Loss Of Tumor Suppressor Research Articles

Biallelic Dicer1 Loss Mediated by aP2-Cre Drives Angiosarcoma.

Angiosarcoma is an aggressive vascular sarcoma with an extremely poor prognosis. Because of the relative rarity of this disease, its molecular drivers and optimal treatment strategies are obscure. DICER1 is an RNase III endoribonuclease central to miRNA biogenesis, and germline DICER1 mutations result in a cancer predisposition syndrome, associated with an increased risk of many tumor types. Here, we show that biallelic Dicer1 deletion with aP2-Cre drives aggressive and metastatic angiosarcoma independent of other genetically engineered oncogenes or tumor suppressor loss. Angiosarcomas in aP2-Cre;Dicer1Flox/- mice histologically and genetically resemble human angiosarcoma. miR-23 target genes, including the oncogenes Ccnd1 as well as Adam19, Plau, and Wsb1 that promote invasiveness and metastasis, were enriched in mouse and human angiosarcoma. These studies illustrate that Dicer1 can function as a traditional loss-of-function tumor suppressor gene, and they provide a fully penetrant animal model for the study of angiosarcoma development and metastasis. Cancer Res; 77(22); 6109-18. ©2017 AACR.

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation.

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

PTEN in the maintenance of genome integrity: From DNA replication to chromosome segregation.

Faithful DNA replication and accurate chromosome segregation are the key machineries of genetic transmission. Disruption of these processes represents a hallmark of cancer and often results from loss of tumor suppressors. PTEN is an important tumor suppressor that is frequently mutated or deleted in human cancer. Loss of PTEN has been associated with aneuploidy and poor prognosis in cancer patients. In mice, Pten deletion or mutation drives genomic instability and tumor development. PTEN deficiency induces DNA replication stress, confers stress tolerance, and disrupts mitotic spindle architecture, leading to accumulation of structural and numerical chromosome instability. Therefore, PTEN guards the genome by controlling multiple processes of chromosome inheritance. Here, we summarize current understanding of the PTEN function in promoting high-fidelity transmission of genetic information. We also discuss the PTEN pathways of genome maintenance and highlight potential targets for cancer treatment.

Advances in Pathobiology of Primary Central Nervous System Lymphoma.

Objective:Primary central nervous system lymphoma (PCNSL) is a specific type of non-Hodgkin lymphoma with poor prognosis. The rare incidence of this disease and difficulty to obtain sufficient tissue material impede deep research into PCNSL. However, application of modern molecular techniques makes it possible to find biological characteristics exclusive to PCNSL. Therefore, we systematically reviewed the latest research progress on biological characteristics and pathogenesis of PCNSL.Data Sources:The data analyzed in this review were from the articles listed in PubMed database.Study Selection:Articles focusing on the biology of PCNSL at the cytogenetic or molecular level were reviewed, including clinical, basic research, and review articles.Results:With respect to histopathology, perivascular growth pattern and reactive perivascular T-cell infiltration are regarded as typical histopathological manifestations of tumor cells in PCNSL. Moreover, tumor cells of PCNSL predominantly express an activated B-cell-like phenotype, including CD10− BCL-6+ MUM1+, CD10− BCL-6− MUM1+, and CD10− BCL-6− MUM1−. On the molecular level, some molecular and genetic alterations may contribute to malignant transformation, including mutations of proto-oncogenes and tumor suppressor genes, gains and losses of genetic material, as well as aberrant activation of some important signaling pathways, such as nuclear factor-κB and JAK/STAT pathway.Conclusions:The integrated molecular mechanisms involved in pathogenesis of PCNSL are not well understood. The important biomarkers indicating prognosis are not identified. Multicenter studies should be carried out to elucidate pathogenesis of PCNSL to find novel and effective therapeutic strategies.

P53 loss does not permit escape from BrafV600E-induced senescence in a mouse model of lung cancer.

Lung cancer arises through the acquisition of a number of genetic lesions, with a preponderance of activating mutations in the canonical mitogen-activated protein kinase (MAPK) cascade (RTK-RAS-RAF-MEK). BrafV600E expression induces benign lung adenomas that fail to progress to adenocarcinoma because of oncogene-induced senescence (OIS). BrafV600E expression, coupled with simultaneous p53 ablation, permits bypass of senescence and progression to lung adenocarcinoma. However, spontaneous human tumors sustain mutations in a temporally separated manner. Here, we use a mouse lung cancer model where oncogene activation (BrafV600E expression) and tumor suppressor loss (p53 ablation) are independently controlled through the actions of Flp and Cre recombinase, respectively. We show that p53 loss before OIS is permissive for the transition from lung adenoma to adenocarcinoma. In contrast, p53 loss after senescence is established fails to enable escape from senescence and disease progression. This study demonstrates that BrafV600E induced senescence is irreversible in vivo and suggests that therapy-induced senescence would halt further tumor progression.

Gleason Score 7 Prostate Cancers Emerge through Branched Evolution of Clonal Gleason Pattern 3 and 4.

Purpose: The molecular features that account for the distinct histology and aggressive biological behavior of Gleason pattern 4 (Gp4) versus Gp3 prostate cancer, and whether Gp3 tumors progress directly to Gp4, remain to be established.Experimental Design: Whole-exome sequencing and transcriptome profiling of laser capture-microdissected adjacent Gp3 and cribiform Gp4 were used to determine the relationship between these entities.Results: Sequencing confirmed that adjacent Gp3 and Gp4 were clonal based on multiple shared genomic alterations. However, large numbers of unique mutations in the Gp3 and Gp4 tumors showed that the Gp4 were not derived directly from the Gp3. Remarkably, the Gp3 tumors retain their indolent-appearing morphology despite acquisition of multiple genomic alterations, including tumor suppressor losses. Although there were no consistent genomic alterations that distinguished Gp3 from Gp4, pairwise transcriptome analyses identified increased c-Myc and decreased p53 activity in Gp4 versus adjacent clonal Gp3 foci.Conclusions: These findings establish that at least a subset of Gp3 and aggressive Gp4 tumors have a common origin, and support a branched evolution model wherein the Gp3 and Gp4 tumors emerge early from a common precursor and subsequently undergo substantial divergence. Genomic alterations detectable in the Gp3 may distinguish these tumors from truly indolent Gp3. Screening for a panel of these genomic alterations in men who have prostate biopsies showing only Gp3 (Gleason score 6, Gs6) may allow for more precise selection of men who can be safely managed by active surveillance versus those who may benefit from further intervention. Clin Cancer Res; 23(14); 3823-33. ©2017 AACR.

Highlights of This Issue

Osteosarcoma (OS) is a highly aggressive bone tumor of childhood and adolescence for which alternative therapeutic options are urgently needed. Baglio and colleagues demonstrate that osteosarcoma cells release TGFβ-rich extracellular vesicles that educate mesenchymal stem cells to support tumor progression through IL6 secretion. Therapeutic blockade of IL6R prevents lung metastasis formation induced by tumor-educated MSCs in an orthotopic xenograft mouse model of osteosarcoma. OS patients have activated TGFβ and stroma-dependent IL6 signaling and display elevated circulating levels of EV-associated TGFβ. This study provides the basis for the use of IL6 and TGFβ blocking agents as new therapeutic strategy for osteosarcoma.Robust preclinical models of pediatric acute lymphoblastic leukemia (ALL) are essential in prioritizing novel drugs for clinical evaluation. Jones and colleagues developed bioluminescence imaging (BLI)-enabled ALL patient-derived xenografts (PDXs), and compared BLI with traditional flow cytometric methods in evaluating preclinical drug efficacy. BLI monitored remission with greater range than PB measurements and allowed measurement of leukemia in discrete organs. Correlating BLI and flow cytometry data enabled noninvasive assessment of minimal residual disease, as well as the establishment of novel scoring parameters. This article highlights the utility of BLI in enabling stringent evaluation of drug efficacy in ALL PDXs, ensuring that only the most promising agents are put forward for clinical assessment.Implementation of primary high-risk HPV testing in cervical screening results in better protection against cervical (pre)cancer. However, its specificity for clinically meaningful disease is lower than that of current cytology-based screening. Therefore, molecular markers that identify clinically relevant lesions would reduce over-referral and overtreatment. Following a genome-wide approach, Verlaat and colleagues identified three novel methylation markers located at a chromosomal gain at 3q, which showed an excellent clinical performance for detection of cervical (pre)cancer in cervical scrapes. These markers enable the detection of advanced precursor lesions with a high short-term progression risk to cancer. These findings could greatly improve cervical screening.Many Gleason pattern 3 (Gp3) prostate cancers detected on biopsy are indolent and do not require further therapy, but a subset of these patients will have or develop higher-grade cancers. Sowalsky and colleagues establish that a subset of Gp3 tumors emerge early from a common precursor lesion that also can progress to Gp4. These Gp3 tumors have molecular features, including tumor suppressor losses that may distinguish them from indolent Gp3, and which may be used clinically as biomarkers to identify patients with only Gp3 on biopsy who are at increased risk of having occult Gp4.

Androgen induces G3BP2 and SUMO-mediated p53 nuclear export in prostate cancer.

The androgen receptor (AR) has a central role in prostate cancer progression, particularly treatment-resistance disease including castration-resistant prostate cancer. Loss of the p53 tumor suppressor, a nuclear transcription factor, is also known to contribute to prostate malignancy. Here we report that p53 is translocated to the cytoplasm by androgen-mediated induction of G3BP2, a newly described direct target gene of AR. G3BP2 induces both cell cycle progression and blocks apoptosis. Translocation of p53 is regulated by androgen-dependent sumoylation mediated by the G3BP2-interacting SUMO-E3 ligase, RanBP2. G3BP2 knockdown results in reduced tumor growth and increased nuclear p53 accumulation in mouse xenograft models of prostate cancer with or without long-term androgen deprivation. Moreover, strong cytoplasmic p53 localization is correlated clinically with elevated G3BP2 expression and predicts poor prognosis and disease progression to the hormone-refractory state. Our findings reveal a new AR-mediated mechanism of p53 inhibition that promotes treatment-resistant prostate cancer.

Abstract LB-015: Dissecting TGF beta-induced lethal EMT in Kras-driven pancreatic cancer

Abstract Pancreatic cancer, mostly pancreatic ductal adenocarcinoma (PDAC), is a devastating malignancy with an extremely poor prognosis. New insights are urgently needed into the tumorigenesis process and the tumor suppression mechanisms of pancreatic cancer. Genetic alterations in PDAC occur in a temporal sequence, usually initiating with activating mutations of the KRAS (~95%), and progressing by loss of tumor suppressors, including INK4A/ARF, TP53 and SMAD4/DPC4. Our recent work reveals that in mouse KrasG12D-driven PDAC cells, TGFβ induces a robust epithelial-to-mesenchymal transition (EMT) response, which leads to cell apoptosis. To determine the KrasG12D-dependent mechanism of priming pancreatic epithelial cells to be sensitive to TGFβ-induced lethal EMT, we utilize systematic approaches to integrate the transcriptome profile and Smad2/3 binding patterns to the genome to identify candidate mediators that regulate TGFβ-induced lethal EMT. To identify TGFβ gene response changes mediated by KrasG12D, we perform RNA-Seq analysis of inducible Tet-On KrasG12D pancreatic epithelial cells (PECs) treating with TGFβ or SB505124. Systematic analyses reveal that mutant KrasG12D, but not KrasWT, enables robust TGFβ gene responses of Snai1, Has2, Has3 and IL11. The massive induction of these genes is crucial for TGFβ-induced lethal EMT. Smad2/3 ChIP-Seq analysis reveals that transcription factor binding motifs of Rreb1 are specially associated with these KrasG12D-enhanced Smad2/3 binding sites. Furthermore, depletion of Rreb1 greatly impairs TGFβ-induced apoptosis. In summary, oncogenic KrasG12D drives a cell state change and sensitizes premalignant PECs to respond to TGFβ-induced lethal EMT. This function switch of TGFβ is potentially mediated through Kras-activated Rreb1 as a specific DNA binding partner of Smads. Citation Format: Jie Su. Mem. Dissecting TGF beta-induced lethal EMT in Kras-driven pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-015. doi:10.1158/1538-7445.AM2017-LB-015

Abstract 3978: B7-H1/PD-L1 soluble expression in multiple cancer subtypes: High sensitivity measurement by immunoassay

Abstract Aberrant expression of the B7-H1/PD-L1 immunomodulatory molecule is evident in many forms of cancer. The evidence for increased protein levels of B7-H1/PD-L1 comes largely from immunohistochemistry analysis of surgical and biopsy specimens. While several studies attempt to correlate B7-H1/PD-L1 expression with poor prognosis, there are studies that conclude no correlation based on technical difficulties associated with detecting B7-H1/PD-L1 protein expression on the cell surface. We were able to quantifiably measure soluble B7-H1/PD-L1 in conditioned medium from tumor cell cultures using two new immunoassays, a Quantikine® ELISA and a Simple Plex™ Assay. We show that breast, prostate, glioma, and non-Hodgkin’s lymphoma cells express various levels of B7-H1/PD-L1 which correlate to their PTEN or proliferation status. This is in agreement with previous findings showing that increases in B7-H1/PD-L1 expression levels are frequently coincident with loss of the PTEN tumor suppressor or increased activation of the phosphoinositide 3-kinase (PI 3-K) pathway. The androgen-responsive LNCaP prostate cancer cell line does not exhibit measurable B7-H1/PD-L1 in any instance, while the androgen-independent prostate cancer cell line PC-3 expresses measurable B7-H1/PD-L1 that is further inducible with PMA treatment. In all cell lines included in our study, incubation with PMA increases the level of B7-H1/PD-L1 released into the culture media, while pretreatment with the PI 3-K inhibitor, LY294002, abrogates this increase. Importantly, the Simple Plex platform is able to quantify very low levels of B7-H1/PD-L1 that are below detection by ELISA. Our data suggest that combining the Quantikine ELISA and Simple Plex platforms is a powerful strategy for reliably and quantitatively assessing soluble B7-H1/PD-L1 levels. Citation Format: Justin Haworth, Rainer Grant, Samuel Thayer, Melissa Floren, Nathan Steere, Isabel O'Brien, Paul Younge, Michael Anderson, Greta Wegner, Kathryn Brumbaugh. B7-H1/PD-L1 soluble expression in multiple cancer subtypes: High sensitivity measurement by immunoassay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3978. doi:10.1158/1538-7445.AM2017-3978

Abstract 794: Potential role of DLL4 in uveal melanoma vascular mimicry

Abstract Uveal melanoma is the most common malignancy of the eye. Thanks to gene array analysis it is possible to classify uveal melanoma in Class 1 (low metastasis risk) and Class 2 (high metastasis risk) tumor. This classification will ultimately determine the tumor treatment, risk of metastasis and patient surveillance. Progression to metastasis remains by far the greatest problem in uveal melanoma and is associated with loss of BAP1 tumor suppressor. Bioinformatic analyses of RNA-Seq indicated that pro-angiogenic genes such as DLL4, VEGFA, VEGFC and HIF1a are overexpressed in Class 2 compared to Class 1 uveal melanoma while angiogenic inhibitors such as ZFP36L1, HIF1AN, VEGFB, VHL and HIF3A are downregulated. Further, we found that DLL4 is among the 5 most highly overexpressed genes associated with BAP1 loss in clinical specimens and in uveal melanoma cell lines induced to deplete BAP1. DLL4 is a Notch ligand known to regulate endothelial cells, bone marrow endothelial cell progenitors and angiogenesis. We hypothesize that DLL4 contributes to vascular mimicry in uveal melanoma. To test this hypothesis, we will test uveal melanoma cell lines induced to deplete BAP1 using shRNA in cell culture-based and in vivo models. The results of this research have the potential to elucidate the mechanism by which vascular mimicry occurs in uveal melanoma. Citation Format: Julia Escandon, Matthew G. Field, Stefan Kurtenbach, Jeffim Kuznetzov, Christina L. Decatur, J William Harbour. Potential role of DLL4 in uveal melanoma vascular mimicry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 794. doi:10.1158/1538-7445.AM2017-794

Abstract 1256: Non-steroidal anti-inflammatory drugs induce ER stress and have an immunomodulatory role in the suppression of colorectal tumorigenesis

Abstract Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths in the United States. A number of epidemiological studies, clinical trials, and animal studies have demonstrated the chemopreventive effect of non-steroidal anti-inflammatory drugs (NSAIDs) on CRC. However, the exact mechanism by which NSAIDs suppress colorectal tumorigenesis has remained unclear. Our previous study showed that NSAID-induced death receptor signaling, along with loss of the gatekeeper APC tumor suppressor, triggers a synthetic lethal interaction and apoptotic death in an otherwise normal cell. In this study, we determined how NSAIDs induce death receptor signaling, and found a critical role of the endoplasmic reticulum (ER) stress response. Treating CRC cells with the NSAIDs sulindac or indomethacin upregulated C/EBP homologous transcription factor (CHOP), a key mediator of the ER stress response that is required for the induction of death receptor 5 (DR5) by NSAIDs. Blocking ER stress abolished the apoptotic effect of NSAIDs in CRC cells and in normal colonic epithelial cells with knockdown of APC, and also suppressed the chemopreventive activity of NSAIDs in APCMin/+ mice. Furthermore, we identified plasma membrane translocation of calreticulin in cells undergoing NSAID-induced apoptosis, suggesting the involvement of immunomodulation in this tumor suppression. Our results provide novel insight into the chemopreventive mechanism of NSAIDs, which may help design more effective chemopreventive strategies and agents. Citation Format: Rochelle E. Fletcher, Lin Zhang, Brian Leibowitz, Jian Yu. Non-steroidal anti-inflammatory drugs induce ER stress and have an immunomodulatory role in the suppression of colorectal tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1256. doi:10.1158/1538-7445.AM2017-1256

Restoration of tumor suppression in vivo by systemic delivery of chemically-modified PTEN mRNA nanoparticles.

11582 Background: The onset and maintenance of cancer frequently involves gain of oncogenic function along with loss of tumor suppression. PTEN is a well-characterized tumor suppressor gene that is lost or mutated in many human cancers including ~50% of metastatic castration-resistant prostate cancer (mCRPC). Reintroduction of functional PTEN for mCRPC treatment has proven difficult. Methods: PTEN mRNA was synthesized by in vitro transcription method and modified with ARCA capping and enzymatic polyadenylation, and then substituted with Pseudo-UTP, 5’-Methyl-CTP. A robust self-assembly approach was employed to prepare PTEN mRNA nanoparticles (NPs) using cationic lipid-like compound G0-C14 and PLGA polymer coated with lipid-PEG shell. PTEN expression in tumors and PI3K-AKT pathway were confirmed by IHC and western blot, respectively. Apoptosis was checked by flow cytometry and Tunel assays. In vivo toxicity was studied by hematologic and histologic tests, and immune response. Results: We successfully restored PTEN mRNA to PTEN-null prostate cancer (PCa) cells via systemic delivery of mRNA NPs. These mRNA NPs are stable in serum, demonstrate minimal toxicity, and provide highly effective transfection in PCa cells (substantially higher HA-PTEN expression than plasmid PTEN transfection) and PCa xenograft tumors, leading to ~85% inhibition of tumor cell growth in vitro and in vivo. We also confirm mRNA NP-mediated systemic restoration of PTEN function in PTEN-null PCa and delineate its tumor suppression through inhibition of the PI3K-AKT pathway and enhancement of apoptosis. Conclusions: The work provides proof of principle for the systemic reintroduction of mRNA-based tumor suppressor genes to tumors in vivo. Because PTEN loss is frequent in late-stage PCa, this approach may have feasibility in this patient population. Considering the strong potential of mRNA therapy and the lack of systemic studies of in vivo mRNA transfection of tumors, this study sheds light on the useful application of NP-mediated mRNA delivery for validating tumor suppressors (e.g., PTEN) as a therapeutic target in cancer treatment where loss of a tumor suppressor contributes to the underlying genetic mechanism of cancer.

Circulating tumor cells (CTCs) N-terminal androgen receptor expression to identify patients (pts) with castrate resistant prostate cancer (CRPC) who are more sensitive to chemotherapy.

5034 Background: Loss of the retinoblastoma tumor suppressor (RB) function was identified as a major means to develop CRPC; the expression of the androgen receptor (AR) is under stringent RB control; and tumors devoid of RB function are hypersensitive to treatment with chemotherapy. Exploratory analysis evaluated baseline N-terminal AR expression in CTCs in men with chemotherapy-naïve CPRC and correlated to changes in PSA, leading us to inquire if this biomarker may identify pts sensitive to chemotherapy. Methods: In a multicenter phase II randomized trial of approved doses of abiraterone acetate/prednisone (AA-Arm 1) or combination AA and standard doses of cabazitaxel (AA/CBZ-Arm 2). Patients on AA received CBZ upon progression. Baseline CTCs were obtained on all pts and expression of N-terminal AR expression was performed by Epic Sciences. Positive AR N-terminal expression (AR+) was based on the presence of at least 1 CTC or CK- cell with AR N-terminal signal expression above the 3.0 positivity threshold. Serial PSAs were determined at baseline and every 3 weeks with routine labs and imaging every 12 weeks. Results: To date, 42 of 80 pts have been enrolled: 22 pts to AA, and 20 pts to AA/CBZ. Both regimens were well tolerated with 8/42 (19%) pts experiencing treatment-related grade 3 or 4 toxicities. Blood from 35 patients underwent CTC analysis. Seventy-seven percent of pts (27/35) had detectable CTCs; 11 of 35 pts (31%) had AR overexpression. Of the pts with AR+ CTCs, 1/5 pts treated with AA, and 5/6 pts treated with AA/CBZ had a PSA decline > 50% from baseline. Conclusions: Real-time CTC analysis of N-terminal AR expression was feasible and data suggests that this may identify a cohort of pts who may benefit from the combination of CBZ with AA. Further studies are ongoing to evaluate whether cellular heterogeneity and RB expression in CTCs play a role in identifying pts who would benefit from chemotherapy. The trial is coordinated by the Prostate Cancer Clinical Trials Consortium, LLC and funded by Sanofi US Services Inc. and Prostate Cancer Foundation. Clinical trial information: NCT02218606.

Insulin-like growth factor 2 axis supports the serum-independent growth of malignant rhabdoid tumor and is activated by microenvironment stress.

Malignant rhabdoid tumors (MRTs) are rare, lethal, pediatric tumors predominantly found in the kidney, brain and soft tissues. MRTs are driven by loss of tumor suppressor SNF5/INI1/SMARCB1/BAF47. The prognosis of MRT is poor using currently available treatments, so new treatment targets need to be identified to expand treatment options for patients experiencing chemotherapy resistance. The growth hormone insulin-like growth factor 2 (IGF2) signaling pathway is a promising target to overcome drug resistance in many cancers. Here, we evaluated the role of IGF2 axis in MRT cell proliferation. We showed that microenvironment stress, including starvation treatment and chemotherapy exposure, lead to elevated expression of IGF2 in the SNF5-deficient MRT cell line. The autocrine IGF2, in turn, activated insulin-like growth factor 1 receptor (IGF1R), insulin receptor (INSR), followed by PI3K/AKT pathway and RAS/ERK pathway to promote cancer cell proliferation and survival. We further demonstrated that impairment of IGF2 signaling by IGF2 neutralizing antibody, IGF1R inhibitor NVP-AEW541 or AKT inhibitor MK-2206 2HCl treatment prevented MRT cell growth in vitro. Taken together, our characterization of this axis defines a novel mechanism for MRT cell growth in the microenvironment of stress. Our results also demonstrated the necessity to test the treatment effect targeting this axis in future research.

Branched chain amino acid metabolism and cancer: the importance of keeping things in context.

Because non-homeostatic proliferation increases anabolic demand, tumor cells reprogram metabolism in ways that support growth. Although tumor cells retain some metabolic flexibility, the constitutive activation of oncogenes and mutation or loss of tumor suppressors limits their metabolic choices and creates nutrient dependencies not present in their normal counterparts (1-8). Identifying and targeting these differences in metabolic wiring will likely be an effective means to limit tumor growth while sparing normal cells. Clearly, different oncogenic mutations activate distinct downstream gene expression programs that drive metabolic reprogramming in ways that favor certain biosynthetic routes. At the same time, oncogenic events occur in the divergent epigenetic landscapes associated with different tissues of origin. As normal lung and pancreatic cells from the same individual contain identical genomes yet exhibit markedly different gene expression patterns, it stands to reason that tumor cells with shared oncogenic drivers but different tissue origins would do the same.

45P - Targeting the vulnerability of RB tumor suppressor loss in triple negative breast cancer

45P - Targeting the vulnerability of RB tumor suppressor loss in triple negative breast cancer

Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells

Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. DNA2 nuclease is an evolutionarily conserved essential enzyme in replication and HR repair. Here we demonstrate that DNA2 is overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90–95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA double strand break end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anticancer drugs by targeting druggable nuclease DNA2. We propose DNA2 inhibition as new strategy in cancer therapy by targeting replication stress, a molecular property of cancer cells that is acquired as a result of oncogene activation instead of targeting currently undruggable oncoprotein itself such as KRAS.

Abstract A25: Sensing stress in cancer: a novel therapy targeting protein synthesis through the unfolded protein response in prostate cancer development

Abstract Distinct oncogenic lesions within components of the Myc or PI3K pathways hijack the translation machinery, to establish an aberrant “cancer proteome”, that drives cancer cell growth and tumor development. Invariably, however, increases in the protein synthesis capacity of cancer cells impose a cellular stress that requires an adaptive or evasive response in order for cancer cells to survive and continue proliferating. One of the key cytoprotective stress response pathways is known as the unfolded protein response (UPR), which is engaged when an excess of unfolded/misfolded proteins accumulate within the lumen of the endoplasmic reticulum. How the specific arms of UPR are coupled to increased protein synthesis in cancer etiology and drug response is still poorly understood. In this study we established a new genetic mouse model to address the role of protein synthesis-dependent activation of the UPR in prostate cancer initiation and maintenance promoted by loss of the PTEN tumor suppressor and overexpression of the Myc oncogene. We show that in this epithelial cancer model, Myc hyperactivation and PTEN loss synergize to dramatically stimulate PERK and IRE-1α activity, two major signaling arms of the UPR pathway. Distinct mechanisms transduce adaptive signals under conditions of ER stress: PERK control of protein synthesis through phosphorylation of the key translation initiation factor eIF2α and IRE1α-mediated splicing of the XBP-1 mRNA to produce an active and stable XBP1 transcription factor (XBP1s). Importantly, we observed that UPR activation is associated with formation of prostatic intraepithelial neoplasia (PIN) and invasive carcinoma. Strikingly, the pharmacologic inhibition of eIF2α phosphorylation leads to tumor inhibition. We have further established patient derived xenograft (PDX) models from primary and metastatic human prostate tumors to extend the role of UPR in specific steps of human tumor development. We have also developed a series of novel mouse and human prostate cancer cell culture systems to define the role of localized translation of ER-associated ribosomes during oncogenic transformation. Together this work provides critical insight into a fundamental mechanism by which the augmented protein synthesis requirement of cancer cells is coupled to an adaptation of ER cellular stress, reflecting a new cancer-specific vulnerability that is targetable. Citation Format: Crystal S. Conn, Hao G. Nguyen, Yae Kye, John T. Cunningham, Charles Truillet, Michael Evans, Tony L. Huynh, Peter Walters, Davide Ruggero. Sensing stress in cancer: a novel therapy targeting protein synthesis through the unfolded protein response in prostate cancer development. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A25.

Abstract P1-08-01: Regulation of estrogen receptor-α by NF1

Abstract Background. Although great strides have been made in targeting the ER pathway for treating ER+ breast cancer, relapse and death is common and is closely linked to resistance to ER-targeting agents. As a result, the majority of deaths from breast cancer still come from ER+ tumors. To discover drivers for endocrine resistance, we have sequenced tumor DNAs from a cohort of >600 patients treated with 5-year tamoxifen (Tam) monotherapy with a median 10.4 years follow up. Our preliminary data show that the worst outcome mutations (Hazard Ratio of ∼3 for relapse) were mostly those of the Neurofibromatosis type 1 (NF1) gene (encoding Neurofibromin), with nonsense/frame shift mutations creating early stop codons. Germline NF1mutations cause neurofibromatosis type 1, a common inherited disorder that predisposes individuals to both benign and malignant tumors of the nervous system, as well as an increased risk for breast cancer. Analysis of DNA sequencing data has also shown that the NF1 gene is mutated in a wide range of common cancers (e.g., melanoma, lymphoma, and cancers of the lung, breast, and colon). Thus, NF1-deficiency underlies the formation and/or progression of a large number of cancers, so that the development of therapies targeted to NF1-deficient malignancies would have broad impact. These observations support the hypothesis that NF1 gene inactivation is associated with aggressive tumor behaviors, such as endocrine therapy resistance in breast cancer. The key focus of this study is to define how the NF1 protein neurofibromin, regulates endocrine therapy resistance. Although neurofibromin is best known as a negative regulator for Ras, our data show that it may have other functions. Method. Our data suggest that many of the identified nonsense/frame shift create a NF1 null state; thus, we have used gene-silencing to recapitulate the effects of such NF1 mutations on the activities of ER+ breast cancer cells. NF1+ and NF1– ER+ breast cancer cells were grown in defined media to measure how estradiol (E2) and Tam impact their growth, transforming activities, and gene expression. The binding between neurofibromin and components of the ER transcriptional pathway was measured biochemically and using the mammalian two-hybrid system. Results. Our data showed that NF1-silenced cells use Tam as an agonist and can grow with very little E2, and these activities are driven by enhanced recruitment of ER to the ERE, leading to efficient expression of many classic ER-responsive genes. Expressing the NF1-GAP domain does not restore normal responses to Tam and E2 in NF1-silenced cells, suggesting that neurofibromincan regulate ER activity in a Ras-independent manner. To investigate the possibility that neurofibromin can directly regulate ER, we found that it can bind ER; furthermore, neurofibromin was more strongly recruited to the ERE by Tam than by E2. Conclusion. Our data support a model whereby neurofibromin acts like a co-repressor for ER. As such,NF1 loss may result in more aggressive tumor behaviors by activating, not only the Ras pathways, but also the ER transcriptional pathways. Simultaneous activation of two powerful oncogenic pathways by the loss of a single tumor suppressor may explain why neurofibromin is such a potent tumor suppressor lost in a wide range of cancers. Citation Format: Zheng Z-Y, Cakar B, Lavere P, Cao J, Yao J, Singh P, Lei JT, Toonen JA, Haricharan S, Anurag M, Shah K, Kavuri M, Chan DW, Chen X, Gutmann DH, Foulds CE, Ellis MJ, Chang EC. Regulation of estrogen receptor-α by NF1 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-01.