bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract PR01: Loss of the hippo pathway scaffold “Kibra” in a mouse model of human claudin-low breast cancer
Authors: Knight Jennifer F. , Lesurf Robert , Saleh Sadiq M. , Davis Ryan R. , Zhao Hong , Zuo Dongmei , Cardiff Robert D. , Gregg Jeff , Hallett Michael , Park Morag
Published: May 2015

Human claudin-low breast cancers belong to the triple negative subclass of the disease. Triple negative breast cancers (TNBCs) cannot be treated with targeted therapies that exist for estrogen receptor and Her2-positive breast cancers, emphasizing the need for new therapeutic targets. We generated a mouse model which recapitulates key features of the claudin-low subtype (Knight et al., PNAS 2013). In this model, expression of the Met receptor tyrosine kinase synergizes with loss of the tumor suppressor gene p53 (MMTV-Met;Trp53fl/+;MMTV-Cre) in driving mammary tumorigenesis. MMTV-Met;Trp53fl/+;Cre mammary tumors have a predominately spindloid pathology, with low expression of cell junction molecules and epithelial markers, and high expression of an epithelial-to-mesenchymal transition (EMT) signature.

Use of array-CGH to assess the genomic landscape of MMTV-Met;Trp53fl/+;Cre claudin-low tumors identified amplification of the endogenous Met locus, suggestive of oncogene addiction. Consistent with this, primary cells derived from these tumors were dependent upon Met signaling for proliferation and survival. Furthermore, Met inhibition in these cells was able to partially restore cell-cell junctions.

In addition to Met amplification, a consistent genomic event in MMTV-Met;Trp53fl/+;Cre tumors was loss of a chromosome 11 region, syntenic with human chromosome 5q, loss of which is a frequent event in human TNBC. Located within this region is the gene Wwc1, also known as Kibra, which encodes a scaffold protein known to positively regulate the Hippo tumor suppressor pathway. To understand the contribution of Kibra loss to claudin-low biology, we performed Kibra RNA interference in MMTV-Met (wildtype p53) solid carcinoma cells, in which Kibra expression is retained. Knockdown of Kibra led to weakening of cell-cell junctions and colony dispersal, consistent with EMT induction.

This work supports that Kibra loss and consequential disruption of the Hippo pathway, may play a significant role in claudin-low biology. Our work now focuses on re-induction of Hippo signaling in human and mouse claudin-low tumor cells to increase our understanding of the role of this pathway in tumor promotion and to provide new possibilities for therapeutic intervention in TNBCs.

This abstract is also presented as Poster A010.

Citation Format: Jennifer F. Knight, Robert Lesurf, Sadiq M. Saleh, Ryan R. Davis, Hong Zhao, Dongmei Zuo, Robert D. Cardiff, Jeff Gregg, Michael Hallett, Morag Park. Loss of the hippo pathway scaffold “Kibra” in a mouse model of human claudin-low breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr PR01.
Abstract PR06: Overcoming resistance to PI3K inhibitors in breast cancer: Results of a pooled shRNA screen and combinatorial drug screen
Authors: Vora Sadhna , Elkabets Moshe , Kim Nayoon , Costa Carlotta , Liu Manway , Xiaoyan Li , Cao Alex , Schlegel Robert , Scaltriti Maurizio , Baselga Jose , Huang Alan , Engelman Jeffrey A.
Published: May 2015

The phosphatidylinositol 3-kinase (PI3K) pathway is deregulated across a broad spectrum of human cancers. Therefore, there have been strong efforts to inhibit this pathway, with many novel targeted agents, including isoform-specific PI3K inhibitors, pan-PI3K inhibitors, and dual PI3K/mTOR inhibitors, entering the clinic. Although initial responses have been observed in PIK3CA and HER2 amplified cancers, resistance frequently emerges, thereby limiting the benefit from these drugs. We developed several PI3K inhibitor resistant cell lines by culturing sensitive PIK3CA mutant cells in gradually escalating doses of PI3K inhibitor until resistance emerges, typically over a period of several months. We then used a drug screen utilizing ~40 targeted agents to identify drugs that synergize with PI3K inhibitors in the resistant cells. Subsequently, to elucidate mechanisms of resistance to PI3K inhibitors, we used a pooled shRNA screen to identify hairpins that sensitize the resistant cells to PI3K inhibition. These two complementary screens have identified mTORC as a likely mediator of resistance to PI3K inhibition: mTORC inhibitors synergize with PI3K inhibitors in the resistant cells, and furthermore, hairpins directed against mTORC (FRAP1) sensitize resistant cells to PI3K inhibition. Other promising therapeutic targets identified by these screens that may contribute to resistance to PI3K inhibitors include IGF1R and AKT.

This abstract is also presented as Poster B069.

Citation Format: Sadhna Vora, Moshe Elkabets, Nayoon Kim, Carlotta Costa, Manway Liu, Li Xiaoyan, Alex Cao, Robert Schlegel, Maurizio Scaltriti, Jose Baselga, Alan Huang, Jeffrey A. Engelman. Overcoming resistance to PI3K inhibitors in breast cancer: Results of a pooled shRNA screen and combinatorial drug screen. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr PR06.
Abstract A18: A novel, human-compatible mouse model for preclinical research on prolactin in breast cancer
Authors: Christensen Heather R. , Murawsky Michael K. , Gregerson Karen A.
Published: May 2015

Over 90% of all human breast cancers express receptors for prolactin (PRL). This mitogenic hormone has been associated with an increased risk for breast cancer (as supported by epidemiological and molecular biology research). Despite this evidence, knowledge of the precise mechanisms of action for PRL in human breast cancer is limited. This is likely due to the lack of a suitable experimental model for studying the potential role of PRL in breast cancer growth and metastasis in vivo. For other reproductive hormones, the research model that has proven to be critical for preclinical studies of cancer is the grafting of human cancer cells into immunodeficient mice. Currently, no such xenograft model exists for the study of human PRL (hPRL) in breast cancer. The primary reason for this is the significant structural differences between rodent and hPRL. The mouse PRL (mPRL) hormone does not activate the hPRL receptor. Thus, grafted human cancer cells cannot respond to circulating PRL produced by the mouse host. To address this void in the repertoire of research tools, we have generated a novel set of mouse strains that have been “humanized” with regard to the structural gene, tissue expression, and secretory patterns of PRL. These mice were created using bacterial artificial chromosome technology and have been crossed onto the mouse PRL knockout background, resulting in mice that synthesize and secrete hPRL in the absence of mPRL. Our characterization studies demonstrate that the hPRL within the mice is: (1) expressed in a human-like pattern (including local production in the mammary gland), (2) secreted in a physiological fashion, and (3) controlled by known regulators (estrogen / dopamine). Further, we have shown that the hPRL produced in the mice is fully bioactive at the hPRL receptor within human breast cancer cells (T47D). We propose that these innovative models will be superior tools for examining the effects of hPRL on the growth and metastasis of human breast cancer xenografts. To this end, we have bred these mice onto the NOD/SCID immunodeficient background to allow for such preclinical studies of PRL in cancer. These novel mouse lines represent a major advancement in the research models available for the study of human breast cancer. As such, we expect that their use will have a significant impact on future diagnosis, prognosis and treatment of hormone-dependent breast cancers.

Citation Format: Heather R. Christensen, Michael K. Murawsky, Karen A. Gregerson. A novel, human-compatible mouse model for preclinical research on prolactin in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A18.
Abstract A21: Transgenic Onco-Pig cells mimic human cancer
Authors: Collares Tiago , Seixas Fabiana K. , Rund Laurie A. , Hu Wenping , Rodrigues Fernanda M. , Liang Ying , Singh Kuldeep , Counter Cristopher M. , Schook Lawrence B.
Published: May 2015

In order to take full advantage of the potential for new therapeutics and biotechnology applications, there is an urgent need for new animal models supporting cancer research. Pigs share many genetic and physiological similarities with humans. Our previous studies have shown that overexpression of several human oncogenes led to tumor development in pigs. Transgenic pigs were engineered to contain oncogenic KRASG12D and dominant-negative p53R167H, downstream of a LoxP-polyA (STOP)-LoxP sequence (LSL) and CAG promoter, such that exposure to Cre-recombinase induces their expression in desired tissues. Fibroblast cell strains generated from four such clones were infected with an adenovirus vector (Ad-Cre-GFP) encoding Cre recombinase and GFP protein or control vector (Ad-GFP) with GFP alone. Cells were analyzed for cell migration rates, cell proliferation, growth in soft agar, tumor development in immunodeficient mice, histopathological and characterization tumor and RT-PCR from tumor. All four cell strains expressed KRASG12D and p53R167H mRNA, transformed phenotypes such as increased cell migration rates, increase cell proliferation, and growth in soft agar. Migration rates in a wound assay were significantly different (184 vs 67 at 24 hr (p ≤ 0.01). CFSE assay determined that CRE cells divided twice as many times as control GFP cells in a 73 hr period (p ≤ 0.01). Additionally, GFP cells were unable to form colonies in soft agar, while each of the CRE cell lines formed over 100 colonies (p ≤ 0.01). CRE cell lines produced tumors in the mice (13/14) while no tumors developed from the GFP lines. Histopathological analyses revealed the tumors to be sarcomas, which were non-encapsulated, densely cellular and locally infiltrative with marked cellular and nuclear pleomorphism. Genetic analysis of tumor DNA showed the presence of CAG, KRASG12D and p53R167H transgene sequences. RT-PCR of tumor cDNA confirmed expression of the oncogenic KRASG12D and dominant-negative p53R167H mRNA from each tumor; therefore, demonstrating that induction of the transgenes in these porcine cells triggered a tumorigenic phenotype. Our next step will be to monitor tumor incidence following site-specific transgene induction in these Onco-pigs that can then be challenged with new anticancer therapies and interventional strategies.

Citation Format: Tiago Collares, Fabiana K. Seixas, Laurie A. Rund, Wenping Hu, Fernanda M. Rodrigues, Ying Liang, Kuldeep Singh, Cristopher M. Counter, Lawrence B. Schook. Transgenic Onco-Pig cells mimic human cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A21.
Abstract A40: Pancreatic cancer patient-derived orthotopic xenograft (PDOX™) is effectively targeted by Salmonella typhimurium A1-R
Authors: Hiroshima Yukihiko , Zhao Ming , Maawy Ali , Zhang Yong , Katz Matthew H.G. , Fleming Jason B. , Uehara Fuminari , Miwa Shinji , Yano Shuya , Momiyama Masashi , Suetsugu Atsushi , Chishima Takashi , Tanaka Kuniya , Bouvet Michael , Endo Itaru , Hoffman Robert M.
Published: May 2015

The aim of this study is to determine the efficacy of tumor-targeting Salmonella typhimurium A1-R (A1-R) (Cancer Disc. 2, 588-590, 2012) on pancreatic cancer PDOX™ compared to standard chemotherapy. The PDOX™ was grown in transgenic nude red fluorescent protein (RFP) mice in order that the PDOX™ acquire RFP-expressing stroma in order to label the tumor. The RFP-PDOX™ were then orthotopically transplanted to non-transgenic nude mice in order to image tumor growth and drug efficacy. The RFP labeled stroma are stable upon passage. A1-R treatment significantly reduced tumor weight as well as tumor fluorescence area compared to untreated control (p = 0.011), with comparable efficacy of gemcitabine, cisplatin and 5-FU. A1-R had the highest efficacy compared to any of the chemotherapy drugs tested based on Evan's histological response criteria. The present report demonstrates Salmonella typhimurium A1-R was effective for human pancreatic PDOX™ and is a promising therapy for this highly treatment-resistant disease.

Citation Format: Yukihiko Hiroshima, Ming Zhao, Ali Maawy, Yong Zhang, Matthew H.G. Katz, Jason B. Fleming, Fuminari Uehara, Shinji Miwa, Shuya Yano, Masashi Momiyama, Atsushi Suetsugu, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. Pancreatic cancer patient-derived orthotopic xenograft (PDOX™) is effectively targeted by Salmonella typhimurium A1-R. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A40.
Abstract A44: Directing personalized breast cancer treatment with pathway signatures
Authors: Jhan Jing-Ru , Andrechek Eran
Published: May 2015

One of the major limitations in the treatment of breast cancer is the patient to patient heterogeneity of the tumors. This is evidenced by the well-known intrinsic subtypes of breast cancer, including luminal A and B, basal, claudin-low, HER2 and normal like. If one considers the hormonal status (ER / PR) of the breast cancers in addition to the intrinsic subtypes, then additional classifications are apparent. Indeed, recent work has suggested that there are approximately 17 subtypes of breast cancer. With these various types of breast cancer, clinicians are faced with the challenge of determining the optimal treatment strategy for the individual breast cancer patient. When treatment options are considered in this way, it becomes readily apparent that breast cancer, and cancer in general, needs to be treated with a personalized approach. We believe that one mechanism to address the requirement for personalized therapy is to employ cell signaling pathway signatures to differentiate the tumors into treatment groups. As a proof of principle experiment, we have used pathway signatures in a mouse model of breast cancer. The signatures clearly predict various subtypes of cancer in the MMTV-Myc model system with elevation of the EGFR, TNF and Ras pathway in one subtype of tumor whereas these pathways are not predicted to be active in a second subtype of cancer from the same model system. In the second tumor subtype, there is a high predicted activity for other pathways, including Myc, Stat3 and AKT. To demonstrate that these predictive signatures can be used to guide clinical therapy, we then took an approach where we targeted each of the pathways in a combinatorial approach with small molecule inhibitors in transplantable tumors from the initial tumor subtypes. Thus, we have two therapies, each tailored to a specific subtype of a mouse model breast cancer. Safety trials for the combination of the three drugs in each treatment arm revealed that there were no significant side effects when inhibitors were used at the low doses designed for use in therapy. Importantly, we have shown that the predicted combination therapy is effective in blocking tumor growth in the tumor type that is predicted to be responsive while it is ineffective in blocking tumor growth in the other subtype of tumors. These results clearly demonstrate in a proof of principle experiment that using pathway signatures is a viable mechanism for identifying and directing treatment for breast cancer.

Citation Format: Jing-Ru Jhan, Eran Andrechek. Directing personalized breast cancer treatment with pathway signatures. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A44.
Abstract B14: Gain of function contribution of mutant AKT-E17K to lung and mammary tumorigenesis in mouse models
Authors: Malanga Donatella , Belmonte Stefania , Belmonte Stefania , Marco Carmela De , Paciello Orlando , Baldi Alfonso , Viglietto Giuseppe
Published: May 2015

The hotspot Akt1-E17K mutation that results in a glutamic acid to lysine substitution in the seventeenth amino acid position in the pleckstrin homology domain of Akt1 occurs in approximately 0.6-2% of human lung cancers and in 6-8% of human breast cancer. To determine whether this Akt1 variant is a bona-fide activating mutation and to investigate the mechanistic basis of the Akt1 gain of function dependent mechanisms in lung and breast tumorigenesis we generated a transgenic Cre-inducible murine strain in the ROSA26 (R26) locus (R26-Akt1-E17K mice). To analyse the effects of mutant Akt1 in lung epithelium, we made use of two different systems to activate the transgene into pulmonary cells: intranasal instillation of an Adenovirus carrying the Cre recombinase (Ad-Cre) and the mating of R26-Akt1-E17K mice with Thyroid transcription factor-1 (TTF-1)-Cre mice that allow expression of the transgenes in lung epithelium. We report that in murine lung epithelium, mutant Akt1-E17K induces bronchial and/or bronchiolar hyperplastic lesions that at low frequency progress to frank carcinoma and accelerates tumor formation induced by chemical carcinogens. To assess the potential role of Akt1 as an oncogene in mammary epithelium, we examined the consequences of the mutant Akt1-E17K in the mammary gland of mice by mating of R26-Akt1-E17K mice with Mouse mammary tumor virus (MMTV)-Cre mice. We provide strong evidences that the expression of mutant Akt1-E17K in the mammary gland, driven by the MMTV promoter, was sufficient to produce a tumoral response in mice starting from 5-6 months of age. Transgenic mice developed invasive and in situ carcinoma in 45% of the cases and, in agreement with these results, mice that express activated Akt1 in the mammary gland showed poor survival

Citation Format: Donatella Malanga, Stefania Belmonte, Stefania Belmonte, Carmela De Marco, Orlando Paciello, Alfonso Baldi, Giuseppe Viglietto. Gain of function contribution of mutant AKT-E17K to lung and mammary tumorigenesis in mouse models. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B14.
Abstract B20: Mouse models of Ewing's sarcoma and fusion-related sarcoma: Correct phenotypes and cell-of-origin
Authors: Tanaka Miwa , Nakamura Takuro
Published: May 2015

Tumor-specific fusion genes associated with chromosomal translocations play an important role in development of bone and soft tissue sarcoma. Clarification of tumorigenic processes by fusion genes is valuable to understand disease mechanisms and to develop molecular targeted therapies. We have recently established ex vivo mouse models for Ewing's sarcoma (ES) by introducing EWS-FLI1 into mouse embryonic cells. The ES model enabled us to analyze tumorigenic processes from the initial step to the highly malignant stage, and to select important genetic pathways in the processes. The knockdown of the EWS-FLI1 target genes and members of the important signaling pathways significantly suppressed cell proliferation of ES in vitro. Importance of such pathways were further confirmed by using specific inhibitors for PARP, EZH2 or Wnt/β-catenin pathway both in vitro and in vivo. Moreover, Cre-loxp-mediated knockout of EWS-FLI1 showed that mouse ES growth is fusion gene-dependent. In addition, we have succeeded to generate synovial sarcoma, alveolar soft part sarcoma and Ewing-like sarcoma by introducing SYT-SSX1, ASPL-TFE3 or CIC-DUX4, respectively. These results indicate essential roles of fusion oncogenes in sarcomagenesis and tumor maintenance, and the epigenetic status of cell-of-origin. Our platform will allow us to explore and evaluate novel targeted therapies.

Citation Format: Miwa Tanaka, Takuro Nakamura. Mouse models of Ewing's sarcoma and fusion-related sarcoma: Correct phenotypes and cell-of-origin. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B20.
Abstract IA21: Tumor heterogeneity and cell-of-origin of mouse small cell and non-small cell lung cancer
Authors: Berns Anton
Published: May 2015

Small cell lung cancer (SCLC) is one of the most lethal human malignancies, due to its high metastatic potential and chemo-resistance upon relapse. Using the Rbf/f;p53f/f mouse model for SCLC, we found that the tumors are often composed of phenotypically different cells, characterized by mesenchymal and neuroendocrine markers. These cells often share a common origin. Crosstalk between these cells can endow the neuroendocrine component with metastatic capacity, illustrating the potential relevance of tumor cell heterogeneity in dictating functional tumor properties. Also specific genetic lesions appear to be associated with metastatic potential.

Interestingly, the two cell types can also interconvert raising the question of their cell-of-origin. To investigate this in more detail, we inactivated Trp53 and Rb1 in distinct cell types in the adult lung by targeting Cre-recombinase expression to Clara, neuroendocrine, and alveolar type II cells using adenoviral vectors. We could show that neuroendocrine cells serve as the predominant although not the exclusive cell of origin of SCLC.

In contrast, mutant Kras-driven adenocarcinomas (one of the NSCLC subtypes) originates primarily from alveolar type II cells. However, in LSL-mutant-Kras;p53flox/flox mice also other cell types gave effectively rise to adenomas and adenocarcinomas. Our data indicate that both cell type specific features and the nature of the oncogenic lesion(s) are critical factors in determining the tumor initiating capacity of lung (progenitor) cells. Furthermore, the cell-of-origin appears to influence the malignant properties of the resulting tumors.

Citation Format: Anton Berns. Tumor heterogeneity and cell-of-origin of mouse small cell and non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA21.
Abstract PR04: Linking development and cancer modeling in zebrafish identifies putative therapeutic targets in medullary thyroid cancer
Authors: Villefranc Jacques A. , Alazar Aida , Houvras Yariv
Published: May 2015

Medullary thyroid cancer (MTC) is a neuroendocrine cancer that arises from malignant transformation of calcitonin-producing C-cells. A majority of MTC is associated with somatically acquired mutations in the proto-oncogene Rearranged during transfection (RET), resulting in constitutive activation of downstream effectors of the ERK and AKT signaling pathways. While surgical treatment may be curative in patients with localized MTC, a significant fraction of patients harbor residual disease and ultimately relapse. Much of the research on MTC has converged on inhibition of RET, however, studies suggest that other signaling pathways may contribute to disease. As a result, there is a growing need to identify novel targets for MTC.

The zebrafish is well regarded as a model to study of development due its genetic tractability, conservation of pathways and rapid organ development; characteristics that also make it a fitting model to study human cancers. In this study we take advantage of these traits to 1) perform an in vivo chemical screen to identify modulators of C-cell development in zebrafish and 2) develop an in vivo oncogenic RET model in zebrafish to test the putative ‘hits’ from our screen. We hypothesize that the nexus of development and cancer modeling in the zebrafish will lead to therapies of potential use in treating MTC. We screened compounds modulating pathways involved in development, kinase signaling and chromatin modification for their ability to influence C-cell development and found that separate inhibition of the FGF and GSKβ; signaling pathways results in marked down-regulation of markers associated with C-cell differentiation. In parallel, we have introduced human oncogenic RET into an analogous cell type of neural crest origin in zebrafish which results in their early transformation, and are testing the ability of the identified ‘hits’ to suppress transformation of these cells. Our results implicate the FGF and GSKβ; pathways in C-cell development and encourage further investigation of their potential as therapeutic targets for MTC. Moreover, our studies highlight the use of zebrafish as model to identify novel targeted therapies in cancer.

This abstract is also presented as Poster B44.

Citation Format: Jacques A. Villefranc, Aida Alazar, Yariv Houvras. Linking development and cancer modeling in zebrafish identifies putative therapeutic targets in medullary thyroid cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR04.
Abstract PR07: RNAi mouse models: Revolutionizing drug discovery in vivo
Authors: Premsrirut Prem , Dow Lukas , Hannon Gregory , Zuber Johannes , Lowe Scott , Zender Lars , Fellmann Christof
Published: May 2015

RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi tools including RNAi transgenic mice remains a significant limitation. One main hurdle is the identification of potent RNAi triggers, or short hairpin RNAs (shRNAs), that will induce stable and regulated gene silencing. Due to the lack of understanding of the requirements for shRNA biogenesis and target suppression, many predicted shRNAs fail to efficiently induce gene suppression. We have developed a “Sensor assay” that enables the biological identification of effective shRNAs at large scale and show that our assay reliably identifies potent shRNAs that are surprisingly rare and predominantly missed by existing algorithms. In addition, we have engineered a new miRNA scaffold, miR-E, that is more efficiently processed and thus produces more potent knockdown of target genes than our previous miR30 system. By combining our sensored miR-E based shRNAs with high efficiency ES cell targeting, we have developed a fast, scalable pipeline for the production of shRNA transgenic mice with reversible gene silencing. We show that RNAi can cause sufficient knockdown to recapitulate the phenotypes of knockout mice, particularly in cancer models. More importantly, unlike traditional knockout models, RNAi has the powerful advantage of reversibility, since the endogenous gene remains intact. Using this system, we generated a number of inducible RNAi transgenic lines and demonstrate how this approach can identify predicted phenotypes and also unknown functions for well-studied genes. In addition, through regulated gene silencing we are able to mimic drug therapy in mice without the actual drug molecule, allowing us to determine the therapeutic value and/or toxic effects associated with systemic gene suppression. Using this approach, we have been able to pinpoint potential toxicities associated with gene inhibition, results that will guide drug development to avoid target failures which will likely cause harmful and intolerable effects in patients. In a model of hepatocellular carcinoma, we demonstrate that short-term inhibition of a ribosomal protein is sufficient to induce stable cell cycle arrest of liver tumor cells. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene – mice with enormous predictive power that will shape our development of better tolerated therapies.

This abstract is also presented as Poster B30.

Citation Format: Prem Premsrirut, Lukas Dow, Gregory Hannon, Johannes Zuber, Scott Lowe, Lars Zender, Christof Fellmann. RNAi mouse models: Revolutionizing drug discovery in vivo. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR07.
Abstract A06: NORE1A is a double-barreled Ras senescence effector linking Ras to p53 and Rb
Authors: Donninger Howard , Calvisi Diego , Barnoud Thibaut , Schmidt M. Lee , Clark Geoffrey J.
Published: May 2015

In addition to driving growth and transformation, activated forms of Ras are potent inducers of oncogene induced senescence (OIS). OIS appears to be a major barrier that must be overcome to permit Ras driven tumorigenesis. The signaling pathways utilized by Ras to induce senescence, and how they are subverted during tumor development remain poorly characterized.

NORE1A is a member of the RASSF family of tumor suppressors. It binds directly to activated Ras via the Ras effector domain and acts as a Ras death effector. Frequent loss of NORE1A expression is observed in many tumor types and hereditary genetic defects in NORE1A predispose carriers to cancer. We have found that NORE1A can connect Ras to the induction of p21CIP1 and cell cycle arrest. Therefore, we sought to determine if NORE1A might play a role in Ras induced senescence.

We now show that NORE1A is a potent mediator of Ras induced senescence. Knockdown of NORE1A suppresses the ability of Ras to induce senescence in multiple cell systems and enhances Ras driven transformation. We have identified two novel signaling pathways activated by Ras/NORE1A. First, NORE1A forms a Ras regulated, endogenous complex with the kinase HIPK2. HIPK2 can phosphorylate p53 to induce apoptosis or recruit acetyltransferases to acetylate p53 to induce senescence signaling. NORE1A suppresses HIPK2 apoptotic post-translational modifications of p53 but activates pro-senescence post-translational modifications. NORE1A also binds and destabilizes mdm2 to enhance the stability of nuclear p53. Primary human tumors show a close correlation between the expression levels of NORE1A and acetylated p53.

In addition to p53, we show that NORE1A also links Ras to the regulation of Retinoblastoma (Rb) protein. NORE1A promotes a potent, Ras dependent stabilization of the Rb protein. It also promotes dephosphorylation of RB, an activating event.

Thus, NORE1A is a double-barreled Ras senescence effector which connects Ras to two of the most important senescence regulating tumor suppressors in the cell.

Loss of NORE1A activity in tumors is usually due to epigenetic inactivation or aberrant protein degradation by calpains. Both of these mechanisms, in principal, may be subject to clinical intervention to restore NORE1A function. This may provide a novel approach to antagonizing Ras driven tumors.

Citation Format: Howard Donninger, Diego Calvisi, Thibaut Barnoud, M. Lee Schmidt, Geoffrey J. Clark. NORE1A is a double-barreled Ras senescence effector linking Ras to p53 and Rb. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A06. doi: 10.1158/1557-3125.RASONC14-A06
Abstract A08: Mechanistic dissection of Ral GTPase signaling in driving KRAS-dependent pancreatic cancer growth
Authors: Gentry Leanna R. , Martin Timothy D. , Reiner David J. , Der Channing J.
Published: May 2015

The high frequency (95%) of KRAS mutations in pancreatic ductal adenocarcinomas (PDAC), together with strong evidence for the KRAS dependency of PDAC growth, makes the K-Ras oncoprotein an attractive therapeutic target. However, to date the development of direct inhibitors of K-Ras has not been successful, with most efforts focused on targeting components of K-Ras downstream effector signaling. While the pharmaceutical industry has centered on inhibitors of the Raf or PI3K effector signaling pathways, there is substantial evidence that K-Ras-dependent cancer growth must rely on the activities of other effector pathways. Of those pathways, the Ral guanine nucleotide exchange factor RalGEF) effector pathway, leading to activation of the Ras-like (Ral) small GTPases is well-validated to drive PDAC growth. Interestingly, while the two Ral isoforms share significant sequence identity (82%) as well as the same set of effectors, our studies found that they have strikingly different roles in PDAC growth. While RalA but not RalB is necessary for PDAC anchorage-independent growth in vitro and tumorigenic growth in vivo, RalB is the critical driver of PDAC invasion in vitro and metastasis in vivo. The mechanistic basis for their different biological roles in cancer remains to be elucidated. We hypothesize that their divergent C-terminal sequences and the posttranslational modifications that they signal for result in distinct subcellular localizations that then contribute to Ral isoform-selective effector interactions that promote distinct biological consequences in PDAC. To evaluate our hypothesis, we initiated studies to determine the effector(s) that mediates RalA-dependent anchorage-independent growth and tumorigenesis in vivo, the effector(s) that mediates RalB-dependent invasion and metastasis in vivo; and the role of the C-terminal hypervariable domain in causing distinct RalA- and RalB-dependent phenotypes and effector utilization. We found that both RalA and RalB subcellular localization is dependent on the C-terminal CAAX tetrapeptide signaled modifications by Rce1 and ICMT. We also determined that RalB preferentially activated mTORC1 to regulate pancreatic tumor cell Matrigel migration, through the Sec5 effector and regulation of exocyst function.

Citation Format: Leanna R. Gentry, Timothy D. Martin, David J. Reiner, Channing J. Der. Mechanistic dissection of Ral GTPase signaling in driving KRAS-dependent pancreatic cancer growth. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A08. doi: 10.1158/1557-3125.RASONC14-A08
Abstract A11: Oncogenic Ras signaling involves sustained perinuclear localization of the effector kinases p-ERK1/2 and CK2 via KSR-1 and endosomal trafficking
Authors: Lee Sook , Basu Sandip K. , Walia Vijay , Morrison Deborah K. , Johnson Peter F.
Published: May 2015

In primary cells oncogenic Ras can induce cellular senescence, an important tumor suppression mechanism that must be bypassed in tumor cells. Oncogene-induced senescence is regulated in part by the transcription factor C/EBPβ, which is post-translationally activated by Ras signaling. We previously reported that C/EBPβ activation by H-RasV12 is suppressed in immortalized and transformed cells by a novel 3'UTR-dependent mechanism termed 3'UTR regulation of protein activity, or UPA. UPA acts by restricting the Cebpb mRNA to a peripheral cytoplasmic region, which is spatially distinct from a perinuclear compartment containing the C/EBPβ-activating kinase, p-ERK1/2. UPA can thereby block C/EBPβ phosphorylation/activation and suppress its pro-senescence functions in tumor cells. Here we identify CK2 as another C/EBPβ kinase that undergoes perinuclear relocalization in RasV12-expressing cells. Accordingly, CK2-mediated phosphorylation of C/EBPβ was inhibited by the Cebpb 3'UTR in tumor cells but not primary cells. The MAPK signaling scaffold KSR-1 and endocytic trafficking were both required for RasV12-induced activation of C/EBPβ and perinuclear targeting of p-ERK and CK2. CK2 co-localized and interacted with KSR-1 and Rab11 (a marker of recycling endosomes), while p-ERK appeared to be associated with a different endosomal population. RasV12 expression led to sustained perinuclear localization of p-ERK and CK2 in both transformed and senescent cells. In contrast, growth factors (GFs) only transiently induced this response, which occurred with delayed kinetics (4-6 hr) coinciding with transitory activation of C/EBPβ. Our results indicate that oncogenic Ras signaling involves constitutive activation of the delayed GF signaling phase, characterized by restriction of the effector kinases p-ERK and CK2 to perinuclear endosomes. In this compartment they can access critical targets such as C/EBPβ in a UPA-regulated manner to promote either transformation or senescence.

Citation Format: Sook Lee, Sandip K. Basu, Vijay Walia, Deborah K. Morrison, Peter F. Johnson. Oncogenic Ras signaling involves sustained perinuclear localization of the effector kinases p-ERK1/2 and CK2 via KSR-1 and endosomal trafficking. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A11. doi: 10.1158/1557-3125.RASONC14-A11
Abstract A20: ARHGEF2 inhibition impairs cell growth and viability in KRAS-dependent cancer cells
Authors: Vargas Maria Jose Sandi , Kent Oliver A. , Normand Josee , Rottapel Robert
Published: May 2015

Sporadic tumours carrying activating mutations of the RAS family of GTPases are characterized by a progressive and multiple step tumorigenesis. Among the three isoforms, K-RAS is the most commonly mutated (Prior et al., 2012). ARHGEF2 is a microtubule-associated guanine nucleotide exchange factor (GEF) for the Rho family of small GTPases whose expression is up-regulated (more than 30 times) upon Ras neoplastic transformation in Rat fibroblasts (Zuber et al., 2000). A truncated version of the protein can transform NIH 3T3 cells (Whitehead et al., 1995) and induce tumour formation in nude mice (Brecht et al., 2005). We recently identified ARHGEF2 as a transcriptional target of the RAS/MAPK pathway and its expression contributes to cell survival and growth in RAS-transformed cells (Cullis et al., in revision). We aimed to determine if ARHGEF2 is required for tumour cell viability in different KRAS mutant cancer cell lines. We used an RNAi approach using two different ARHGEF2 and KRAS shRNAs to monitor cell growth and viability in six different cancer cell lines harbouring a KRAS mutation: four pancreatic adenocarcinomas (PL45, Panc 02.03, Panc 04.03 and KP4), one endometrial adenocarcinoma (HEC-1-A) and one colorectal adenocarcinoma (LS 174T). We found that ARHGEF2 is required for cell growth and survival in half of the cell lines analyzed. We established an ARHGEF2 and KRAS dependency index for those cell lines and further analysis allowed us to determine a positive correlation (correlation coefficient of 0.64, p value <0.03) between ARHGEF2 and KRAS dependency. In conclusion, our findings suggest that: i/ ARHGEF2 plays a role in the tumorigenesis and survival of KRAS dependent cell lines, but further in vitro and in vivo studies are required; ii/ ARHGEF2 inhibition could be a potential therapeutic target in tumours harbouring KRAS mutations.

References:

Prior, IA et al. A comprehensive survey of Ras mutations in cancer. Cancer Res. 2012.

Zuber, J. et al. A genome-wide survey of RAS transformation targets. Nat Gen. 2000.

Whitehead, I et al. Expression cloning of lfc, a novel oncogene with structural similarities to guanine nucleotide exchange factors and to the regulatory region of protein kinase C. J Biol Chem. 1995.

Brecht, M. et al. Activation of gef-h1, a guanine nucleotide exchange factor for RhoA, by DNA transfection. Int J Cancer. 2005.

Cullis, J. et al. The Rho GEF-H1 is required for RAS oncogene-diven pancreatic cancer. In revision.

Citation Format: Maria Jose Sandi Vargas, Oliver A. Kent, Josee Normand, Robert Rottapel. ARHGEF2 inhibition impairs cell growth and viability in KRAS-dependent cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A20. doi: 10.1158/1557-3125.RASONC14-A20
Abstract A31: Activity-based protein profiling reveals adaptive response to pharmacological MEK inhibition in KRAS mutant non-small cell lung cancer
Authors: Kim Jae-Young , Fang Bin , Welsh Eric A. , Kinose Fumi , Li Jiannong , Eschrich Steven A. , Koomen John M. , Haura Eric B.
Published: May 2015

KRAS mutations account for development of approximately 30% of lung adenocarcinomas. While RAF-MEK-ERK signaling cascade is a key regulator of RAS-mediated proliferation and survival, the clinical efficacy of MEK inhibitors is still underwhelming in RAS-driven cancers. We treated KRAS mutant non-small cell lung cancer (NSCLC) cell lines with two MEK inhibitors (AZD6244 and MEK162) currently in clinical development. In all cell lines tested (N=11), we failed to observe significant effects on cell viability or induction of apoptosis. Because increasing evidence indicates that cancer cells undergo adaptive proteomic rewiring in response to kinase inhibitors, we performed activity-based protein profiling (ABPP) after treating 11 KRAS mutant NSCLC cell lines with MEK inhibitors in order to identify global enzymatic changes potentially involved in these adaptive processes. Our ABPP approach employs desthiobiotin-ATP probe, which enables selective labeling of active sites in ATP-binding proteins including kinases, chaperones and metabolic enzymes. Labeled proteins were subsequently purified and analyzed by mass spectrometry-based proteomics technique. Our ABPP results revealed that MEK inhibitors increased levels of multiple receptor tyrosine kinases (RTK) including EGFR and MET. MEK inhibitors induced MET phosphorylation and EGF-mediated AKT phosphorylation was markedly enhanced in the presence of MEK inhibitors, indicating increased RTK activity in adaptive response to MEK inhibition. We also found that MEK inhibition led to increased multiple heat shock proteins including HSP90 and HSP70, suggesting the possibility of chaperone-mediated adaptive resistance. Finally, MEK inhibitors globally increased enzymes involved in glucose utilization pathways implicating metabolic alterations in adaptive response to MEK inhibition. Collectively, our systematic activity-based proteomic profiling approach provides novel insights into adaptive resistance mechanisms to MEK inhibitors, suggesting rational combination treatment strategies for KRAS mutant NSCLC. Updated works will be presented.

Citation Format: Jae-Young Kim, Bin Fang, Eric A. Welsh, Fumi Kinose, Jiannong Li, Steven A. Eschrich, John M. Koomen, Eric B. Haura. H. Activity-based protein profiling reveals adaptive response to pharmacological MEK inhibition in KRAS mutant non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A31. doi: 10.1158/1557-3125.RASONC14-A31
Abstract A33: Enhancement of thymidine phosphorylase downregulation by erlotinib enhances cytotoxicity affected by tamoxifen in human non-small cell lung cancer cells
Authors: Lin Yun-Wei , Chiu Hsien-Chun , Syu Jhan-Jhang , Jian Yun-Ting , Chen Chien-Yu
Published: May 2015

Tamoxifen is a triphenylethylene nonsteroidal antiestrogen used worldwide as an adjuvant chemotherapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen also could induce cytotoxicity in non-small cell lung cancer (NSCLC) cells have not been proved. In this study, tamoxifen treatment inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with tamoxifen decreased TP mRNA and protein levels in an AKT inactivation-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vectors significantly rescued the decreased TP protein and mRNA levels in tamoxifen-treated NSCLC cells. In contrast, a combination of PI3K inhibitors (LY294002 or wortmannin) further decreased the TP expression and cell viability induced by tamoxifen. Moreover, knocking down TP expression by transfection with small interfering RNA of TP enhanced the cytotoxicity and growth inhibition of tamoxifen. Erlotinib (Tarceva, OSI-774), an orally available inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for clinical use in the NSCLC therapy. Compared to each single treatment, the combining tamoxifen with erlotinib resulted in a synergistically inhibited the cell viability and cell growth, accompanied with the reduction of phospho-AKT, phosph-ERK1/2, and TP protein levels. An enhancement of AKT or ERK1/2 activation by transfecting the cancer cells with constitutively active AKT or MKK1/2 expression vectors significantly restored the two drugs combination-reduced TP protein levels as well as cell viability. Taken together, our results suggest that the down-modulation of AKT and ERK1/2-mediated TP expression by erlotinib represents a key factor in enhancing the cytotoxic effects of tamoxifen in NSCLC cells.

Citation Format: Yun-Wei Lin, Hsien-Chun Chiu, Jhan-Jhang Syu, Yun-Ting Jian, Chien-Yu Chen. Enhancement of thymidine phosphorylase downregulation by erlotinib enhances cytotoxicity affected by tamoxifen in human non-small cell lung cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A33. doi: 10.1158/1557-3125.RASONC14-A33
Abstract A40: Targeting TBK1 promotes apoptosis in MEK-inhibitor resistant mutant NRAS melanoma cells
Authors: Vu Ha Linh , Aplin Andrew E.
Published: May 2015

Melanoma is a devastating form of skin cancer. Fifteen to twenty percent of melanoma patients have an activating mutation in the GTPase, NRAS. Despite advances in the targeted inhibitor treatment for mutant BRAF melanoma patients, the options for mutant NRAS patients remain poor. TANK-binding kinase 1 (TBK1) is an atypical IκB kinase family member that acts downstream of the RAS effector RalGEF but the role of TBK1 in melanoma is not known. We found that NRAS overexpression in wild-type NRAS melanoma cells increased TBK1 phosphorylation. In a panel of NRAS mutant melanoma cells, we characterized sensitivity to MEK inhibition in 2D as well as 3D cell cultures that mimic the dermal microenvironment. We show that there is a differential response to MEK inhibition in this panel. TBK1 inhibition, through either siRNA transfection or pharmacological inhibitors, cooperated with MEK inhibition to promote apoptosis. These effects were particularly noted in those NRAS mutant cell lines that are insensitive to MEK inhibition. By contrast, this effect was absent in melanoma cell lines wild type for NRAS. These results identify TBK1 as a downstream effector for NRAS mutant melanoma and as a potential target for use in combinatorial therapy in this subset of patients for whom current therapeutic options are limited.

Citation Format: Ha Linh Vu, Andrew E. Aplin. Targeting TBK1 promotes apoptosis in MEK-inhibitor resistant mutant NRAS melanoma cells. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A40. doi: 10.1158/1557-3125.RASONC14-A40