α1 isoform of AMPK (AMPK-α1) occurred in striatal neurons of humans and mice with HD. Overactivation of AMPK in the striatum caused brain atrophy, facilitated neuronal loss, and increased formation of Htt aggregates in a transgenic mouse model (R6/2) of HD. Such nuclear accumulation of AMPK-α1 was activity dependent. Prevention of nuclear translocation or inactivation of AMPK-α1 ameliorated cell death and down-regulation of Bcl2 caused by mutant Htt (mHtt). Conversely, enhanced expression of Bcl2 protected striatal cells from the toxicity evoked by mHtt and AMPK overactivation. These data demonstrate that aberrant activation of AMPK-α1 in the nuclei of striatal cells represents a new toxic pathway induced by mHtt.

Rationale: Although a cardioprotective function of target of rapamycin (TOR) signaling inhibition has been suggested by pharmacological studies using rapamycin, genetic evidences are still lacking. We explored adult zebrafish as a novel vertebrate model for dissecting signaling pathways in cardiomyopathy. Objective: We generated the second adult zebrafish cardiomyopathy model induced by doxorubicin. By genetically analyzing both the doxorubicin and our previous established anemia-induced cardiomyopathy models, we decipher the functions of TOR signaling in cardiomyopathies of different etiology. Methods and Results: Along the progression of both cardiomyopathy models, we detected dynamic TOR activity at different stages of pathogenesis as well as distinct effects of TOR signaling inhibition. Nevertheless, cardiac enlargement in both models can be effectively attenuated by inhibition of TOR signaling through short-term rapamycin treatment. To assess the long-term effects of TOR reduction, we used a zebrafish target of rapamycin ( ztor ) mutant identified from an insertional mutagenesis screen. We show that TOR haploinsufficiency in the ztor heterozygous fish improved cardiac function, prevented pathological remodeling events, and ultimately reduced mortality in both adult fish models of cardiomyopathy. Mechanistically, these cardioprotective effects are conveyed by the antihypertrophy, antiapoptosis, and proautophagy function of TOR signaling inhibition. Conclusions: Our results prove adult zebrafish as a conserved novel vertebrate model for human cardiomyopathies. Moreover, we provide the first genetic evidence to demonstrate a long-term cardioprotective effect of TOR signaling inhibition on at least 2 cardiomyopathies of distinct etiology, despite dynamic TOR activities during their pathogenesis.

Regulatory T cells (T reg cells) are characterized by the expression of the forkhead lineage-specific transcription factor Foxp3, and their main function is to suppress T cells. While evaluating T reg cells, we identified a population of Foxp3-positive cells that were CD11b+F4/80+CD68+, indicating macrophage origin. These cells were observed in spleen, lymph nodes, bone marrow, thymus, liver, and other tissues of naive animals. To characterize this subpopulation of macrophages, we devised a strategy to purify CD11b+F4/80+Foxp3+ macrophages using Foxp3-GFP mice. Analysis of CD11b+F4/80+Foxp3+ macrophage function indicated that these cells inhibited the proliferation of T cells, whereas Foxp3− macrophages did not. Suppression of T cell proliferation was mediated through soluble factors. Foxp3− macrophages acquired Foxp3 expression after activation, which conferred inhibitory properties that were indistinguishable from natural Foxp3+ macrophages. The cytokine and transcriptional profiles of Foxp3+ macrophages were distinct from those of Foxp3− macrophages, indicating that these cells have different biological functions. Functional in vivo analyses indicated that CD11b+F4/80+Foxp3+ macrophages are important in tumor promotion and the induction of T reg cell conversion. For the first time, these studies demonstrate the existence of a distinct subpopulation of naturally occurring macrophage regulatory cells in which expression of Foxp3 correlates with suppressive function.

Purpose Preoperative aromatase inhibitor (AI) treatment promotes breast-conserving surgery (BCS) for estrogen receptor (ER) –positive breast cancer. To study this treatment option, responses to three AIs were compared in a randomized phase II neoadjuvant trial designed to select agents for phase III investigations. Patients and Methods Three hundred seventy-seven postmenopausal women with clinical stage II to III ER-positive (Allred score 6-8) breast cancer were randomly assigned to receive neoadjuvant exemestane, letrozole, or anastrozole. The primary end point was clinical response. Secondary end points included BCS, Ki67 proliferation marker changes, the Preoperative Endocrine Prognostic Index (PEPI), and PAM50-based intrinsic subtype analysis. Results On the basis of clinical response rates, letrozole and anastrozole were selected for further investigation; however, no other differences in surgical outcome, PEPI score, or Ki67 suppression were detected. The BCS rate for mastectomy-only patients at presentation was 51%. PAM50 analysis identified AI-unresponsive nonluminal subtypes (human epidermal growth factor receptor 2 enriched or basal-like) in 3.3% of patients. Clinical response and surgical outcomes were similar in luminal A (LumA) versus luminal B tumors; however, a PEPI of 0 (best prognostic group) was highest in the LumA subset (27.1% v 10.7%; P = .004). Conclusion Neoadjuvant AI treatment markedly improved surgical outcomes. Ki67 and PEPI data demonstrated that the three agents tested are biologically equivalent and therefore likely to have similar adjuvant activities. LumA tumors were more likely to have favorable biomarker characteristics after treatment; however, occasional paradoxical increases in Ki67 (12% of tumors with > 5% increase after therapy) suggest treatment-resistant cells, present in some LumA tumors, can be detected by post-treatment profiling.

AbstractJAK-STAT signaling is involved in the regulation of cell survival, proliferation, and differentiation. JAK tyrosine kinases can be transiently activated by cytokines or growth factors in normal cells, whereas they become constitutively activated as a result of mutations that affect their function in tumors. Specifically, the JAK2V617F mutation is present in the majority of patients with myeloproliferative disorders (MPDs) and is implicated in the pathogenesis of these diseases. In the present study, we report that the kinase CK2 is a novel interaction partner of JAKs and is essential for JAK-STAT activation. We demonstrate that cytokine-induced activation of JAKs and STATs and the expression of suppressor of cytokine signaling 3 (SOCS-3), a downstream target, are inhibited by CK2 small interfering RNAs or pharmacologic inhibitors. Endogenous CK2 is associated with JAK2 and JAK1 and phosphorylates JAK2 in vitro. To extend these findings, we demonstrate that CK2 interacts with JAK2V617F and that CK2 inhibitors suppress JAK2V617F autophosphorylation and downstream signaling in HEL92.1.7 cells (HEL) and primary cells from polycythemia vera (PV) patients. Furthermore, CK2 inhibitors potently induce apoptosis of HEL cells and PV cells. Our data provide evidence for novel cross-talk between CK2 and JAK-STAT signaling, with implications for therapeutic intervention in JAK2V617F-positive MPDs.

Abstract Background: Neoadjuvant endocrine therapy (NET) trials serve to inform design and selection of agents for larger adjuvant endocrine trials. The neoadjuvant aromatase inhibitor (AI) trial ACOSOG Z1031 randomized the treatment of postmenopausal women with ER rich (Allred 6-8) clinical stage 2/3 breast cancer to either EXE, ANA and LET. The results are therefore of interest in the context of new data that compares these AIs in the adjuvant setting. The opportunity to develop predictive biomarker tests for NET sensitivity was also a key study objective in order guide future selection of patients for inclusion in studies that compare NET versus neoadjuvant chemotherapy (NCT). The value of a baseline PAM50 intrinsic subtype test for NET patient selection was therefore an embedded objective of the study. Methods: Of the 374 women who began study treatment on Z1031, pretreatment biopsy specimen for central IHC testing for ER and Ki67 was available on 304 patients. The PAM50 intrinsic subtype test (PMCID: 2667820) could be conducted on 170 baseline samples. The Preoperative Endocrine Prognostic Index (PEPI) an approach to predicting long term outcomes based on pathological stage and surgical specimen ER and Ki67 status was also conducted because patients with a PEPI of zero (T1/2 N0, ER+, Ki67<=2.7%) have been found to have an excellent long term prognosis and therefore unlikely to benefit from chemotherapy (PMCID: 255670). Results: The degree of Ki67 suppression, the absolute level of Ki67 in the surgical specimen and the PEPI results were not significantly different by treatment arm (Kruskal-Wallis tests p >0.05). Thirty Percent (95%CI: 19.6-42.9%) of women with pre-AI LumA subtype and 12% (95%CI: 6.4-20.0%) of women with pre-AI LumB subtype had a PEPI of zero predicting excellent prognosis. Multivariate logistic modeling found that the likelihood of a PEPI score of zero is 3.2 fold higher (95%CI: 1.43 7.12) among 66 women with a pre-AI LumA subtype compared to 100 women with a pre-AI LumB subtype. Additional baseline variables (clinical T and N stage, HER2 status, pre-AI Ki-67 level (< = 10% vs. >10%) were not found to modify the chance of a PEPI of zero. Baseline PAM50 Subtype versus PEPI Conclusion: The results of Z1031 show no differences in clinical or biologic response between LET, EXE, and ANA in the neoadjuvant setting; predicting that no difference in efficacy will be seen between these agents in adjuvant studies. Pre-AI LumA versus LumB status, defined by the PAM50 subtype, identifies patients who are more likely to be in the most favorable PEPI score following neoadjuvant AI therapy. Patients with pretreatment LumA status may, therefore, not be suitable for a randomized trial of NET versus NCT since approximately one third of these patients may be able to avoid chemotherapy altogether. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S1-2.

Abstract Defective microRNA (miRNA) biogenesis contributes to the development and progression of epithelial ovarian cancer (EOC). In this study, we examined the hypothesis that single nucleotide polymorphisms (SNP) in miRNA biogenesis genes may influence EOC risk. In an initial investigation, 318 SNPs in 18 genes were evaluated among 1,815 EOC cases and 1,900 controls, followed up by a replicative joint meta-analysis of data from an additional 2,172 cases and 3,052 controls. Of 23 SNPs from 9 genes associated with risk (empirical P < 0.05) in the initial investigation, the meta-analysis replicated 6 SNPs from the DROSHA, FMR1, LIN28, and LIN28B genes, including rs12194974 (G>A), an SNP in a putative transcription factor binding site in the LIN28B promoter region (summary OR = 0.90, 95% CI: 0.82–0.98; P = 0.015) which has been recently implicated in age of menarche and other phenotypes. Consistent with reports that LIN28B overexpression in EOC contributes to tumorigenesis by repressing tumor suppressor let-7 expression, we provide data from luciferase reporter assays and quantitative RT-PCR to suggest that the inverse association among rs12194974 A allele carriers may be because of reduced LIN28B expression. Our findings suggest that variants in LIN28B and possibly other miRNA biogenesis genes may influence EOC susceptibility. Cancer Res; 71(11); 3896–903. ©2011 AACR.

Abstract The mitochondrial transcription factor A (TFAM) is required for mitochondrial DNA (mtDNA) replication and transcription. Disruption of TFAM results in heart failure and premature aging in mice. But very little is known about the role of TFAM in cancer development. Here, we report the identification of frequent frameshift mutations in the coding mononucleotide repeat of TFAM in sporadic colorectal cancer (CRC) cell lines and in primary tumors with microsatellite instability (MSI), but not in microsatellite stable (MSS) CRC cell lines and tumors. The presence of the TFAM truncating mutation, in CRC cells with MSI, reduced the TFAM protein level in vivo and in vitro and correlated with mtDNA depletion. Furthermore, forced overexpression of wild-type TFAM in RKO cells carrying a TFAM truncating mutation suppressed cell proliferation and inhibited RKO cell-induced xenograft tumor growth. Moreover, these cells showed more susceptibility to cisplatin-induced apoptosis due to an increase of cytochrome b (Cyt b) expression and its release from mitochondria. An interaction assay between TFAM and the heavy-strand promoter (HSP) of mitochondria revealed that mutant TFAM exhibited reduced binding to HSP, leading to reduction in Cyt b transcription. Collectively, these data provide evidence that a high incidence of TFAM truncating mutations leads to mitochondrial copy number reduction and mitochondrial instability, distinguishing most CRC with MSI from MSS CRC. These mutations may play an important role in tumorigenesis and cisplatin-induced apoptotic resistance of most microsatellite-unstable CRCs. Cancer Res; 71(8); 2978–87. ©2011 AACR.