Development Of Therapy Resistance Research Articles

Immuno-PET Imaging of HER3 in a Model in which HER3 Signaling Plays a Critical Role

HER3 is overexpressed in various carcinomas including colorectal cancer (CRC), which is associated with poor prognosis, and is involved in the development of therapy resistance. Thus, an in vivo imaging technique is needed to evaluate the expression of HER3, an important therapeutic and diagnostic target. Here, we report successful HER3 PET imaging using a newly generated anti-human HER3 monoclonal antibody, Mab#58, and a mouse model of a HER3-overexpressing xenograft tumor. Furthermore, we assessed the role of HER3 signaling in CRC cancer tissue-originated spheroid (CTOS) and applied HER3 imaging to detect endogenous HER3 in CTOS-derived xenografts. Cell binding assays of 89Zr-labeled Mab#58 using the HER3-overexpressing cell line HER3/RH7777 demonstrated that [89Zr]Mab#58 specifically bound to HER3/RH7777 cells (Kd = 2.7 nM). In vivo biodistribution study in mice bearing HER3/RH7777 and its parent cell xenografts showed that tumor accumulation of [89Zr]Mab#58 in HER3/RH7777 xenografts was significantly higher than that in the control from day 1 to day 4, tending to increase from day 1 to day 4 and reaching 12.2 ± 4.5%ID/g. Radioactivity in other tissues, including the control xenograft, decreased or remained unchanged from day 1 to day 6. Positron emission tomography (PET) in the same model enabled clear visualization of HER3/RH7777 xenografts but not of RH7777 xenografts. CTOS growth assay and signaling assay revealed that CRC CTOS were dependent on HER3 signaling for their growth. In PET studies of mice bearing a CRC CTOS xenograft, the tumor was clearly visualized with [89Zr]Mab#58 but not with the 89Zr-labeled control antibody. Thus, tumor expression of HER3 was successfully visualized by PET with 89Zr-labeled anti-HER3 antibody in CTOS xenograft-bearing mice, a model that retains the properties of the patient tumor. Non-invasive targeting of HER3 by antibodies is feasible, and it is expected to be useful for cancer diagnosis and treatment.

Production, Purification, and Characterization of ¹⁵N-Labeled DNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements.

Oxidatively induced DNA damage is caused in living organisms by a variety of damaging agents, resulting in the formation of a multiplicity of lesions, which are mutagenic and cytotoxic. Unless repaired by DNA repair mechanisms before DNA replication, DNA lesions can lead to genomic instability, which is one of the hallmarks of cancer. Oxidatively induced DNA damage is mainly repaired by base excision repair pathway with the involvement of a plethora of proteins. Cancer tissues develop greater DNA repair capacity than normal tissues by overexpressing DNA repair proteins. Increased DNA repair in tumors that removes DNA lesions generated by therapeutic agents before they became toxic is a major mechanism in the development of therapy resistance. Evidence suggests that DNA repair capacity may be a predictive biomarker of patient response. Thus, knowledge of DNA-protein expressions in disease-free and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. Our laboratory has developed methodologies that use mass spectrometry with isotope dilution for the measurement of expression of DNA repair proteins in human tissues and cultured cells. For this purpose, full-length (15)N-labeled analogs of a number of human DNA repair proteins have been produced and purified to be used as internal standards for positive identification and accurate quantification. This chapter describes in detail the protocols of this work. The use of (15)N-labeled proteins as internal standards for the measurement of several DNA repair proteins in vivo is also presented.

Detailed imaging and genetic analysis reveal a secondary BRAF(L505H) resistance mutation and extensive intrapatient heterogeneity in metastatic BRAF mutant melanoma patients treated with vemurafenib.

Resistance to treatment is the main problem of targeted treatment for cancer. We followed ten patients during treatment with vemurafenib, by three-dimensional imaging. In all patients, only a subset of lesions progressed. Next-generation DNA sequencing was performed on sequential biopsies in four patients to uncover mechanisms of resistance. In two patients, we identified mutations that explained resistance to vemurafenib; one of these patients had a secondary BRAF L505H mutation. This is the first observation of a secondary BRAF mutation in a vemurafenib-resistant patient-derived melanoma sample, which confirms the potential importance of the BRAF L505H mutation in the development of therapy resistance. Moreover, this study hints toward an important role for tumor heterogeneity in determining the outcome of targeted treatments.

Epithelial Plasticity in Cancer: Unmasking a MicroRNA Network for TGF-β-, Notch-, and Wnt-Mediated EMT.

Epithelial-to-mesenchymal transition (EMT) is a reversible process by which cancer cells can switch from a sessile epithelial phenotype to an invasive mesenchymal state. EMT enables tumor cells to become invasive, intravasate, survive in the circulation, extravasate, and colonize distant sites. Paracrine heterotypic stroma-derived signals as well as paracrine homotypic or autocrine signals can mediate oncogenic EMT and contribute to the acquisition of stem/progenitor cell properties, expansion of cancer stem cells, development of therapy resistance, and often lethal metastatic disease. EMT is regulated by a variety of stimuli that trigger specific intracellular signalling pathways. Altered microRNA (miR) expression and perturbed signalling pathways have been associated with epithelial plasticity, including oncogenic EMT. In this review we analyse and describe the interaction between experimentally validated miRs and their target genes in TGF-β, Notch, and Wnt signalling pathways. Interestingly, in this process, we identified a “signature” of 30 experimentally validated miRs and a cluster of validated target genes that seem to mediate the cross talk between TGF-β, Notch, and Wnt signalling networks during EMT and reinforce their connection to the regulation of epithelial plasticity in health and disease.

Causes of Chromosomal Instability.

The majority of human cancer cells are highly aneuploid harboring chromosome numbers deviating from the modal number of 46. In cancer, aneuploidy is a consequence of an increased rate of whole chromosome missegregation during mitosis, a process known as chromosomal instability (CIN). In fact, CIN is a hallmark of human cancer and is thought to contribute to tumorigenesis, tumor progression, and the development of therapy resistance by providing a high genetic variability that might foster rapid adaptation processes. However, the molecular mechanisms that cause chromosome missegregation in cancer cells are still poorly understood. So far, several mechanisms underlying CIN have been proposed and some of them are indeed detectable in human cancer cells exhibiting CIN. Examples include, for instance, weakened spindle checkpoint signaling, supernumerary centrosomes, defects in chromatid cohesion, abnormal kinetochore-microtubule attachments and increased spindle microtubule dynamics. Here, the mechanisms leading to CIN in human cancer cells are summarized.

Development of an Ultra-Deep Sequencing Method on Ion Torrent PGM to Detect Mutations in a Panel of Genes Relevant to Disease Progression and Drug Resistance in Chronic Lymphocytic Leukaemia

Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia in the Western world. The disease is characterised by a great variability in clinical course and response to treatment. Acquired somatic mutations in genes related to multiple signal pathways have recently emerged as important factors contributing to this heterogeneity. These mutations accumulate with disease progression or development of therapy resistance. However, with conventional methods they are very difficult to be detected in a single test, particularly when the size of the affected cell population is small. Therefore, there is a clinical need for a rapid, reliable, affordable and highly sensitive sequencing method to monitor these mutations from an early stage of this disease.Herein we developed an ultra-deep next generation sequencing approach based on Ion Torrent PGM to sequence a total of 246 exons of 15 genes including TP53, ATM, BIRC3, NOTCH1, SF3B1, MYD88, LRP1B, SAMHD1, FBXW7, POT1, HISTIH1E, XPO1, CHD2, PCLO and ZFPM2. These genes were selected because mutations in them have been reported to involve at least 5% of CLL patients and associate with poor outcome of CLL. Serial CLL samples were collected at multiple time points from diagnosis to disease progression/drug resistance from a cohort of 33 patients. Initially, target DNA in samples taken at advanced disease stages, without or with mutations in TP53 as detected and quantified by FASAY assay and Sanger sequencing, was enriched and amplified using Agilent HaloPlex probes, with 4604 amplicons for a sequenceable size of 135.91 Kbp of each sample. On each Ion 318 Chip (1 GB output), barcoded DNA enrichment preparations from 4 patients were sequenced by the PGM. For confirmation, the same patient samples were tested repeatedly in an independent experiment starting from DNA preparation. The sequence data were aligned to a human reference genome (Hg19); variants were called by the Torrent Variant Caller (v4.0-6) and visualised with the IGV. Results showed that the target coverage was 99.999% and an average coverage depth 3941x. Importantly, all of the known TP53 mutations were repeatedly detected at expected frequencies. In addition, extra mutations at lower levels (5% - 20% alleles) in multiple genes, including TP53 and SF3B1, were detected.In a sensitivity test of this method, DNA samples from 5 cases, each with a known % of mutation in TP53measured by FASAY assay and Sanger sequencing, were pooled together and then mixed with a wild-type DNA sample to serially dilute these mutations to 20% - 0.2% alleles before target enrichment. With an average coverage depth of 1843x (range: 1610 - 2187) for these 5 target locations, each mutation was readily detected at 20%, 5% and 1%, with the average quality scores being 1497.3, 61.2 and 29.4, respectively. However, only 3 out the 5 mutations could be detected at the level of 0.2%.Taken together, we have successfully developed a sensitive next generation sequencing method for detecting mutations in a CLL gene panel. This allows us to monitor multiple mutations affecting as low as 1% of alleles in CLL samples throughout the disease course and possibly discover clinically useful biomarker(s) for CLL progression and resistance to therapy. DisclosuresNo relevant conflicts of interest to declare.

Quantitative Profiling of Chromatome Dynamics Reveals a Novel Role for HP1BP3 in Hypoxia-induced Oncogenesis

In contrast to the intensely studied genetic and epigenetic changes that induce host cell transformation to initiate tumor development, those that promote the malignant progression of cancer remain poorly defined. As emerging evidence suggests that the hypoxic tumor microenvironment could re-model the chromatin-associated proteome (chromatome) to induce epigenetic changes and alter gene expression in cancer cells, we hypothesized that hypoxia-driven evolution of the chromatome promotes malignant changes and the development of therapy resistance in tumor cells. To test this hypothesis, we isolated chromatins from tumor cells treated with varying conditions of normoxia, hypoxia, and re-oxygenation and then partially digested them with DNase I and analyzed them for changes in euchromatin- and heterochromatin-associated proteins using an iTRAQ-based quantitative proteomic approach. We identified a total of 1446 proteins with a high level of confidence, including 819 proteins that were observed to change their chromatin association topology under hypoxic conditions. These hypoxia-sensitive proteins included key mediators of chromatin organization, transcriptional regulation, and DNA repair. Furthermore, our proteomic and functional experiments revealed a novel role for the chromatin organizer protein HP1BP3 in mediating chromatin condensation during hypoxia, leading to increased tumor cell viability, radio-resistance, chemo-resistance, and self-renewal. Taken together, our findings indicate that HP1BP3 is a key mediator of tumor progression and cancer cell acquisition of therapy-resistant traits, and thus might represent a novel therapeutic target in a range of human malignancies.

Abstract 3423: The genomic evolution of prostate cancer under the selective pressure of anti-androgen therapy

Abstract Background The implementation of novel technologies such as array comparative genomic hybridization (aCGH) and next-generation sequencing has led to a deeper understanding of the genomic nature of cancer. However, these analyses have classically been done without respecting intra-tumor heterogeneity. Here, we applied a methodology that allows us analyzing the genomic profile of distinct tumor populations from individual tumors and their clonal evolution during the progression to castration-resistant disease, thus enabling the discovery of novel genetic alterations in the course of the development of therapy resistance. Methods Matched pre- and post- hormone treated fresh frozen and/or formalin fixed prostate cancer samples were selected from our biobank. Clonal tumor populations were flow-sorted according to their nuclear DNA content. Sorted tumor populations were subjected to whole genome CGH and to full exome sequencing analyses by the use of Agilent SurePrint 2x400k microarrays and the SureSelect All Exon Kit, respectively. Target aberrations were evaluated using a prostate cancer tissue micro array (TMA) and fluorescence in situ hybridization as well as immunohistochemistry. Results The de-convolution of the genomic evolution in the investigated tumors shows that genetic aberrations and mutations are newly occurring under the selective pressure of anti-androgen therapy. More importantly, judging on the temporal dynamics of changes in allelic frequencies, we cannot only observe events that become more abundant, but also cancer associated mutations that are negatively selected for. We are currently following up several of these targets, of which at least one shows significant association with overall survival and differences in its expression pattern between hormone naïve and castration resistant cancer. Conclusions Genomic profiling of distinct clonal tumor populations during prostate cancer progression allows for analysis of intra-tumoral heterogeneity and the underlying clonal evolution. Importantly, this approach identifies genomic aberrations that were selected for under the pressure of hormone ablation therapy. Citation Format: Joël Roman Gsponer, Tanja Dietsche, Alexander Rufle, Thomas Lorber, Darius Juskevicius, Valeria Perrina, Elisabeth Lenkiewicz, Tobias Zellweger, Alexander Bachmann, Michael T. Barrett, Cyrill A. Rentsch, Christian Ruiz, Lukas Bubendorf. The genomic evolution of prostate cancer under the selective pressure of anti-androgen therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3423. doi:10.1158/1538-7445.AM2014-3423

Abstract A95: Intermittent, but not chronic, hypoxia increased chromosomal copy number variations of prostate cancer cells.

Abstract Background: Intra-tumoral hypoxia as a potential mechanism for therapy resistance has been well documented in prostate cancer. Screening drugs under hypoxic conditions, however, has not been adopted for drug discovery. Part of this is due to the difficulty to recapitulate the heterogeneity of intra-tumoral hypoxia in vitro. Indeed, a spatio-temporal irregularity of the microvascular network of a growing tumor creates regions that are either chronically (diffusion-limited) or intermittently (perfusion-limited) hypoxic. If genomic instability is the fundamental driving force for development of therapy resistance, the hypoxic culture condition with the most genomic instability would serve as an alternative in vitro model for drug testing. We therefore accessed chromosomal copy number variations (CCNV) in prostate cancer cells grew under intermittent as well as chronic hypoxia. Method: Metastatic prostate cancer cell lines, PC3 and DU145, were grown under normoxia (21% oxygen), chronic hypoxia (0.2% oxygen), or intermittent hypoxia (16 hours of 0.2% oxygen alternating with 8 hours of 21% oxygen) for total of 3 weeks. PC3 cells were also treated with the 1 µM poly(ADP-ribose) polymerase (PARP) inhibitor, veliparib under intermittent or chronic hypoxia conditions for 3 weeks. Genomic DNA was extracted for molecular karyotyping with the nanoString nCounter human karotyping platform, which covers 24 human chromosomes with 338 probes. DNA from human foreskin fibroblast (HFF) grew under normal culture condition (21% oxygen) was used as a reference. Results: Both PC3 and DU145 cells continue to proliferate under intermittent as well as chronic hypoxia. Compared to untreated PC3 cells, daily treatment with PARP inhibitor under chronic or intermitent hypoxia for 3 weeks did not affect the proliferation or survival of the PC3 cells. Compared to HFF, frequent CCNVs were observed in PC3 and DU145 cells. This is consistent with the known complicated karyotype of these 2 cell lines. When compared to PC3 or DU145 cells cultured under normoxia, CCNVs were enhanced under intermittent hypoxia, but not chronic hypoxia. Daily treatment with PARP inhibitor did not alter CCNVs under either chronic hypoxia or intermittent hypoxia. Conclusions: Intermittent hypoxia is probably a more relevant model to study genomic instability and drug resistance than chronic hypoxia. Neither chronic hypoxia nor intermittent hypoxia sensitizes prostate cancer cells to PARP inhibitor, veliparib. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A95. Citation Format: Jingsong Zhang, Xue Wang. Intermittent, but not chronic, hypoxia increased chromosomal copy number variations of prostate cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A95.

Genomic evolution from primary breast carcinoma to distant metastasis: Few copy number changes of breast cancer related genes

Cancer initiation and progression is characterized by (epi)genetic aberrations. However, little is known about the changes that occur during breast cancer metastasis. In the present study, multiplex ligation-dependent probe amplification was used to compare copy numbers of 21 established oncogenes and tumor suppressor genes between 55 primary breast cancer samples and corresponding distant metastases. Distant breast cancer metastases generally showed similar gene copy number aberrations compared to their corresponding primary tumors. The few genes that showed differences between primary tumor and metastasis (PRDM14, MED1, CCNE1, TRAF4, MTDH, CDH1) have been implicated in the development of therapy resistance.

Histone deacetylase inhibitors for the treatment of breast cancer: recent trial data

Epigenetic modification has recently been recognized as an important factor in the development of therapy resistance in cancer. Hence, histone deacetylase inhibitors as potential epigenetic modifiers are an emerging class of novel cancer therapeutics and have been extensively studied. Here, we review the role of histone deacetylase inhibitors in the treatment of breast cancer as single agents as well as in combination with chemotherapeutic, hormonal, and targeted agents.

Polo-Like Kinase 1: A Novel Target for the Treatment of Therapy-Resistant Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma (NHL) which is one of the most aggressive lymphomas. Despite recent improvements in therapies, the development of therapy-resistance is still a major problem; therefore, in order to understand the molecular basis of therapy-resistance, stable therapy-resistant MCL cell lines have been established by us. Based on the gene expression profiles of these cell lines, Polo-like kinase 1 (PLK1) was chosen as a therapeutic target. In this paper, we demonstrate a significant antilymphoma effect of targeting PLK1 in therapy-resistant MCL cells and primary MCL cells from refractory patients. PLK1 knockdown with the antisense oligonucleotide (ASO)/or small molecule inhibitor BI2536 showed significantly decreased proliferation and increased apoptosis in therapy-resistant MCL cell lines and MCL primary cells. Additionally, the direct protein-protein interaction partners of PLK1 were mapped using ingenuity pathway and confirmed the level of association of these partners with PLK1 based on their expression changes following PLK1 knockdown using real-time PCR. Results suggest that PLK1 is a viable target for the treatment of therapy-resistant MCL.

The kinase RSK2 modulates the sensitivity of ovarian cancer cells to cisplatin

Platinum-based chemotherapy (e.g. cisplatin, carboplatin) is standard of care for many types of cancer including ovarian cancer, however, the efficacy of treatment is hampered by the development of therapy resistance. The mechanisms behind platinum resistance are not completely understood. Here, we have investigated the role of the family of p90 Ribosomal S6 kinases (RSK), important downstream mediators of ERK1/2, in the response to cisplatin chemotherapy. Strikingly, whereas treatment with cisplatin did not alter the levels of RSK1 in response to cisplatin treatment, the structurally related RSK2 protein was downregulated in an ovarian cancer cell line (A2780). Furthermore, we found that knockdown of RSK2, in contrast to knockdown of RSK1, gave rise to enhanced cisplatin sensitivity in a cisplatin sensitive as well as a cisplatin-resistant A2780 cell line. These results indicate that RSK2 is regulated in response to cisplatin treatment, and this downregulation may contribute to the cytotoxic action of cisplatin. Since RSK2 is frequently amplified in a growing number of cancers, this may have implications for the sensitivity of these tumours to platinum-based cytotoxics.

Abstract 3281: Development of a novel transgenic mouse model for prostate cancer

Abstract Prostate cancer (PCa) is the most common cancer and the second leading cause of cancer death in American men. Androgen-deprivation therapy (ADT) is a common and effective therapy for advanced / metastatic PCa. However, most initially regressed PCa will relapse and become castration-resistant PCa (CRPC). Currently, there is no cure for CRPC. Chemotherapy, including agents such as Docetaxel, is a standard therapy for CRPC. However, it only modestly extends patient survival. Therefore, castration resistance and chemoresistance of PCa are huge medical problems for PCa treatment. To overcome castration resistance and chemoresistance, it is crucial to delineate the underlying molecular mechanisms. c-Myc is the most significantly amplified oncogene in human PCa, and its overexpression is very common in PCa as early as prostatic intraepithelial neoplasia (PIN). These indicate its critical roles in PCa progression and the development of therapy-resistance. Transgenic models are widely used in cancer research. Accordingly, Hi-MYC transgenic model has been created using an enhanced probasin promoter to drive c-Myc overexpression in prostate epithelia. These mice develop invasive prostate carcinomas that share molecular features with human PCa. However, since the probasin promoter activity is androgen-dependent, these MYC prostate tumors lose c-Myc oncogene expression upon castration. As a result, the tumor regression in these androgen-depleted Hi-MYC mice represents the mixed effects of both an artificial direct effect from loss of oncogene expression and a potential real effect from tumor cellular responses to androgen-ablation that are relevant to human PCa. Finally, Hi-MYC mice do not develop CRPC tumors after castration. Therefore, the Hi-MYC model, along with other similarly created Myc-based transgenic models for PCa, cannot be used to study CRPC, the lethal form of PCa. To study CRPC and chemoresistance of CRPC, we have developed a novel transgenic model (CAG-SMIL) for human PCa. This model allows us to specifically turn on c-Myc oncogene expression in prostate epithelia after crossing CAG-SMIL mice with PB-Cre4 mice. In addition, once turned on, the expression of c-Myc transgene will be driven by an enhanced beta-actin promoter that is androgen-independent. Therefore, after castration of the prostate tumor bearing PB-Cre4/CAG-SMIL males, we will be able to, for the first time, concisely study c-Myc signaling pathway 1) in castration induced prostate tumor regression, 2) in the recurrence of CRPC tumors, and 3) in the development of chemoresistance in CRPC tumors. Finally, we have incorporated an enhanced luciferase reporter into our transgenic model. This will allow a real-time in vivo bioluminescence imaging (BLI imaging) for prostate tumor progression, tumor response to various therapeutic agents, as well as tumor relapse after the development of therapy (castration and chemotherapy) resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3281. doi:1538-7445.AM2012-3281

Abstract 816: Functional analysis of miRNA in chemotherapy resistant neuroblastoma

Abstract Neuroblastoma is a paediatric cancer of the sympathetic nervous system, accounting for 15 % of childhood cancer related deaths. Treatment of neuroblastoma (NB) patients involves intensive rounds of multimodal chemotherapy, with initial responses often being positive, but with a high likelihood of relapse and development of therapy resistance. The aim of this project is to identify miRNAs associated with resistance to chemotherapeutics in NB, and elucidate the molecular mechanisms involved. In order to understand miRNA involvement in NB drug resistance, two MYCN amplified (KellyCis83, CHP-212Cis100) and one MYCN single copy cell line (SK-N-ASCis24) resistant to cisplatin (CDDP) were developed using ‘drug pulse selection’. An increase in resistance to CDDP was confirmed by examination of IC50s before and after selection. Expression of 770 miRNAs was analyzed using Taqman arrays and drug resistant profiles were compared to those of parental lines. 41, 51 and 72 miRNAs were differentially expressed in KellyCis83, SKNASCis24 and CHP-212Cis100 respectively. A total of 8 miRNA exhibited the same pattern of differential expression in both KellyCis83 and SKNASCis24, 6 miRNA in KellyCis83 and CHP-212Cis100, and 8 miRNA in SKNASCis24 and CHP-212Cis100. 2 miRNA were commonly differentially expressed across all three cell lines. Assessment of cellular proliferation revealed resistant variants KellyCis83 and CHP-212Cis100 grown in the presence of low-dose CDDP consistently outgrew cells grown in normal media. This increase in growth is not observed in the parental cell lines. MiRNA expression profiles of cells challenged with 0.2 μM CDDP were obtained and 31 miRNAs differentially expressed in KellyCis83 and CHP-212Cis100 identified. Many miRNAs identified have been previously associated with drug resistance, confirming the efficacy of this approach. For example, in the panel of 8 miRNA common to KellyCis83 and SK-N-ASCis24, all miRNA are predicted to target at least one of two key copper transporters involved in CDDP transport; hCtr1 and ATP7A. Each cell line was also analyzed using aCGH to identify DNA copy number alterations. A region of focal gain containing NAIP (NLR family, apoptosis inhibitory protein) was identified in SKNASCis24, and a 15-fold increase of NAIP mRNA was confirmed using qPCR. NAIP inhibits caspases-3, 7 and 9 resulting in increased cell survival. This discovery was of interest because 13 of the 33 miRNAs down-regulated in SK-N-ASCis24 are predicted to target NAIP. SiRNA depletion of NAIP mRNA led to increased sensitivity to etoposide, but not CDDP in SK-N-ASCis24. However, both drugs have different mechanisms of action and it is possible that NAIP has a greater influence over the function of etoposide in our model. In conclusion, we have identified miRNA associated with drug resistance in NB, and are now working to elucidate the mechanism of miRNA involvement in the process of multidrug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 816. doi:1538-7445.AM2012-816

Abstract 806: Extracellular matrix metalloprotease inducer (EMMPRIN) and CD44 protein complexes are exclusively formed in basal- and normal-like breast cancer cell lines

Abstract Extracellular Matrix Metalloprotease Inducer (EMMPRIN) is a multifunctional glycoprotein that is involved in tumor cell invasion and metastasis, and is a predictor for tamoxifen resistance in breast cancer. EMMPRIN is implicated in development of therapy resistance by forming a functional transporter complex through interaction with CD44 and monocarboxylate transporter 4. Presence of EMMPRIN transporter protein complexes may therefore be a better predictor for therapy resistance and a direct target for drugs. In this study, we aimed to identify EMMPRIN-CD44 protein interactions in a large panel of breast cancer cell lines. EMMPRIN-CD44 protein interaction was investigated in a panel of 41 breast cancer cell lines, which represent the luminal (n=12), ErbB2 (n=13), basal (n=7), and normal-like (n=9) intrinsic subtypes, using in situ Proximity Ligation Assay (isPLA). In addition, protein expression of both EMMPRIN and CD44 in the cell lines was evaluated by immunohistochemistry (IHC). IHC and isPLA read-out was performed by bright field microscopy and 0-1-2-3 scoring was applied to none-weak-moderate-strong staining (IHC) or interaction (isPLA), respectively. Moderate and strong EMMPRIN expression was observed in 50% of luminal, 36% of ErbB2, 86% of basal, and 100% of normal-like cell lines, and moderate to high CD44 expression was detected in 64%, 83%, 86%, and 100% in luminal, ErbB2, basal, and normal-like cell lines, respectively. Interestingly, moderate to strong EMMPRIN-CD44 interaction was exclusively detected in basal (64%) and normal-like (67%) cell lines, whereas luminal and ErbB2 cells lines had no detectible, or only very weak, EMMPRIN-CD44 interaction (≥square = 23.5, P<0.0001). Our results indicate that EMMPRIN-CD44 protein complexes are formed exclusively in basal and normal-like breast cancer cell lines. These breast cancer subtypes are associated with an aggressive phenotype and poor patient survival. We speculate that active EMMPRIN-CD44 transporters are involved in drug resistance, resulting in poor clinical outcome, and will investigate this in more detail in clinical breast cancer specimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 806. doi:1538-7445.AM2012-806

P2-02-07: Regulation of mTOR Signaling by Proto-Oncogene PELP1.

Abstract BACKGROUND: Despite positive effects of hormonal therapy, initial or acquired resistance to endocrine therapies frequently occurs. Emerging evidence suggests that ER action is complex and requires functional interactions with coregulators. In addition, ER also participates in extra-nuclear signaling events in the cytoplasm and growth factor cross talk with ER is implicated in the development of therapy resistance. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays an important role on cell growth and proliferation. Proline, Glutamic-acid and Leucine-rich Protein 1 (PELP1) is an ER coregulator that functions in nuclear as well as extra-nuclear actions. PELP1 functions as a proto-oncogene and is a prognostic indicator of shorter breast cancer specific survival. The objective if this study is to examine whether PELP1 participates in ER-growth factor signaling cross talk via mTOR pathway. METHODS: To test this hypothesis, we have used ER-positive model cells (MCF7, ZR75) along with PELP1 over expressing (MCF7-PELP1, ZR75-PELP1) and PELP1 knock down cells (MCF7-PELP1 shRNA, ZR-75-PELP1 shRNA). Estrogen and Heregulin were used as signal inducers. Rapamycin that inhibit mTORC1 and AZD8055, an inhibitor of both mTORC1 and mTORC2 activities were used as pharmacological inhibitors to block mTOR pathway. The affect of blocking mTOR axis on PELP1 mediated oncogenic functions was determined using ERE and E2F reporter gene assays, cell proliferation (MTT) and anchorage independent assays. Cell cycle progression was monitored by flow cytometry. PELP1 modulation of mTOR signaling was validated using siRNA approach in MCF7, ZR75 cells and/or by PELP1 overexpression in PELP1-shRNA clones and evaluated the role of identified components for changes in the mTOR signaling using Western analysis. IHC analysis of mTOR signaling components was performed using tissues collected from PELP1 overexpressing and PELP1 siRNA liposome treated tumors. RESULTS: Over expression of PELP1 enhanced the estrogen and heregulin mediated cell proliferation in breast cancer cells. Knock down of PELP1 with siRNA significantly reduced the activation of mTOR signaling components (p70S6K and 4E-BP1). Accordingly, over expression of PELP1 in the breast cancer cells correlated with increased mTOR signaling. Pharmacological inhibition of mTOR substantially reduced PELP1 mediated ER coactivation functions in ERE reporter gene assays. Rapamycin (10−7M) or AZD8055 (10−8M) abolished PELP1 mediated growth advantage in estrogen and heregulin stimulated breast cancer cells. Further, combinatorial treatment of mTOR inhibitors sensitized PELP1 overexpressing cells to tamoxifen therapy. IHC studies using xenograft tissues with PELP1 overexpression or down regulation, showed correlation of PELP1 expression with the activation of mTOR signaling components. CONCLUSIONS: Our results suggest that PELP1 driven oncogenic functions involve PELP1 modulation of mTOR signaling and blockage of mTOR signaling render PELP1 driven tumors highly sensitive to therapeutic inhibition. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-07.

Drug Resistance: Challenges to Effective Therapy

The success of current treatment strategies is limited by the development of therapy resistance as evidenced by recurrence of the primary tumor or distant metastasis. Eradication of primary and metastatic disease requires interventions at both the cancer cell and tumor microenvironment levels. In this review, we will discuss mechanisms that are intrinsic to cancer cells, and those that are mediated by the tumor microenvironment as contributors to drug resistance. Mechanisms contributing to multidrug resistance phenotype and the challenges facing molecular targeted therapy are discussed. The DNA damage tolerance pathway confers tolerance to a variety of structurally and functionally unrelated drugs. A rationale for targeting the DNA damage tolerance pathway as a novel tool for overcoming drug resistance is discussed. We have also addressed the need for employing clinically relevant model systems for performing drug sensitivity evaluations. These model systems must take into account the three-dimensional organization and in vivo relationship of tumor with its microenvironment. Such integrative efforts would not only yield a more global understanding of the tumor- and microenvironment-derived mechanisms involved in emergence of drug resistance but would also provide novel therapeutic targets that will disrupt the interactions between the tumor cells and its microenvironment.

Abstract 733: A therapeutic opportunity in melanoma: Targeting CXCR1/2-dependent signaling attenuates therapy resistance

Abstract Advanced melanoma is one of the most intractable tumors, due to its insensitivity to all current treatment. Systemic chemotherapy is still considered the mainstay of treatment for advanced melanoma with limited survival impact. Failure of most agents is attributed to development of therapy resistance. Our previous findings demonstrate that CXCL-8 is a critical mediator in tumor growth, invasion, and angiogenesis. Two high affinity receptors for CXCL-8, CXCR1 and CXCR2, are differentially expressed on melanoma and endothelial cells. Based on these observations, we hypothesize that targeting CXCR1/2-dependent signaling will attenuate chemotherapy resistance and will synergistically enhance chemotherapeutic response for malignant melanoma. We used human melanoma cell lines A375P (low metastatic) and A375SM (highly metastatic), which express low and high levels of CXCL-8 respectively. First, we evaluated the role of CXCR1/2 ligands in chemo-resistance. CXCL-1 and -8 mRNA and protein expression was analyzed by quantitative realtime PCR and enzyme-linked immunosorbant assay, following treatment of dacarbazine and temozolomide. A significant increase in the CXCL-1 and -8 levels was observed in A375P and A375SM cells which was dependent on concentration and duration of chemotherapeutic treatment. In order to test whether blocking CXCR1/2 expression using genetic knock-down modulates chemotherapeutic responses, we used A375SM cells expressing different levels of CXCR1 and CXCR2. Our data suggest that knockdown of CXCR1 significantly enhanced the cytotoxic effect of dacarbazine in vitro. However, we did not observe a similar response in melanoma cells knocked down for CXCR2. Taken together, our preliminary data demonstrate that melanoma cells that survive chemotherapy express higher levels of CXCR1/2 ligands and targeting the CXCR1/2-dependent pathway might be a potential therapeutic approach to be combined with chemotherapy in advanced melanoma. 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 733. doi:10.1158/1538-7445.AM2011-733

Abstract P5-06-12: Roscovitine Confers Tumor Suppressive Effect on Therapy-Resistant Breast Tumor Cells

Abstract Background: Current clinical strategies for treating hormonal breast cancer involve the use of antiestrogens (AE) that block ERα functions, and aromatase inhibitors (AI) that decrease local and systemic estrogen production. Both of these strategies improve outcomes for ER+ve breast cancer; however development of therapy resistance is a major clinical problem. Emerging data implicate ER-signaling cross talk, and alteration in ER subtypes as the major causes of resistance. Current evidence suggest that ERa increases proliferation, while ERα decreases cell proliferation and the ratio between ERα and ERα is the driving force for tumor cell proliferation. Further, most downstream events in the resistance pathways converge upon modulation of cell cycle regulatory proteins; the most conspicuous of which is the activation of Cyclin Dependent Kinase 2 (CDK2) pathway. Roscovitine is one of the most frequently studied andused CDK2 inhibitor. In this study, we examined whether Roscovitine confers tumor suppressive advantage to therapy resistant breast epithelial cells by inhibiting CDK functions. Methods: We tested the effect roscovitine using three different therapy resistant model cells; (a) MCF-7-Tam (acquired Tamoxifen resistance model); (b) MCF-7-LTLT-ca (acquired Letrozole resistance model); (c) MCF-7-HER2 that exhibit Tamoxifen resistance (ER-growth factor signaling cross talk model). Cells were treated with Roscovitine (0-30 uM) for 24 hours and proliferation was measured after 48h using a luminescent cell viability assay (Promega). CDK activity, expression of ERα, β subtypes, ER-coregulators, and expression of ER target genes was measured by Western and PCR assays. To test the efficacy of Roscovitine in vivo, pre-clinical nude mice models bearing xenografts of therapy resistant cells were subjected to orally available Roscovitine (100mg/kg/tid/oral) for 10 days or treated with vehicle and tumor growth was monitored for a further 15 days. Results: The results showed that Roscovitine (10uM) significantly inhibited the growth of all three therapy resistant cells in cell proliferation and foci formation assays (P<0.05). FACS analysis revealed that Roscovitine treatment increased proportion of cells in G2-M phase. Therapy resistant cells treated with Roscovitine showed decreased CDK activity and low cyclin D levels indicating decreased cell proliferation. Interestingly, these studies also revealed an unexpected discovery that CDK inhibitor Roscovitine has potential to alter the ratio of Estrogen receptors with preferential upregulation of ERα with concomitant down regulation of ERα, ER-coregulators including AIB1 and PELP1. Results from xenograft studies showed that Roscovitine substantially reduced growth of therapy resistant tumors. Conclusions: Our results suggest that Roscovitine can be used to inhibit the growth of therapy resistant cells. Since many advanced and therapy resistant tumors exhibit loss or reduced expression of ERα, dual action of Roscovitine will provide a novel drug to inhibit both CDKs and to increase the expression of tumor suppressor ERα, thus has potential to serve as a double-edged sword to interfere with the resistance mechanisms. Supported by DOD Fellowship W81XWH-09-1-0010 (BCN) and NIH grant CA095681(RV). Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-12.