Cancer Therapeutic Response Research Articles

Mcl-1 Mediates TWEAK/Fn14-Induced Non–Small Cell Lung Cancer Survival and Therapeutic Response

Insensitivity to standard clinical interventions, including chemotherapy, radiotherapy, and tyrosine kinase inhibitor (TKI) treatment, remains a substantial hindrance towards improving the prognosis of patients with non-small cell lung cancer (NSCLC). The molecular mechanism of therapeutic resistance remains poorly understood. The TNF-like weak inducer of apoptosis (TWEAK)-FGF-inducible 14 (TNFRSF12A/Fn14) signaling axis is known to promote cancer cell survival via NF-κB activation and the upregulation of prosurvival Bcl-2 family members. Here, a role was determined for TWEAK-Fn14 prosurvival signaling in NSCLC through the upregulation of myeloid cell leukemia sequence 1 (MCL1/Mcl-1). Mcl-1 expression significantly correlated with Fn14 expression, advanced NSCLC tumor stage, and poor patient prognosis in human primary NSCLC tumors. TWEAK stimulation of NSCLC cells induced NF-κB-dependent Mcl-1 protein expression and conferred Mcl-1-dependent chemo- and radioresistance. Depletion of Mcl-1 via siRNA or pharmacologic inhibition of Mcl-1, using EU-5148, sensitized TWEAK-treated NSCLC cells to cisplatin- or radiation-mediated inhibition of cell survival. Moreover, EU-5148 inhibited cell survival across a panel of NSCLC cell lines. In contrast, inhibition of Bcl-2/Bcl-xL function had minimal effect on suppressing TWEAK-induced cell survival. Collectively, these results position TWEAK-Fn14 signaling through Mcl-1 as a significant mechanism for NSCLC tumor cell survival and open new therapeutic avenues to abrogate the high mortality rate seen in NSCLC. The TWEAK-Fn14 signaling axis enhances lung cancer cell survival and therapeutic resistance through Mcl-1, positioning both TWEAK-Fn14 and Mcl-1 as therapeutic opportunities in lung cancer.

MicroRNAs in cancer therapeutic response: Friend and foe.

Cancer initiation and development engage extremely complicated pathological processes which involve alterations of a large number of cell signaling cascades and functional networks in temporal and spatial orders. During last decades, microRNAs (miRNAs), a class of non-coding RNAs, have emerged as critical players in cancer pathogenesis and progression by modulating many pathological aspects related to tumor development, growth, metastasis, and drug resistance. The major function of miRNAs is to post-transcriptionally regulate gene expression depending on recognition of complementary sequence residing in target mRNAs. Commonly, a particular miRNA recognition sequence could be found in a number of genes, which allows a single miRNA to regulate multiple functionally connected genes simultaneously and/or chronologically. Furthermore, a single gene can be targeted and regulated by multiple miRNAs. However, previous studies have demonstrated that miRNA functions are highly context-dependent, which leads to distinct pathological outcomes in different types of cancer as well as at different stages by alteration of the same miRNA. Here we summarize recent progress in studies on miRNA function in cancer initiation, metastasis and therapeutic response, focusing on breast cancer. The varying functions of miRNAs and potential application of using miRNAs as biomarkers as well as therapeutic approaches are further discussed in the context of different cancers.

Genetic Variants in the Fibroblast Growth Factor Pathway as Potential Markers of Ovarian Cancer Risk, Therapeutic Response, and Clinical Outcome

The fibroblast growth factor (FGF) and FGF receptor (FGFR) axis plays a critical role in tumorigenesis, but little is known of its influence in ovarian cancer. We sought to determine the association of genetic variants in the FGF pathway with risk, therapeutic response, and survival of patients with ovarian cancer. We matched 339 non-Hispanic white ovarian cancer cases with 349 healthy controls and genotyped them for 183 single-nucleotide polymorphisms (SNPs) from 24 FGF (fibroblast growth factor) and FGFR (fibroblast growth factor receptor) genes. Genetic associations for the main effect, gene-gene interactions, and the cumulative effect were determined. Multiple SNPs in the FGF-FGFR axis were associated with an increased risk of ovarian cancer. In particular, FGF1 [fibroblast growth factor 1 (acidic)] SNP rs7727832 showed the most significant association with ovarian cancer (odds ratio, 2.27; 95% CI, 1.31-3.95). Ten SNPs were associated with a reduced risk of ovarian cancer. FGF18 (fibroblast growth factor 18) SNP rs3806929, FGF7 (fibroblast growth factor 7) SNP rs9920722, FGF23 (fibroblast growth factor 23) SNP rs12812339, and FGF5 (fibroblast growth factor 5) SNP rs3733336 were significantly associated with a favorable treatment response, with a reduction of risk of nonresponse of 40% to 60%. Eleven SNPs were significantly associated with overall survival. Of these SNPs, FGF23 rs7961824 was the most significantly associated with improved prognosis (hazard ratio, 0.55; 95% CI, 0.39-0.78) and was associated with significantly longer survival durations, compared with individuals with the common genotype at this locus (58.1 months vs. 38.0 months, P = 0.005). Survival tree analysis revealed FGF2 rs167428 as the primary factor contributing to overall survival. Significant associations of genetic variants in the FGF pathway were associated with ovarian cancer risk, therapeutic response, and survival. The discovery of multiple SNPs in the FGF-FGFR pathway provides a molecular approach for risk assessment, monitoring therapeutic response, and prognosis.

Imaging VCAM-1 as an Indicator of Treatment Efficacy in Metastatic Ovarian Cancer

The inability to successfully treat women with ovarian cancer is due in large part to the advanced stage of disease at diagnosis, the development of platinum resistance, and the lack of sensitive methods to monitor tumor progression and response to treatment. Vascular cell adhesion molecule-1 (VCAM-1) is expressed on the mesothelium of ovarian cancer patients. We investigated VCAM-1 expression as a marker of peritoneal metastasis and tumor response to platinum-based chemotherapy. Peritoneal or omental biopsies obtained from women diagnosed with stage I, stage II, or stage III/IV ovarian cancer were evaluated by immunohistochemistry. The effects of carboplatin on mesothelial VCAM-1 expression were determined in cultured cells by Western blot. Radiolabeled VCAM-1-specific peptide imaging probes and SPECT were used in a mouse model of ovarian cancer peritoneal metastasis to identify VCAM-1 as a viable imaging target. VCAM-1 expression correlated with tumor stage. All specimens from stage I patients were negative, whereas 29% of stage II patients and 73% of stage III/IV patients were positive. Although most women with advanced stage disease expressed VCAM-1, the incidence of expression was reduced among women who received neoadjuvant chemotherapy, suggesting a role for chemotherapy in regulating VCAM-1 expression. Treatment of mesothelial cells in culture with carboplatin resulted in a transient decrease in VCAM-1 expression 4 h after treatment that returned to baseline within 16-24 h. In vivo imaging of VCAM-1 also demonstrated an acute decrease in expression 4 h after carboplatin administration that recovered within 48 h in mice harboring platinum-resistant tumors. Chronic VCAM-1 expression reflected the effect of platinum-based treatment on tumor burden. Specifically, carboplatin treatment of mice with platinum-sensitive tumors showed reduced VCAM-1 expression, which correlated with reduced tumor burden; mice with platinum-resistant tumors retained elevated VCAM-1 expression and tumor burden after treatment. Clinically relevant VCAM-1-specific imaging probes identify VCAM-1 expression as an indicator of ovarian cancer peritoneal metastasis and therapeutic response to platinum-based agents. These observations support testing the utility of VCAM-1 imaging probes to monitor treatment response in ovarian cancer patients, thus providing the potential to improve management of women with this disease.

Optical Detection and Virotherapy of Live Metastatic Tumor Cells in Body Fluids with Vaccinia Strains

Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV). In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs) in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease.

Abstract 1376: Exercise alters breast cancer phenotype through distinct reductions in host-derived proinflammatory growth factor ligands.

Abstract It is becoming increasingly recognized that host (systemic) and tumor cell release of proinflammatory growth factors are critical determinants of cancer progression and therapeutic response in patients with solid tumors. Thus, strategies that can attenuate systemic and/or tumor production of proinflammatory growth factors may offer an effective approach to improve therapeutic outcomes following a cancer diagnosis. The present study tests the central hypothesis that exercise modulates systemic levels of key growth factor ligands that, in turn, inhibit the activity of critical downstream cell signaling pathways to effectively inhibit tumor progression. To address this question, we took advantage of sera collected from a clinical trial examining the efficacy of supervised exercise training, relative to sedentary control, in patients with early or advanced solid tumors. Multiplex ELISA analysis showed that exercising patients (N=23) had significant reductions in circulating concentrations of interleukin (IL)-4, MIP1-β (macrophage inflammatory protein-1β), vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-α), and hepatocyte growth factor (HGF) in comparison with patients randomized to sedentary control (N=21). Exposure of estrogen receptor positive (ER+) and Triple-Negative (ER-/PR-/HER2-) distinct human breast cancer cell lines (MCF-7 and MDA-MB-231) to serum from exercised breast cancer patients led to marked alterations in cellular phenotype as shown by increases in proliferation, migration, and apoptosis, compared with exposure to serum from control patients. In vitro ‘add-back’ experiments using recombinant growth factors in concentrations consistent with that observed in the clinical trial, revealed that HGF produced similar alterations in tumor proliferation and apoptosis as that observed with serum from exercising patients. Co-culturing of human breast cancer cells with exercise serum and a neutralizing antibody against HGF, led to increases in proliferation and decreases in apoptosis in comparison to exercise serum alone. These results suggest that growth factor ligand deprivation may play a critical role in mediating the effects of exercise on tumor cellular phenotype. As such, our findings may provide initial insight into the potential mechanisms underlying recent observations showing higher levels of exercise correlate with more favorable disease outcomes in early breast cancer patients. More generally, this study indicates the widespread potential of exercise to modulate growth factor-driven signaling and by extension, tumor progression and possibly innate or acquired resistance to therapy. Citation Format: Oliver Glass, Brant A. Inman, Kerry S. Courneya, John R. Mackey, Erik Nelson, Zachary Hartman, Lee W. Jones. Exercise alters breast cancer phenotype through distinct reductions in host-derived proinflammatory growth factor ligands. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1376. doi:10.1158/1538-7445.AM2013-1376

Molecular Pathways: Toll-like Receptors in the Tumor Microenvironment—Poor Prognosis or New Therapeutic Opportunity

Numerous reports have described Toll-like receptor (TLR) expression in the tumor microenvironment as it relates to cancer progression, as well as their involvement in inflammation. While TLRs mediate immune surveillance, clinical studies have associated TLR expression in the tumor with poor patient survival, indicating that TLR expression may affect cancer treatment and survival. This review will examine mechanisms in which TLR activation upregulates protumorigenic pathways, including the induction of inducible nitric oxide synthase (iNOS2) and COX2, which in turn increase TLR expression and promote a feed-forward loop leading to tumor progression and the development of more aggressive tumor phenotypes. These propagating loops involve cancer cell, stroma, and/or immune cell TLR expression. Because of abundant TLR expression in many human tumors, several TLR agonists are now in clinical and preclinical trials and some have shown enhanced efficacy when used as adjuvant with radiation, chemotherapy, or cancer vaccines. These findings suggest that TLR expression influences cancer biology and therapeutic response, which may involve specific interactions within the tumor microenvironment, including mediators of inflammation such as nitric oxide and the arachidonic acid signaling pathways.

Calcium-permeable ion channels in control of autophagy and cancer

Autophagy, or cellular self-eating, is a tightly regulated cellular pathway the main purpose of which is lysosomal degradation and subsequent recycling of cytoplasmic material to maintain normal cellular homeostasis. Defects in autophagy are linked to a variety of pathological states, including cancer. Cancer is the disease associated with abnormal tissue growth following an alteration in such fundamental cellular processes as apoptosis, proliferation, differentiation, migration and autophagy. The role of autophagy in cancer is complex, as it can promote both tumor prevention and survival/treatment resistance. It's now clear that modulation of autophagy has a great potential in cancer diagnosis and treatment. Recent findings identified intracellular calcium as an important regulator of both basal and induced autophagy. Calcium is a ubiquitous secondary messenger which regulates plethora of physiological and pathological processes such as aging, neurodegeneration and cancer. The role of calcium and calcium-permeable channels in cancer is well-established, whereas the information about molecular nature of channels regulating autophagy and the mechanisms of this regulation is still limited. Here we review existing mechanisms of autophagy regulation by calcium and calcium-permeable ion channels. Furthermore, we will also discuss some calcium-permeable channels as the potential new candidates for autophagy regulation. Finally we will propose the possible link between calcium permeable channels, autophagy and cancer progression and therapeutic response.

Hormonally responsive breast cancer cells in a microfluidic co-culture model as a sensor of microenvironmental activity.

Breast cancer cell growth and therapeutic response are manipulated extrinsically by microenvironment signals. Despite recognition of the importance of the microenvironment in a variety of tumor processes, predictive measures that incorporate the activity of the surrounding cellular environment are lacking. In contrast, tumor cell biomarkers are well established in the clinic. Expression of Estrogen Receptor-alpha (ERα) is the primary defining feature of hormonally responsive tumors and is the molecular target of therapy in the most commonly diagnosed molecular subtype of breast cancer. While a number of soluble factors have been implicated in ERα activation, the complexity of signaling between the cellular microenvironment and the cancer cell implies multivariate control. The cumulative impact of the microenvironment signaling, which we define as microenvironmental activity, is more difficult to predict than the sum of its parts. Here we tested the impact of an array of microenvironments on ERα signaling utilizing a microfluidic co-culture model. Quantitative immunofluorescence was employed to assess changes in ERα protein levels, combined with gene expression and phosphorylation status, as measures of activation. Analysis of microenvironment-induced growth under the same conditions revealed a previously undescribed correlation between growth and ERα protein down-regulation. These data suggest an expanded utility for the tumor biomarker ERα, in which the combination of dynamic regulation of ERα protein and growth in a breast cancer biosensor cell become a read-out of the microenvironmental activity.

Pregnane xenobiotic receptor in cancer pathogenesis and therapeutic response

Pregnane xenobiotic receptor (PXR) is an orphan nuclear receptor that regulates the metabolism of endobiotics and xenobiotics. PXR is promiscuous and unique in that it is activated by a diverse group of xenochemicals, including therapeutic anticancer drugs and naturally-occurring endocrine disruptors. PXR has been predominantly studied to understand its regulatory role in xenobiotic clearance in liver and intestine via induction of drug metabolizing enzymes and drug transporters. PXR, however, is widely expressed and has functional implications in other normal and malignant tissues, including breast, prostate, ovary, endometrium and bone. The differential expression of PXR and its target genes in cancer tissues has been suggested to determine the prognosis of chemotherapeutic outcome. In addition, the emerging evidence points to the implications of PXR in regulating apoptotic and antiapoptotic as well as growth factor signaling that promote tumor proliferation and metastasis. In this review, we highlight the recent progress made in understanding the role of PXR in cancer, discuss the future directions to further understand the mechanistic role of PXR in cancer, and conclude with the need to identify novel selective PXR modulators.

18F]Fluorodeoxyglucose Positron Emission Tomography–Computed Tomography in Breast Cancer: When… and When Not?

Diagnostic imaging plays an important role in the care of patients with breast cancer and is used for breast cancer detection, diagnosis, staging, and therapeutic response evaluation. Advances in imaging technology, especially relatively newer technologies such as magnetic resonance imaging (MRI) and positron emission tomography without or with integrated computed tomography (PET or PET/CT) provide powerful diagnostic tools, but also generate questions and controversy regarding where and when to use these new modalities. Early studies of new imaging approaches most often come from centers that are scientifically invested in the development of the technology. Typically, small and highly selected patient populations are evaluated, and these preliminary studies often indicate excellent diagnostic performance. Later on, when large multicenter trials are performed with more clinically representative patient populations, the new test invariably performs less well. Nevertheless, the excitement surrounding early results from a new imaging technology and the increasing public access to these early results fuel the desire by patients for access to new forms of diagnostic imaging. Physicians and patients share the wish for diagnostic certainty in excluding cancer spread at the time of diagnosis and may reason that a purely diagnostic procedure, even if untested, can do no harm. These factors, combined with the challenge and expense of carrying out larger scale diagnostic imaging trials, can lead to the early adoption of new imaging studies into clinical practice, often before their performance and optimal use have been fully determined. However, the use of a new imaging technology in populations of patients unlikely to benefit from its use can add anxiety, expense, unnecessary procedures, and delay in care. Equally important, inappropriate use in the wrong patient population can obfuscate benefit in other clinical settings where the imaging might be helpful. These dilemmas have been aptly demonstrated in the application of [F]fluorodeoxyglucose (FDG) PET and PET/CT to early breast cancer staging. Early studies of FDG PET for breast cancer axillary staging (reviewed in Eubank et al) demonstrated high sensitivity and specificity (85%–90% or better for both) and created considerable enthusiasm for FDG PET as a noninvasive method for detecting axillary nodal metastases. However, these studies were heavily biased towards patients with advanced disease, often with larger and more aggressive cancers in which the disease burden in the lymph nodes was larger and better suited for detection by FDG PET compared with early-stage disease. A subsequent multicenter trial cast doubt on these early studies, and more recent single-center trials performed in the era of sentinel lymph node mapping showed that, compared with sentinel lymph node biopsy, the sensitivity of FDG PET and PET/CT for axillary nodal metastases was as low as 20%–40%. In the article that accompanies this editorial, Pritchard et al add to this body of evidence. In a prospective, four-center study of 325 patients in Ontario with stages I and II breast cancer and clinically negative axilla who underwent FDG PET and PET/CT for axillary nodal staging, the authors found nearly 100% specificity, but sensitivity of only 24%, for FDG PET. In addition, in 13 patients who were suspected of having distant metastases by FDG PET, 10 had false-positive findings, and only three were confirmed to have stage IV disease by biopsy or clinical follow-up. Although there were some minor limitations to the study, including a lack of central PET scan review and the inclusion of some patients scanned with older technology, the Pritchard study confidently shows both FDG PET/CT’s limited sensitivity for axillary nodal metastases and limited yield for distant disease in early-stage breast cancer. The results for distant metastasis detection echo earlier studies of systemic staging using CT and bone scan that showed a low prevalence of distant metastases (5% or less in stage I breast cancer) and a notable number of false-positive findings. Although there have been some exceptions, the majority of recent studies and systematic reviews are in agreement with the results of the Pritchard study and suggest a low diagnostic yield for FDG PET/CT in patients with stage I and early stage II breast cancer. This evidence underlies the strong recommendation in the current National Comprehensive Cancer Network (NCCN) consensus guidelines that systemic staging, including FDG PET/CT, is not indicated for early-stage breast cancer in the absence of signs or symptoms suggesting metastasis. Despite existing evidence and guidelines, there is anecdotal evidence that the use of imaging for systemic staging in early-stage breast cancer in the medical community is quite variable. Recent evidence from the ADVICE consortium in the Puget Sound region shows two trends relevant to the work of Pritchard et al. First, the trend in use of JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L S VOLUME 30 NUMBER 12 APRIL 2

BPA and PXR Activation: Human Receptor Is Affected, Mouse Receptor Is Not

Vol. 120, No. 3 News | Science SelectionsOpen AccessBPA and PXR Activation: Human Receptor Is Affected, Mouse Receptor Is Notis accompanied byBisphenol A and Its Analogues Activate Human Pregnane X Receptor Julia R. Barrett Julia R. Barrett Search for more papers by this author Published:1 March 2012https://doi.org/10.1289/ehp.120-a122aCited by:2AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit The high-production-volume chemical bisphenol A (BPA) is a key component in polycarbonate plastics and is used in a wide variety of consumer products. The U.S. general population is widely exposed to BPA, as demonstrated by the chemical’s presence in more than 90% of biological samples tested. BPA has been associated with cardiovascular disease and diabetes in human population studies, but if causality were to be proven, it is doubtful it would arise from BPA’s known weak interaction with estrogen receptors. Researchers previously determined that BPA targets another nuclear receptor, the pregnane X receptor (PXR), and have now determined that the chemical and several related compounds (“analogues”) can induce metabolically important genes via PXR activation [EHP 120(3):399–405; Sui et al.].PXR is activated by numerous endogenous and environmental chemicals. Earlier research established that PXR activation in mice and rats induces genes involved in lipid homeostasis, atherosclerosis, and carcinogenesis. Although PXR is not affected by BPA in rodents, the chemical activates the human receptor and induces target gene expression. This connection suggested a possible mechanism to explain BPA-related cardiovascular disease findings in human populations.In the current study, transfection assays using mouse- and human-derived receptors showed that BPA induces strong, dose-dependent activation of human PXR but not mouse PXR. Computational docking and modeling studies focusing on the structure of human PXR identified key amino acids within the ligand-binding domain that permit BPA to interact with the receptor. These studies also highlighted how species-related differences in this domain can affect how different species respond to specific chemicals.The modeled predictions were confirmed in vitro using site-directed mutagenesis assays that focused on the amino acids critical for BPA docking within PXR. Additionally, several BPA structural analogues were found to activate the PXR. Experiments to determine interactions between BPA and specific analogues showed synergism when BPA was paired with 2-(4'-hydroxyphenyl)-2-phenylpropane (HPP); that is, the effect of the mixture exceeded the sum of the individual chemicals. In a final set of experiments, the ability of BPA and analogues HPP and bisphenol B to separately trigger expression of PXR target genes was tested in a human intestinal cell line. All 3 compounds were found to trigger expression of the metabolic genes CYP3A4, UGT1A1, and MDR1.Although this study did not address questions of risk assessment, its findings suggest that BPA may adversely affect human health via PXR and thus provide a foundation for further exploration of the observed association between BPA and cardiovascular disease. Additionally, given the likelihood that real-life exposures involve chemical mixtures, the demonstrated synergism between BPA and HPP supports the need to include mixtures in in vivo experiments.FiguresReferencesRelatedDetailsCited by Dutta M, Lim J and Cui J (2021) Pregnane X Receptor and the Gut-Liver Axis: A Recent Update, Drug Metabolism and Disposition, 10.1124/dmd.121.000415, 50:4, (478-491), Online publication date: 1-Apr-2022. Pondugula S and Mani S (2013) Pregnane xenobiotic receptor in cancer pathogenesis and therapeutic response, Cancer Letters, 10.1016/j.canlet.2012.08.030, 328:1, (1-9), Online publication date: 1-Jan-2013. Related articlesBisphenol A and Its Analogues Activate Human Pregnane X Receptor3 January 2012Environmental Health Perspectives Vol. 120, No. 3 March 2012Metrics About Article Metrics Publication History Originally published1 March 2012Published in print1 March 2012 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.

Abstract C36: Transcriptome sequencing identifies PCAT-1, a novel lncRNA implicated in prostate cancer progression and therapeutic response to PARP inhibitors

Abstract High-throughput sequencing of polyA+ RNA (RNA-Seq) in human cancer shows remarkable potential to identify both novel disease-specific markers for clinical uses and uncharacterized aspects of tumor biology, particularly long non-coding RNA (lncRNA) species >250 bp in length. To illustrate this approach, we employed RNA-Seq on a cohort of 102 prostate tissues and cell lines and performed ab initio transcriptome assembly to discover unannotated ncRNAs with cancer-specific expression patterns. In total, we observed that ∼20% of the prostate cancer transcriptome represents unannotated, polyadenylated RNA species that were enriched for activate chromatin marks (H3K4me3, RNA polymerase II) defined by ChIP-Seq data. From 1,859 intergenic lncRNAs overall, we nominated 121 such Prostate Cancer Associated Transcripts (PCATs) that collectively performed as well as known biomarkers in differentiating benign, cancerous, and metastatic tissues. Among these, we identified an uncharacterized two-exon, polyadenylated lncRNA, PCAT-1, as a prostate cancer outlier and a prostate-specific driver of cancer cell proliferation. Mechanistically, microarray profiling following modulation of PCAT-1 expression in vitro defined a core set of genes repressed by PCAT-1, identifying this lncRNA as a transcriptional repressor of genes implicated in DNA maintenance processes, such as CENPF and BRCA2. Interestingly, when PCAT-1 expression is low, it is negatively regulated by the Polycomb Repressive Complex 2 (PRC2), which directly binds the PCAT-1 promoter in vitro. Supporting this, human tumor samples with high PCAT-1 or high PRC2 expression were exclusive and defined distinct molecular subtypes distinguished by gene expression signatures of PCAT-1-repressed target genes. PRC2-based repression of PCAT-1 was reversible by inhibition of PRC2. BRCA2 inactivation is known to sensitize cancer cells to small molecular inhibitors of the PARP1 DNA repair enzyme by impairing homologous recombation of double-stranded DNA breaks, suggesting that PCAT-1 expression may sensitize prostate cells to PARP1 inhibitors. To test this, we generated two isogenic overexpression models of PCAT-1, and one stable knockdown model, in prostate cell lines. PCAT-1-overexpressing cell lines (Du145 and RWPE) showed a marked increase in sensitivity to two PARP inhibitors, whereas the stable knockdown cells (LNCaP) showed decreased sensitivity to PARP inhibitors. This was accompanied by a decrease in RAD51 foci formation, a hallmark of DNA repair via homologous recombination, in PCAT-1 overexpressing cells, and an increase in RAD51 foci formation in PCAT-1 knockdown cells. MCF7 breast cancer cells overexpressing PCAT-1 did not recapitulate these phenotypes, supporting our observation that PCAT-1 is a prostate-specific gene. These data define PCAT-1 as a novel prostate cancer-associated lncRNA functionally involved in tumor cell proliferation and sensitivity to PARP inhibitor therapy by suppressing homologous recombination. High PCAT-1 expression may therefore represent a potential biomarker for patient response to PARP targeted therapy. The findings presented herein establish the utility of RNA-Seq to comprehensively identify disease-associated lncRNAs and suggest that clinical translation of these findings may improve the stratification of cancer subtypes. Citation Format: John R. Prensner, Hari K. Iyer, Christopher A. Maher, Felix Feng, Arul M. Chinnaiyan, Matthew K. Iyer, Wei Chen, O. Alejandro Balbin, Saravana M. Dhanasekaran, Qi Cao, Xuhong Cao, Xiaojun Jing, Daniel Robinson. Transcriptome sequencing identifies PCAT-1, a novel lncRNA implicated in prostate cancer progression and therapeutic response to PARP inhibitors [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C36.

Genetic Polymorphisms Underlying Lung Cancer Susceptibility and Therapeutic Response

Recent advances in genome analysis technologies have provided a detailed genome-wide view of cancerous and non-cancerous cells. Lung cancer is largely caused by tobacco smoking, but several studies have implicated inherited genetic factors in disease etiology. Genome-wide association studies (GWASs) using DNA chips have identiˆed loci/genes with polymorphisms that underlie inter-individual diSerences in cancer susceptibility, including single nucleotide polymorphisms (SNPs). CHRNA (cholinergic receptor, nicotinic, alpha), TERT (telomerase reverse transcriptase) and TP63 (tumor protein p63) loci have been linked to lung cancer susceptibility by GWASs. SNPs in TERT and TP63 are preferentially associated with the risk of adenocarcinoma, the commonest histological type of lung cancer aSecting both smokers and non-smokers, whereas those in CHRNA are associated with lung cancer risk irrespective of histological type. An association of functional polymorphisms in DNA repair/metabolic genes with the risk of squamous cell carcinoma, a major histological type developed in smokers, has been suggested, but it remains inconclusive. It was also suggested that an SNP in the TP53 tumor suppressor gene in‰uences the response to platinum-doublet chemotherapy in lung cancer patients. However, analyses have shown that only a subset of SNPs is involved in lung carcinogenesis/therapy. Further GWASs are needed to translate the information on genetic variations into cancer prevention and clinical practice by focusing on speciˆc subtypes of lung cancers or therapeutic responses.

Prostate cancer – a biomarker perspective

Despite early detection and reduced risk of death, prostate cancer still remains the second leading cause of cancer death in American men. There is currently no cure for advanced prostate cancer. The multistage, stochastic and highly heterogeneous nature of prostate cancer, coupled with genetic and epigenetic alterations that occur during disease progression and response to therapy, represent fundamental challenges in our quest to understand and control this complex and prevalent disease. Recent advances in drug development and breakthroughs in omics technologies have renewed our efforts to identify novel biomarkers for prostate cancer prognosis, prediction, and therapeutic response monitoring. In this perspective article, we overview the current status and highlight future prospects of biomarkers for prostate cancer, a disease that affects millions of men worldwide.

Will Chinese ovarian cancer patients benefit from knowing the BRCA2 mutation status?

In Western countries, the mutation status of the BRCA1 and BRCA2 genes is commonly determined for genetic counseling among members of families with a history of breast or ovarian cancer, especially for women of the Ashkenazi Jewish ethnicity. Recent studies in the Cancer Genome Atlas project have demonstrated that BRCA2 mutation carriers are more responsive to platinum-based chemotherapy among high-grade serous ovarian cancer patients. Thus, in Western countries, the mutation status of BRCA1 and BRCA2 is recognized to have an important value with which to assess cancer risk and therapeutic response. However, very limited studies of BRCA1 and BRCA2 mutations and their implications for counseling and therapeutic prediction have been conducted in China. Therefore, a potentially important genetic test that is technically simple has not benefited Chinese women with an increased risk of breast or ovarian cancer. This article summarizes the current progress in the study of BRCA1/2 mutation in China and recommends an increased effort in applying advances in genetic testing to the clinical management of Chinese patients with ovarian cancer.

Between-Patient and Within-Patient (Site-to-Site) Variability in Estrogen Receptor Binding, Measured In Vivo by 18F-Fluoroestradiol PET

Heterogeneity of estrogen receptor (ER) expression may be an important predictor of breast cancer therapeutic response. (18)F-fluoroestradiol PET produces in vivo quantitative measurements of regional estrogen binding in breast cancer tumors. We describe within-patient (site-to-site) and between-patient heterogeneity of lesions in patients scheduled to receive endocrine therapy. In 91 patients with a prior ER-positive biopsy, 505 lesions were analyzed for both (18)F-fluoroestradiol and (18)F-FDG uptake and the (18)F-fluoroestradiol/(18)F-FDG uptake ratio. Standardized uptake values (SUVs) were recorded for up to 16 lesions per patient, of 1.5 cm or more and visible on (18)F-FDG PET or conventional staging. Linear mixed-effects regression models examined associations between PET parameters and patient or lesion characteristics and estimated variance components. A reader study of SUV measurements for 9 scans further examined sources of within-patient variability. Average (18)F-fluoroestradiol uptake and (18)F-fluoroestradiol/(18)F-FDG ratio varied greatly across these patients, despite a history of ER-positive disease: about 37% had low or absent (18)F-fluoroestradiol uptake even with marked (18)F-FDG uptake. (18)F-fluoroestradiol SUV and (18)F-fluoroestradiol/(18)F-FDG ratio measurements within patients with multiple lesions were clustered around the patient's average value in most cases. Summarizing these findings, the intraclass correlation coefficient (proportion of total variation that is between-patient) was 0.60 (95% confidence interval, 0.50-0.69) for (18)F-fluoroestradiol SUV and 0.65 (95% confidence interval, 0.56-0.73) for the (18)F-fluoroestradiol/(18)F-FDG ratio. Some within-patient variation in PET measures (22%-44%) was attributable to interobserver variability as measured by the reader study. A subset of patients had mixed uptake, with widely disparate (18)F-fluoroestradiol SUV or (18)F-fluoroestradiol/(18)F-FDG ratio for lesions in the same scan. (18)F-fluoroestradiol uptake and the (18)F-fluoroestradiol/(18)F-FDG ratio varied greatly between patients but were usually consistent across lesions in the same scan. The average (18)F-fluoroestradiol SUV and (18)F-fluoroestradiol/(18)F-FDG ratio for a limited sample of lesions appear to provide a reasonable summary of synchronous ER expression for most patients. However, imaging the entire disease burden remains important to identify the subset of patients with mixed uptake, who may be at a critical point in their disease evolution.

A database of reaction monitoring mass spectrometry assays for elucidating therapeutic response in cancer

The Quantitative Assay Database (QuAD), http://proteome.moffitt.org/QUAD/, facilitates widespread implementation of quantitative mass spectrometry in cancer biology and clinical research through sharing of methods and reagents for monitoring protein expression and modification. Liquid chromatography coupled to multiple reaction monitoring (LC-MRM) mass spectrometry assays are developed using SDS-PAGE fractionated lysates from cancer cell lines. Pathway maps created using GeneGO Metacore provide the biological relationships between proteins and illustrate concepts for multiplexed analysis; each protein can be selected to examine assay development at the protein and peptide levels. The coupling of SDS-PAGE and multiple reaction monitoring mass spectrometry screening has been used to detect 876 peptides from 218 cancer-related proteins in model systems including colon, lung, melanoma, leukemias, and myeloma, which has led to the development of 95 quantitative assays including stable-isotope-labeled peptide standards. Methods are published online and peptide standards are made available to the research community. Protein expression measurements for heat shock proteins, including a comparison with ELISA and monitoring response to the HSP90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), are used to illustrate the components of the QuAD and its potential utility. This resource enables quantitative assessment of protein components of signaling pathways and biological processes and holds promise for systematic investigation of treatment responses in cancer.

Abstract 2982: Impact of the RB-pathway in breast cancer heterogeneity and therapeutic response

Abstract Molecular heterogeneity in breast cancer is associated with differential prognosis and response to therapy. However, in spite of extensive molecular classification, pathways that can be specifically targeted to understand the basis of disease and improve treatment remain poorly understood within specific subtypes of breast cancer. The retinoblastoma tumor suppressor (RB) pathway is deregulated in most cancers; however varied mechanisms of inactivation have significant impacts on tumor biology. Here novel mechanistic and preclinical models were combined with analyses of tumor specimens to decipher the differential impact of the RB-pathway within breast cancer subtypes and as a specific target for patient stratification and therapeutic targeting. Initial gene expression datasets encompassing over 2000 breast cancer patient samples were used to demonstrate a striking dichotomy in the mechanisms of RB functional inactivation within subtypes of disease. In ER-positive breast cancer, disruption of RB-pathway function was associated with poor prognosis and failure of endocrine therapy. Functional studies demonstrate that such therapeutic failure largely occurs in the presence of an intact Rb gene, as a result of deregulated mitogenic signaling. In such models of hormone refractory disease we demonstrate that pharmacological activation of RB is a potent second-line therapy. In contrast, in ER-negative tumor populations RB inactivation occurs in 40-50% of cases. This event yields complete loss of RB protein, as occurs through genetic and epigenetic mechanisms. Strikingly, in ER-negative disease RB loss is associated with improved response to chemotherapy and longer relapse-free survival. The basis for improved response involves both deregulation of cell cycle checkpoints and apoptotic processes. Ongoing analyses are using synthetic-lethal and combination-therapy approaches to rationally target RB-status in the management of breast cancer. In total, these studies define a clear role for RB-pathway as a determinant of tumor heterogeneity in breast cancer that can be leveraged to more effectively treat breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2982. doi:10.1158/1538-7445.AM2011-2982

Abstract 440: Characterization and comprehensive proteomic analysis of exosomes derived from prostate cancer cell lines

Abstract Background: Exosomes are extracellular nano-vesicles which are secreted upon fusion of multivesicular bodies with the plasma membrane and present in most body fluids. They have been shown to be derived from a multitude of cell types and, while it is postulated that they are important for membrane trafficking as communication vesicles, their relevance in cancer and specifically prostate tumour growth and progression has yet to be determined. Factors such as stage of cancer, cell type and cell cycle could affect the amount and composition of exosomes formed and secreted. While the mechanisms underlying exosome formation and secretion are not fully understood, characterization of exosomes derived from different androgen dependent and independent prostate cancer cell lines (LNCaP, C4-2, VCaP, DU-145, PC-3) versus normal epithelial prostate cell line (RWPE-1) indicate novel information about their possible role in prostate cancer. In Addition proteomic analysis of exosomes derived from different prostate cancer cell lines reflects differential protein content within these entities and could be considered for prognostic potential. Other studies on tumour-related microvesicles suggest the role of these selectively enriched exosomes in cancer progression therefore the presence of differential protein markers in and on prostate cancer cell derived exosomes may yield potential biomarkers for cancer diagnosis and therapeutic response. In this study we characterized exosomes derived from different prostate cancer cells and explain the role of specific proteins present within exosomes which are involved in prostate cancer progression. Methods and Results: Exosomes were purified from the conditioned media(CM) from a panel of five prostate cancer cells and ‘normal’ RWPE-1 cells after 72 hours serum free media treatment. Using differential centrifugation cell debris and protein aggregates were removed from CM. Finally exosomes were isolated in a 30% sucrose cushion using ultracentrifugation. Further analysis using transmission electron microscopy detected the integrity of purified exosomes. Western blot analysis was also used to probe for different exosome markers including Actin, HSP70, HSP90, Rab5, CD9 and CD63. Our proteomic data shows that exosomes derived from specific prostate cancer cells present potential markers of prostate cancer progression. Conclusion: This study highlights a potential for differential protein composition of exosomes as a source of diagnostic biomarkers in addition to therapeutic targets for prostate cancer via non-invasive testing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 440. doi:10.1158/1538-7445.AM2011-440