Role In Therapy Resistance Research Articles

Splicing dysregulation as a driver of breast cancer

Breast cancer is known to be a heterogeneous disease driven by a large repertoire of molecular abnormalities, which contribute to its diverse clinical behaviour. Despite the success of targeted therapy approaches for breast cancer patient management, there is still a lack of the molecular understanding of aggressive forms of the disease and clinical management of these patients remains difficult. The advent of high-throughput sequencing technologies has paved the way for a more complete understanding of the molecular make-up of the breast cancer genome. As such, it is becoming apparent that disruption of canonical splicing within breast cancer governs its clinical progression. In this review, we discuss the role of dysregulation of spliceosomal component genes and associated factors in the progression of breast cancer, their role in therapy resistance and the use of quantitative isoform expression as potential prognostic and predictive biomarkers with a particular focus on oestrogen receptor-positive breast cancer.

Abstract P6-06-04: Targeting replication stress in triple negative breast cancer treatment regimen: An emerging approach

Abstract Triple-negative breast cancers (TNBCs) represent aggressive heterogeneous subtype of breast cancer with poor clinical outcome. TNBCs have been reported to have high levels of replication stress due to i) various oncogene activations (C-myc or EGFR) ii) germline BRCA mutations iii) “BRCAness” in the absence of BRCA mutations in sporadic TNBCs. Replication stress is known to cause genomic instability, promote tumorigenesis and plays a critical role in therapy resistance in TNBCs. Therefore, targeting replication stress has emerged as an effective approach for better TNBC treatment through further downregulation of the remaining checkpoints to induce catastrophic failure of TNBC cells proliferation. Herein, we evaluated the anticancer efficacy of Carbazole Blue (CB), a synthetic analogue of Carbazole, on TNBC cells growth and progression. Our results demonstrated that CB inhibits short and long term viability of TNBC (MDA-MB-231, MDA-MB-468 and BT549) cells in a dose dependent manner without affecting normal mammary epithelial (MCF-10A) cells. In addition, CB treatment significantly reduced proliferation of TNBC cells, as evidenced by the BrdU proliferation assay. Consistent with this, our results further demonstrated that CB treatment induced G1/S cell cycle arrest and apoptosis in TNBCs. Importantly, systemic delivery of CB using nanoparticle-based delivery approach suppressed breast cancer growth without inducing toxicity, in preclinical orthotopic xenograft and PDX mouse models of TNBC. Furthermore, our gene microarray analysis revealed that CB treatment modulates the expression and activity of several genes known to be involved in DNA replication (CDC6, CDT1, MCMs, Claspin, POLE and PCNA) and associated DNA repair machinery such as (XRCC3, Exo1 and RAD51), which play pivotal roles in replication stress. Our results for the first time highlight the potential use of CB as a novel and potent therapeutic agent for treating TNBCs. As exploiting replication stress to treat cancer is gaining major interest, compound/s that may induce replication stress and inhibit DNA repair ability of cancer cells, has immense translational potential. Citation Format: Rajamanickam S, Park JH, Bates K, Timilsina S, Eedunuri VK, Onyeagucha B, Subbarayalu P, Abdelfattah N, Jung KH, Favours E, Mohammad TA, Chen H-IH, Vadlamudi RK, Chen Y, Kaipparettu BA, Arbiser JL, Rao MK. Targeting replication stress in triple negative breast cancer treatment regimen: An emerging approach [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-06-04.

The immunosuppressive cytokine interleukin-4 increases the clonogenic potential of prostate stem-like cells by activation of STAT6 signalling

Interleukin-4 plays a critical role in the regulation of immune responses and has been detected at high levels in the tumour microenvironment of cancer patients, where concentrations correlate with the grade of malignancy. In prostate cancer, interleukin-4 has been associated with activation of the androgen receptor, increased proliferation and activation of survival pathways such as Akt and NF-κB. However, its role in therapy resistance has not yet been determined. Here we investigate the influence of interleukin-4 on primary epithelial cells from prostate cancer patients. Our data demonstrate an increase in the clonogenic potential of these cells when cultured in the presence of interleukin-4. In addition, a Phospho-Kinase Array revealed that in contrast to previously published work, signal transducer and activator of transcription6 (STAT6) is the only signalling molecule activated after interleukin-4 treatment. Using the STAT6-specific inhibitor AS1517499 we could confirm the role of STAT6 in increasing colony-forming frequency. However, clonogenic recovery assays revealed that interleukin-4 does not rescue the effects of either irradiation or docetaxel treatment. We therefore propose that although the interleukin-4/STAT6 axis does not appear to be involved in therapy resistance, it does play a crucial role in the colony-forming abilities of the basal cell population in prostate cancer. IL-4 may therefore contribute to disease relapse by providing a niche that is favourable for the clonogenic growth of prostate cancer stem cells.

Activation of necroptosis to overcome drug resistance in leukemia

The understanding of cell death mechanisms is crucial for the development and application of novel anti-cancer therapies to avoid or circumvent drug-resistance in refractory malignancies. Impairment of apoptotic cell death plays a major role in therapy resistance and relapse of acute lymphoblastic leukemia (ALL) patients. Therefore, efforts are being directed at new agents reactivating apoptosis or inducing alternative cell death pathways such as necroptosis, a regulated form of necrosis. In a recent study published in Science Translational Medicine we show that the IAP (inhibitor of apoptosis proteins) inhibitor birinapant potently induces cell death in patient-derived ALL cells in vitro and in vivo through a receptor-interacting protein kinase 1- (RIP1) dependent mechanism. To define the cell death modality induced downstream of RIP1, we used a multicolor lentiCRISPR approach that allows simultaneous knockout of multiple genes. We observed that apoptosis and necroptosis are induced simultaneously as the inhibition of both pathways is required to restore cell viability upon birinapant treatment. This induction of dual cell death makes birinapant and other IAP inhibitors interesting agents for the treatment of refractory or drug resistant malignancies.

MTOR activity and its prognostic significance in human colorectal carcinoma depending on C1 and C2 complex-related protein expression

AimsTumour heterogeneity and altered activation of signalling pathways play important roles in therapy resistance. The PI3K/Akt/mTOR signalling network is a well-known regulator of several functions that contribute to tumour growth....

Novel function of MDA-9/Syntenin (SDCBP) as a regulator of survival and stemness in glioma stem cells.

Glioblastoma multiforme (GBM) is an aggressive cancer with current therapies only marginally impacting on patient survival. Glioma stem cells (GSCs), a subpopulation of highly tumorigenic cells, are considered major contributors to glioma progression and play seminal roles in therapy resistance, immune evasion and increased invasion. Despite clinical relevance, effective/selective therapeutic targeting strategies for GSCs do not exist, potentially due to the lack of a definitive understanding of key regulators of GSCs. Consequently, there is a pressing need to identify therapeutic targets and novel options to effectively target this therapy-resistant cell population. The precise roles of GSCs in governing GBM development, progression and prognosis are under intense scrutiny, but key upstream regulatory genes remain speculative. MDA-9/Syntenin (SDCBP), a scaffold protein, regulates tumor pathogenesis in multiple cancers. Highly aggressive cancers like GBM express elevated levels of MDA-9 and contain increased populations of GSCs. We now uncover a unique function of MDA-9 as a facilitator and determinant of glioma stemness and survival. Mechanistically, MDA-9 regulates multiple stemness genes (Nanog, Oct4 and Sox2) through activation of STAT3. MDA-9 controls survival of GSCs by activating the NOTCH1 pathway through phospho-Src and DLL1. Once activated, cleaved NOTCH1 regulates C-Myc expression through RBPJK, thereby facilitating GSC growth and proliferation. Knockdown of MDA-9 affects the NOTCH1/C-Myc and p-STAT3/Nanog pathways causing a loss of stemness and initiation of apoptosis in GSCs. Our data uncover a previously unidentified relationship between MDA-9 and GSCs, reinforcing relevance of this gene as a potential therapeutic target in GBM.

Stimulation of cellular senescent processes, including secretory phenotypes and anti-oxidant responses, after androgen deprivation therapy in human prostate cancer

Endocrine resistance is a major problem in prostate cancer. Recent studies suggest that cellular plasticity plays a key role in therapy resistance. Yet little is known about the cellular changes of human prostate cancer after androgen deprivation therapy (ADT). In this study, we investigated cellular senescence, senescence-associated secretory phenotypes (SASPs), and anti-oxidant responses. Hormone ablation upregulated senescence-associated (SA)-β-Gal activity in prostate glands, as well as the expressions of p27KIP1 and p53, in a mouse castration model. In line with this, the expressions of p21CIP1 and p27KIP1 were significantly more upregulated in human non-pathological prostatic glands after ADT than in untreated specimens. In a study of SASP markers, the expressions of IL6 and IL8 were also more upregulated in human non-pathological prostatic glands after ADT than in untreated specimens. IL6, IL8, and MMP2 were expressed more strongly in human prostate cancer specimens resected after ADT than in untreated tumors. Of note, treatment with the anti-oxidant reagent NAC significantly suppressed SA-β-Gal activity in androgen-sensitive human prostate cancer LNCaP cells. In immunohistochemical analyses on anti-oxidant response genes, NRF2 and NQO1 were more upregulated after hormone ablation in human prostate gland and carcinoma specimens after ADT than in untreated specimens or in murine prostate glands after castration. Taken together, these findings suggest that ADT induces cellular senescence processes accompanied by secretory phenotypes and anti-oxidant responses in prostate. These cellular changes may be attractive targets for preventing endocrine resistance in prostate cancer.

EZH2 Protects Glioma Stem Cells from Radiation-Induced Cell Death in a MELK/FOXM1-Dependent Manner

SummaryGlioblastoma (GBM)-derived tumorigenic stem-like cells (GSCs) may play a key role in therapy resistance. Previously, we reported that the mitotic kinase MELK binds and phosphorylates the oncogenic transcription factor FOXM1 in GSCs. Here, we demonstrate that the catalytic subunit of Polycomb repressive complex 2, EZH2, is targeted by the MELK-FOXM1 complex, which in turn promotes resistance to radiation in GSCs. Clinically, EZH2 and MELK are coexpressed in GBM and significantly induced in postirradiation recurrent tumors whose expression is inversely correlated with patient prognosis. Through a gain-and loss-of-function study, we show that MELK or FOXM1 contributes to GSC radioresistance by regulation of EZH2. We further demonstrate that the MELK-EZH2 axis is evolutionarily conserved in Caenorhabditis elegans. Collectively, these data suggest that the MELK-FOXM1-EZH2 signaling axis is essential for GSC radioresistance and therefore raise the possibility that MELK-FOXM1-driven EZH2 signaling can serve as a therapeutic target in irradiation-resistant GBM tumors.

Beyond immune checkpoint blockade: new approaches to targeting host-tumor interactions in prostate cancer: report from the 2014 Coffey-Holden prostate cancer academy meeting.

The 2014 Coffey-Holden Prostate Cancer Academy Meeting, held in La Jolla, CA from June 26 to 29, 2014, was themed: "Beyond Immune Checkpoint Blockade: New Approaches to Targeting Host-Tumor Interactions in Prostate Cancer." Sponsored by the Prostate Cancer Foundation (PCF), this annual, invitation-only meeting is structured as an action-tank, and brought together 72 investigators with diverse academic backgrounds to discuss the most relevant topics in the fields of prostate cancer immunotherapy and the tumor microenvironment. The questions addressed at the meeting included: mechanisms underlying the successes and failures of prostate cancer immunotherapies, how to trigger an effective immune response against prostate cancer, the tumor microenvironment and its role in therapy resistance and tumor metastasis, clinically relevant prostate cancer mouse models, how host-tumor interactions affect current therapies and tumor phenotypes, application of principles of precision medicine to prostate cancer immunotherapy, optimizing immunotherapy clinical trial design, and complex multi-system interactions that affect prostate cancer and immune responses including the effects of obesity and the potential role of the host microbiome. This article highlights the most significant recent progress and unmet needs that were discussed at the meeting toward the goal of speeding the development of optimal immunotherapies for the treatment of prostate cancer.

Cancer stem-like sphere cells induced from de-differentiated hepatocellular carcinoma-derived cell lines possess the resistance to anti-cancer drugs.

BackgroundCancer stem cells (CSCs) are thought to play important roles in therapy-resistance. In this study, we induced cancer stem-like cells from hepatocellular carcinoma (HCC) cell lines using a unique medium, and examined their potential for resistance to anti-cancer drugs.MethodsThe human HCC cell lines SK-HEP-1 (SK), HLE, Hep 3B, and HuH-7 were used to induce cancer stem-like cells with our sphere induction medium supplemented with neural survival factor-1. NANOG and LIN28A were examined as stemness markers. Several surface markers for CSC such as CD24, CD44, CD44 variant, and CD90 were analyzed by flow-cytometry. To assess the resistance to anti-cancer drugs, the MTS assay, cell cycle analysis, and reactive oxygen species (ROS) activity assay were performed.ResultsPoorly differentiated HCC derived SK and undifferentiated HCC derived HLE cell lines efficiently formed spheres of cells (SK-sphere and HLE-sphere), but well-differentiated HCC-derived HuH-7 and Hep 3B cells did not. SK-spheres showed increased NANOG, LIN28A, and ALDH1A1 mRNA levels compared to parental cells. We observed more CD44 variant-positive cells in SK-spheres than in parental cells. The cell viability of SK-spheres was significantly higher than that of SK cells in the presence of several anti-cancer drugs except sorafenib (1.7- to 7.3-fold, each P < 0.05). The cell cycle of SK-spheres was arrested at the G0/G1 phase compared to SK cells. SK-spheres showed higher ABCG2 and HIF1A mRNA expression and lower ROS production compared to parental cells.ConclusionOur novel method successfully induced cancer stem-like cells, which possessed chemoresistance that was related to the cell cycle, drug efflux, and ROS.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-722) contains supplementary material, which is available to authorized users.

Neuropilin-2 and its ligand VEGF-C predict treatment response after transurethral resection and radiochemotherapy in bladder cancer patients.

The standard treatment for invasive bladder cancer is radical cystectomy. In selected patients, bladder-sparing therapy can be performed by transurethral resection (TURBT) and radio-chemotherapy (RCT) or radiotherapy (RT). Our published in vitro data suggest that the Neuropilin-2 (NRP2)/VEGF-C axis plays a role in therapy resistance. Therefore, we studied the prognostic impact of NRP2 and VEGF-C in 247 bladder cancer patients (cN0M0) treated with TURBT and RCT (n = 198) or RT (n = 49) and a follow-up time up to 15 years. A tissue microarray was analyzed by immunohistochemistry. NRP2 expression emerged as a prognostic factor in overall survival (OS; HR: 3.42; 95% CI: 1.48 - 7.86; p = 0.004) and was associated with a 3.85-fold increased risk of an early cancer specific death (95% CI: 0.91 - 16.24; p = 0.066) in multivariate analyses. Cancer specific survival (CSS) dropped from 166 months to 85 months when NRP2 was highly expressed (p = 0.037). Patients with high VEGF-C expression have a 2.29-fold increased risk of shorter CSS (95% CI: 1.03-5.35; p = 0.043) in univariate analysis. CSS dropped from 170 months to 88 months in the case of high VEGF-C expression (p = 0.041). Additionally, NRP2 and VEGF-C coexpression is a prognostic marker for OS in multivariate models (HR: 7.54; 95% CI: 1.57-36.23; p = 0.012). Stratification for muscle invasiveness (T1 vs. T2-T4) confirmed the prognostic role of NRP2 and NRP2/VEGF-C co-expression in patients with T2-T4 but also with high risk T1 disease. In conclusion, immunohistochemistry for NRP2 and VEGF-C has been determined to predict therapy outcome in bladder cancer patients prior to TURBT and RCT.

Mixed‐polarization phenotype of ascites‐associated macrophages in human ovarian carcinoma: Correlation of CD163 expression, cytokine levels and early relapse

Ovarian cancer is typically accompanied by the occurrence of malignant ascites containing large number of macrophages. It has been suggested that these tumor-associated macrophages (TAMs) are skewed to alternative polarization (M2) and thereby play an essential role in therapy resistance and metastatic spread. In our study, we have investigated the nature, regulation and clinical correlations of TAM polarization in serous ovarian cancer. Macrophage polarization markers on TAMs and ascites cytokine levels were analyzed for 30 patients and associated with relapse-free survival (RFS) in a prospective study with 20 evaluable patients. Surface expression of the M2 marker CD163 on TAMs was inversely associated with RFS (p < 0.01). However, global gene expression profiles determined for 17 of these patients revealed a mixed-polarization phenotype unrelated to the M1/M2 classification. CD163 surface expression also correlated with the ascites levels of IL-6 and IL-10 (p < 0.05), both cytokines induced CD163 expression, and their ascites levels showed a clear inverse association with RFS (p < 0.01). These findings define a subgroup of patients with high CD163 expression, high IL-6 and/or IL-10 levels and poor clinical outcome.

Can Global Protein Expression Profiling Determine Prognosis in Diffuse Large B-Cell Lymphoma?

AbstractAbstract 1537 Background.With current rituximab-based treatment about 60 % of patients with diffuse large B-cell lymphoma (DLBCL) can be cured. However, for patients with early relapse or being refractory to such therapy outcome is very poor. The International Prognostic Index (IPI) is still the only prognostic tool used in daily clinical practice to risk stratify DLBCL patients. Yet, IPI has a limited ability to identify patients with risk of early relapse or refractory disease. Thus, there is a great need for reliable biomarkers capable of identifying high-risk patients for whom alternative therapies could be considered. Even though gene expression profiling can differentiate between DLBCL tumor subtypes, several factors influence RNA translation to a final protein. Consequently, there could be a considerable discrepancy between RNA expression levels and the corresponding protein level. Therefore, we hypothesized that global protein expression profiling, using proteomic analysis, could be a tool to provide additional prognostic information. Aims.To compare differences in global protein expression from two groups of DLBCL patients: i) early relapse/refractory patients, and ii) cured patients, in order to find candidate prognostic biomarkers and also potential drug targets. Patients and methods.All de novo DLBCL patients diagnosed in the Gothenburg region between 2004 and 2008 treated with curative intent (R-CHOP) were retrospectively analyzed. Two subgroups were determined; A) patients with progressive disease or relapse within 1 year after completion of treatment and B) patients considered cured, i.e. progression-free with a follow-up of at least 5 years. Five age- and gendermatched patients from each subgroup were selected and protein solutions were extracted from fresh frozen tumor tissue. In parallel, five established DLBCL cell lines were metabolically labeled with 13C6-arginine and 13C6-lysine. Lysates from these labeled cell lines were combined and used as an internal protein standard for relative protein quantification in the so-called Super-SILAC approach. Proteins were separated by SDS-PAGE and in-gel trypsin digested fractions were analyzed by LC-MS using an LTQ-FT/MS instrument. The MaxQuant software was used for data-analysis. Results.In the tumor samples 2523 different proteins were identified, and 1953 of these could be quantified. When comparing the groups, 41 separate proteins with statistically different expression were identified. Furthermore, 18 proteins were solely expressed in the early relapse/refractory group; most of these proteins were associated with cell proliferation (P54, snoRNP GAR1, SCOT), anti-apoptosis (TRAP1, AHA1), cell migration (Caveolin-1, ELMO1) and cell adhesion (VAP-1). We also performed a multivariate analysis (Principal Components Analysis), which produced a panel of 17 proteins that completely separated the groups (Figure 1). [Display omitted] Among the specific proteins separating the groups are e.g. proteins involved in cell proliferation/tumor growth (Erp-72, glutaredoxin, ACTG1, superoxid dismutase 1, DEAD box protein p72), tumor invasion (Moesin), adhesion (hnRNP M receptor) and anti-apoptosis (PTB1, ANT2). Summary/Conclusions.We have identified a substantial amount of proteins with potential prognostic value to identify high-risk DLBCL patients. Some of these proteins could expectedly be found in patients with refractory disease, however for many of them their role in therapy resistance remains to be determined. Further studies of both the individual and clustered proteins may also result in the identification of potential targets for the development of novel therapies for DLBCL. Disclosures:No relevant conflicts of interest to declare.

Clinical significance and prognostic impact of CD133 expression in rectal cancer treated with preoperative radiochemotherapy.

e14161 Background: CD133 has been described as a potential stem cell marker in colorectal cancer and to be associated with higher tumorigenic potential and resistance to radiochemotherapy (RCT). In this study the expression of CD133 was evaluated in pre-RCT tumor biopsies and the corresponding post-RCT surgical specimens of locally advanced rectal cancer patients and correlated to histopathological features and clinical follow-up. Methods: 127 patients with UICC (Union contre le Cancer) II/III rectal cancer treated with preoperative 5-FU based RCT within the German Rectal Cancer Trials were investigated. Pre- and post-RCT CD133 expression levels were determined using immunhistochemistry and correlated with histopathologic parameters, tumor regression grade, cancer recurrence and survival. Results: As compared to pre-RCT biopsies we observed a significantly higher CD133 expression in tumor specimens (p=0.01). There was, however, no correlation for both biopsies and tumor specimens between CD133 expression levels, histopathological features and survival. In matched analyses of corresponding biopsy/tumor pairs, patients with a decreased fraction of CD133+ cells after preoperative RCT showed significantly lower residual tumor stages (p=0.03) and higher histopathological tumor regression (p&lt;0.01). Moreover, these patients had a significantly improved disease-free (DFS) and cancer-specific overall survival (CSS) in uni- (p&lt;0.001) and multivariate analyses (p=0.001), respective. Conclusions: Enrichment of CD133+ cancer cells during preoperative RCT is correlated with minor local tumor response, distant cancer recurrence and survival. Up-regulation of intratumoral CD133 expression – more than pre- and post-RCT CD133 levels – plays an important role in therapy resistance, tumor progression and metastasis in rectal cancers undergoing neoadjuvant RCT.

Abstract 958: PELP1 regulation of mTOR Axis: Role in breast cancer progression and resistance

Abstract Hormonal therapy resistance is a major clinical problem. Emerging evidence suggests that estrogen (ER) participates in extra-nuclear signaling events in the cytoplasm / membrane and such actions may play a role in therapy resistance. Recent studies suggested that mTOR pathway play a critical role in ER-mediated cell proliferation and growth factor signaling crosstalk. Proline, Glutamic-acid and Leucine-rich Protein 1 (PELP1) participates in ER extra-nuclear actions. PELP1 is a prognostic indicator of shorter breast cancer specific survival and PELP1 expression is predominantly in the cytoplasm in a subset of breast tumors. The objective of this application is to test whether cross talk occurs between mTOR-PELP1 signaling axis and whether mTOR targeting drugs can be used to target PELP1 oncogenic functions leading to therapy resistance. We have tested this hypothesis using ER-positive breast cancer cells that over express PELP1 (MCF7-PELP1, ZR75-PELP1) and models cells lacking PELP1 (MCF7-PELP1 shRNA, ZR-75-PELP1 shRNA). Vector transfected ZR75 and MCF7 cells were used as controls. Rapamycin and AZD8055 were used as pharmacological inhibitors to block mTOR pathway. Over expression of PELP1 enhanced the estrogen and Heregulin mediated cell proliferation in breast cancer cells. Rapamycin (10−7M) or AZD8055 (10−8M) treatment significantly reduced PELP1 driven growth in both MCF7 and ZR75 cells that are treated with estrogen. Similarly, Rapamycin and AZD8055 treatment of MCF7 and ZR75 cells significantly reduced PELP1 mediated increase in cell growth of Heregulin stimulated cells. Mechanistic studies using yeast two hybrid screen and reciprocal immunoprecipitations demonstrated that PELP1 directly interacts with mTOR and PP2A. PELP1cyto model cells that uniquely express PELP1 in the cytoplasm, exhibit excessive activation of mTOR signaling pathway upon estrogen and growth factor stimulation. Over expression of PELP1 in the breast cancer model cells increased phosphorylation of mTOR axis components (phos-mTOR, -S6K, -4EBP1,-AKT). Further, Knock down of PELP1 with siRNA significantly reduced the activation of mTOR signaling components upon estrogen and Heregulin stimulation. Immunohistochenistry studies using xenograft tumor tissues with PELP1 overexpression and PELP1cyto driven tumors with PELP1 deregulation showed correlation of PELP1 expression with the activation of mTOR signaling components and that PELP1cyto driven tumors have excessive activation of mTOR signaling components. Combination therapy of Tamoxifen and Rapamycin or AZD8055, sensitized PELP1 deregulated cells to hormonal therapy. Our results suggest that PELP1 driven oncogenic functions involve PELP1 modulation of mTOR signaling and blockade of mTOR signaling render PELP1 driven tumors highly sensitive to therapeutic inhibition. 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 958. doi:1538-7445.AM2012-958

The Role of Bcl-2 Family Proteins in Therapy Responses of Malignant Astrocytic Gliomas: Bcl2L12 and Beyond

Glioblastoma (GBM) is a highly aggressive and lethal brain cancer with a median survival of less than two years after diagnosis. Hallmarks of GBM tumors include soaring proliferative indices, high levels of angiogenesis, diffuse invasion into normal brain parenchyma, resistance toward therapy-induced apoptosis, and pseudopallisading necrosis. Despite the recent advances in neurosurgery, radiation therapy, and the development of targeted chemotherapeutic regimes, GBM remains one of the deadliest types of cancer. Particularly, the alkylating agent temozolomide (TMZ) in combination with radiation therapy prolonged patient survival only marginally, and clinical studies assessing efficacies of targeted therapies, foremost ATP mimetics inhibiting the activity of receptor tyrosine kinases (RTKs), revealed only few initial responders; tumor recurrence is nearly universal, and salvage therapies to combat such progression remain ineffective. Consequently, myriad preclinical and clinical studies began to define the molecular mechanisms underlying therapy resistance of GBM tumors, and pointed to the Bcl-2 protein family, in particular the atypical member Bcl2-Like 12 (Bcl2L12), as important regulators of therapy-induced cell death. This review will discuss the multi-faceted modi operandi of Bcl-2 family proteins, describe their roles in therapy resistance of malignant glioma, and outline current and future drug development efforts to therapeutically target Bcl-2 proteins.

RNAi screening in glioma stem-like cells identifies PFKFB4 as a key molecule important for cancer cell survival

The concept of cancer stem-like cells (CSCs) has gained considerable attention in various solid tumors including glioblastoma, the most common primary brain tumor. This sub-population of tumor cells has been intensively investigated and their role in therapy resistance as well as tumor recurrence has been demonstrated. In that respect, development of therapeutic strategies that target CSCs (and possibly also the tumor bulk) appears a promising approach in patients suffering from primary brain tumors. In the present study, we utilized RNA interference (RNAi) to screen the complete human kinome and phosphatome (682 and 180 targets, respectively) in order to identify genes and pathways relevant for the survival of brain CSCs and thereby potential therapeutical targets for glioblastoma. We report of 46 putative candidates including known survival-related kinases and phosphatases. Interestingly, a number of genes identified are involved in metabolism, especially glycolysis, such as PDK1 and PKM2 and, most prominently PFKFB4. In vitro studies confirmed an essential role of PFKFB4 in the maintenance of brain CSCs. Furthermore, high PFKFB4 expression was associated with shorter survival of primary glioblastoma patients. Our findings support the importance of the glycolytic pathway in the maintenance of malignant glioma cells and brain CSCs and imply tumor metabolism as a promising therapeutic target in glioblastoma.

Molecular Profiling of Direct Xenograft Tumors Established from Human Pancreatic Adenocarcinoma After Neoadjuvant Therapy

Pancreatic adenocarcinoma is among the most resistant of human cancers, yet specific mechanisms of treatment resistance remain poorly understood. Models to study pancreatic cancer resistance remain limited and should reflect in vivo changes that occur within patient tumors. We sought to identify consistent, differentially expressed genes between treatment of naive pancreatic tumors and those exposed to neoadjuvant therapy using a strict, in vivo direct xenograft model system. Over a 42-week period, 12 untreated and treated patient tumors were successfully engrafted into NOD/SCID mice. RNA from each treatment group (5 untreated and 4 treated) was isolated in triplicate and subjected to global gene expression analysis. Consistent gene expression changes with treatment were identified and confirmed using RT-PCR and immunohistochemistry. Engraftment of untreated patient tumors was more frequent than treated tumors (17 of 21 versus 16 of 49, P = .0002) but without differences in observed time until tumor formation. The histology of patient tumors was recapitulated in direct xenograft tumors. Relative to untreated tumors, treated tumors consistently demonstrated more than a 2-fold reduction in TGFβ-R2 mRNA expression and more than a 5-fold increase in IGFBP3 expression (P < .0218) and were confirmed by immunohistochemistry. Engraftment of human pancreatic tumors into immunodeficient mice prior to and following neoadjuvant therapy is possible and provides an in vivo platform for comparison of global gene expression patterns. The decreased TGFβ-R2 expression and increased IGFBP3 expression among direct xenograft tumors derived from treated tumors relative to untreated tumors suggests a role in therapy resistance and warrants further study.

Abstract 693: Evaluating the role of CXCR2 receptor and its ligand in breast cancer therapy resistance

Abstract Breast cancer is the second leading cause of cancer deaths among women after lung cancer, accounting for more than 40,000 deaths each year. Although there has been advancement in the treatment and diagnosis of breast cancer, therapy resistance and relapse are still big hurdles in the way. Recent reports and our preliminary studies suggest that malignant cells that survive the initial chemo- and radiation therapy express higher levels of CXCR2 ligands which might provide a survival benefit to malignant tumors leading to therapy resistance. The specific objective for this study involves targeting CXCR2 receptor and its ligands mediated signaling for therapy resistance in mammary tumor cells. Our hypothesis is that CXCR2 along with its ligands plays an important role in therapy resistance and that targeting this signaling pathway will provide an effective strategy to overcome the problem. We used mammary tumor cells expressing different levels of CXCR2 (Cl66-wt and Cl66-shCXCR2) and examined their response to Paclitaxel and Doxorubicin. We observed significant enhancement of paclitaxel and doxorubicin-mediated toxicity at lower concentrations in Cl66-shCXCR2 cells as compared to Cl66-wt cells. Moreover, we observed an increase in the expression of CXCL1, a CXCR2 ligand, with increasing doses of drugs. However, paclitaxel and doxorubicin induced CXCL-1 levels was significantly lower in Cl66-shCXCR2 cells as compared to Cl66-wt cells. To further validate our findings we selected Cl66 against paclitaxel and doxorubicin resistant cells by treating with increasing doses of these drugs. Cells were maintained in 500nM doxorubicin and 400nM paclitaxel. We analyzed the sensitivity of these cells to doxorubicin and paclitaxel in comparison to wild type parent cells. We observed that resistant cells survive even at higher concentrations (higher than at which they are maintained) of drugs. Although, paclitaxel resistant cells appears to be sensitive at higher doses of paclitaxel when compared to doxorubicin resistant cells in the same set of experiments. Further studies showed an increase in the expression of CXCL1 in the supernatant of these cells after drug treatment. The expression of CXCL1 was higher even at the basal level in the resistant cells in comparison to the parent cells. As it has been shown that expression of CXCR2 ligands increase after chemotherapy, we examined the expression profile of various CXCR2 ligands in these cells at the mRNA level. We observed that these cells express higher levels of CXCL5, CXCL3 and CXCL7, in comparison to parent Cl66 cells. Together, these results demonstrate that i) CXCR2 and its ligand-mediated signaling plays an important role in the drug resistance of mammary tumors cells; ii) that paclitaxel seems to be more effective against mammary tumor cells in comparison to doxorubicin; and iii) targeting CXCR2 expression enhanced chemotherapeutic responses. 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 693. doi:10.1158/1538-7445.AM2011-693

Abstract 4613: Targeting ER-KDM1 axis to sensitize therapy resistant breast cancer cells to hormonal therapy

Abstract Estrogen-induced breast carcinogenesis is characterized by global changes in histone modifications. KDM1 (also known as LSD1) a histone demethylase enzyme, plays a key role in establishing specific histone methylation marks at ERα target gene promoters. Emerging evidence suggests histone methylation imposes ligand dependency; and deregulation of this epigenetic pathway potentially contributes to hormonal independence and adaptive resistance in breast cancer. Our study examined the therapeutic efficacy of pargyline, a KDM1 inhibiting drug, and evaluated the therapeutic benefit to curb growth of therapy resistant breast cancer cells. We used several model cell lines shown to exhibit resistance to hormonal therapy including MCF7-HER2, MCF7-Tam, MCF7-Ca-LTLT, MCF7-PELP1 and parental MCF7 cells as controls. Reporter gene assays showed KDM1 enhanced ERα- mediated transcription in therapy resistant cells. KDM1 functionally interacts with ERα coregulator PELP1 and is co-recruited with PELP1 to ERα target genes. Pargyline treatment substantially inhibited ERα transactivation functions. ChIP analysis revealed distinct activating histone methylation modifications at growth regulatory ERα target genes in aggressively growing and therapy resistant breast cancer cells. Breast cancer models treated with pargyline facilitated reversal of the observed specific modifications and thereby inhibited the growth of breast cancer cells in vitro and in vivo nude mice models. Pargyline also showed significant effect in blocking local estrogen synthesis via alterations in histone methylation modifications at the aromatase promoter. Combinational therapy using three agents that: (a) block ERα's nuclear actions (tamoxifen or letrozole), (b) ERα's extranuclear actions (dasatinib) and (c) ERα's epigenetic modifications (pargyline) showed most promising therapeutic effect compared to single agent therapy on the growth of therapy resistant cells. Our results suggest that histone methylation modifications play a role in therapy resistance and validate the therapeutic potential of pargyline in combinational therapies. Collectively, our results suggests targeting KDM1 axis with pargyline in combination with current endocrine therapies will have better therapeutic effect and may inhibit or delay development of hormonal resistance. This study is funded by Komen grant KG090447. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4613.