bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A49: S6K1 promotes invasiveness of breast cancer cells in a novel model of triple-negative breast cancer metastasis
Authors: Khotskaya Yekaterina B. , Goverdhan Aarthi , Shen Jia , Sarvice Mariano Ponz , Chang Shih-Shin , Hsu Ming-Chuan , Wei Yongkun , Xia Weiya , Steeg Patricia , Yu Dihua , Hung Mien-Chie
Published: May 2015

Introduction: Breast cancer is the second-leading cause of oncology-related death in US women. Despite extensive research, over 30% of breast cancer patients develop metastatic disease, and metastases account for majority of breast cancer-associated morbidity and mortality. Of all invasive breast cancers, patients with tumors lacking expression of the Estrogen and Progesterone hormone Receptors and Human Epidermal growth factor Receptor 2 have the poorest clinical prognosis. These triple-negative tumors (TNBC) represent an aggressive form of the disease that is marked by an early-onset metastasis, a high tumor recurrence rate, and a low overall survival during the first three years post-diagnosis. However, few TNBC mouse models of metastasis currently exist.

Results: We noticed that a well-established MDA-MB-231 TNBC cell line produces rapid and extremely lytic bone lesions in lumbar, sacral and caudal vertebrae, and hind limbs in about 10% of animals injected intravenously. We biopsied one of these bone metastases and established a new metastatic MDA-MB-231-1566 cell line. Following an intravenous injection, MDA-MB-231-1566 cells produce early-onset metastasis to bone in up to 70% of animals with concurrent metastases to lungs, liver, and soft tissues. We demonstrate that 100% of animals injected with MDA-MB-231-1566 cells developed metastasis and had median survival of 60 days vs. 80 days in mice injected with the parental cell line. We also demonstrate that ribosomal protein S6 is highly phosphorylated on Ser235/236 in metastatic TNBC tumors, and that this phosphorylation is indicative of upstream S6 kinase (S6K) activity. Lastly, we provide evidence that targeting S6K with a highly specific inhibitor, PF-4708671, at sub-lethal doses inhibits cell migration without inducing cell death, and thus may provide a potent anti-metastatic adjuvant therapy approach.

Conclusion: We established a new model of rapid TNBC metastases to multiple organs following a simple intravenous injection. We believe this model provides a valuable tool for screening new therapeutics aimed to stop growth of metastases.

Citation Format: Yekaterina B. Khotskaya, Aarthi Goverdhan, Jia Shen, Mariano Ponz Sarvice, Shih-Shin Chang, Ming-Chuan Hsu, Yongkun Wei, Weiya Xia, Patricia Steeg, Dihua Yu, Mien-Chie Hung. S6K1 promotes invasiveness of breast cancer cells in a novel model of triple-negative breast cancer metastasis. [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 A49.
Abstract B17: Stem-like cells selected by serial in vivo orthotopic passage of human pancreatic cancer cells
Authors: Metildi Cristina Angela , Kaushal Sharmeela , Hoffman Robert M. , Bouvet Michael
Published: May 2015

The aim of this study was to determine the stem-like properties of cancer cells after serial orthotopic passages in nude mice. Orthotopic mouse models of human pancreatic cancer cell lines were established by injecting 1x106 BxPC-3 RFP or Panc-1 GFP cells into the pancreas of nude mice. Cell lines were generated from primary pancreatic tumors. 1x106 cells of the passaged cell lines were orthotopically injected into another set of mice and this process was repeated until a decreased host-mouse survival plateau was reached. To evaluate stem-like characteristics, 1x106 cells of the parental (P0) and multiply passaged BxCP-3 RFP (P6) and Panc-1 GFP (P7) cell lines were orthotopically implanted into mice, which were followed until they were premorbid. In a second group of mice, the P0 and P6/7 lines were injected subcutaneously at difference cell concentrations (100, 500, 1000, 10000, 100000 cells) to determine the minimum cell number for tumor initiation. Additional orthotopic models were generated to evaluate differences in tumor progression and sensitivities to chemotherapeutic agent, gemcitabine. The P6 and P7 lines demonstrated more aggressive behavior with regard to tumorigenicity, tumor growth, metastasis, progression, and chemotherapy resistance. A significantly fewer number of P6 passaged cells of BxPC-3 (100 cells, p=0.02) and P7 Panc-1 (500 cells, p=0.005) were required for tumor initiation in mice by 6 weeks compared to their respective P0 lines. The P6/7 lines produced larger tumors at 6 weeks post implantation (p<0.001). Mice harboring P6/7 tumors had a significantly greater degree of cachexia, more ascites, and greater metastatic tumor burden (p≤0.019) at 6 weeks. There was a significantly greater extent of metastatic disease in liver and lung samples from mice with tumors generated from P6/7 lines. The P6/7 lines' average time to metastasis and overall survival was reduced by more than one-half (p<0.001). Both P6/7 lines were less sensitive to gemcitabine (p≤0.009). Thus, serial passaging allows for in vivo selection of more aggressive variants of the human pancreatic cancer cell lines BxPC-3 and Panc-1 which are also resistant to gemcitabine suggesting that stem-like cells are being selected.

Citation Format: Cristina Angela Metildi, Sharmeela Kaushal, Robert M. Hoffman, Michael Bouvet. Stem-like cells selected by serial in vivo orthotopic passage of human pancreatic cancer cells. [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 B17.
Abstract B36: Modifier genes and cancer susceptibility: Lessons from inbred mouse strains
Authors: Nnadi Stephanie , Wang Xiang , Crist Richard C. , Buchberg Arthur M. , Siracusa Linda D.
Published: May 2015

The adenomatous polyposis coli (APC) tumor suppressor gene is mutated in Familial Adenomatous Polyposis (FAP), an inherited disorder that predisposes individuals to developing intestinal polyposis and eventually leads to cancer. In the Min mouse model, the genetic background of mice carrying a mutation in the murine homolog of the APC gene (ApcMin) is critical to the manifestation of tumor phenotypes, as inbred strains have been demonstrated to differ dramatically in their susceptibility to polyposis. Complex trait analyses from several laboratories, including our own, have identified modifier loci that alter intestinal tumor number and size in ApcMin/+ mice, but less than a handful of modifier genes have been identified to date. We review the results of several quantitative trait loci (QTL) studies that have exploited the genomic diversity among inbred mouse strains and report the identification of novel modifier loci that protect against polyposis. We compare the results with cancer susceptibility loci identified through GWAS and other studies in the human genome. The usefulness of using the mouse as a model organism for optimizing complex trait screens for cancer phenotypes and speeding the process of causative gene identification is discussed. Research supported in part by NCI grants to LDS.

Citation Format: Stephanie Nnadi, Xiang Wang, Richard C. Crist, Arthur M. Buchberg, Linda D. Siracusa. Modifier genes and cancer susceptibility: Lessons from inbred mouse strains. [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 B36.
Abstract B41: Paclitaxel and carboplatin loaded pegylated liposomes for ovarian cancer
Authors: Tandel Hemal T.
Published: May 2015

Ovarian cancer is the fifth leading cause of death in women. The current chemotherapy and the standard first line treatment for ovarian cancer is combination of ‘platinum plus taxanes’ which associated with serious side effects such as neutropenia and neurotoxicity due to ‘the non-specific biodistribution’ and ‘low therapeutic index of anticancer agents’. Therefore liposomal formulations are now being developed with the aim to reduce problems related to conventional marketed formulations and increase efficiency of the drugs. The present work focuses on development of long circulating PEGylated liposomal formulation of paclitaxel and carboplatin. Paclitaxel liposomal formulation was prepared with the aim to increase the therapeutic efficiency without use of cremophor EL so as to avoid serious side effects associated with vehicle and carboplatin liposomal formulation was prepared to overcome the side effects, occurrence of resistance to platinum drugs, increase its bioavailability and therapeutic efficacy.

Liposomes of paclitaxel and carboplatin were prepared by ‘thin film hydration’ method with lipids like HSPC, DPPG and cholesterol; and were PEGylated by ‘pre-insertion technique’. The optimized PEGylated liposomes of paclitaxel and carboplatin having the mean average particle size of 93.67 ± 2.1 nm and 96.44 ± 3.13 nm, respectively and hence we can expect maximum accumulation of liposomes in solid tumors and improved therapeutic index of loaded paclitaxel and carboplatin. To study the effect of PEGylation on the release characteristics of drugs, comparative diffusion studies for both the drugs were carried out with marketed formulations, conventional liposomes and PEGylated liposomes. Release of drug about 5% and 9% of paclitaxel and carboplatin respectively was observed in 52 hours from their PEGylated formulations. It was very less compared to their marketed and conventional liposomal formulations. The study reveals that there will be less systemic side effects of drugs after administration with increase bioavailability and therapeutic efficacy.

Citation Format: Hemal T. Tandel, Jr. Paclitaxel and carboplatin loaded pegylated liposomes for ovarian 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 B41.
Abstract B45: Combinatorial activation of PI3-kinase and Ras-Raf signaling in mouse models of advanced prostate cancer
Authors: Wang Jingqiang , Aytes Alvaro , Abate-Shen Cory
Published: May 2015

Concomitant activation of the PI3K and MAP kinase pathways is a hallmark of aggressive prostate cancer. Here, we describe a series of new genetically engineered mouse models of prostate cancer in which PI3K-Akt-mTOR signaling is activated through inducible deletion of PTEN, while MAP kinase signaling is activated through inducible expression of oncogenic mutants of either BRAF or KRAS oncogenes [BRAFV600E (NPB mice) and KRASG12D (NPK mice), respectively]. Activation of PI3K-Akt-mTOR and MAPK signaling pathways in NPB mice results in the formation of lethal prostate tumors that are inherently resistant to castration. These tumors bypass cellular senescence and disseminate to lymph nodes, bone marrow, and lungs where they form overt metastases in approximately 30% of the cases. Molecularly, tumors in the NPB mice cooperate to up-regulate c-MYC oncogene and accordingly, therapeutic treatments with an mTOR inhibitor (rapamycin) and the MEK1/2 inhibitor (PD0325901) results in attenuation of c-MYC levels and reduction primary tumor and metastatic burden. These findings suggest a generalized therapeutic approach to target c-MYC activation in prostate cancer by combinatorial targeting of the PI3K-Akt-mTOR and ERK1/2 MAPK signaling pathways. On the other hand, activation of PI3K-Akt-mTOR and MAP kinase signaling pathways in the NPK mice, results in prostate tumors that are also inherently castration resistant and metastasize to distant organs with 100% penetrance. Our studies show that in the NPK mice, dissemination is an early event in tumorigenesis although overt metastatic growth only occurs in late stage tumors coinciding with the upregulation of the ETS family member ETV4 in the primary tumor. Functional studies have shown that silencing of Etv4 in a metastatic cell line derived from this mouse model (NPK mice) abrogates the metastatic phenotype but does not affect tumor growth. These findings indicate that ETV4 promotes metastasis in prostate tumors that have activation of PI3-kinase and Ras signaling, and therefore, ETV4 represents a potential target of therapeutic intervention for metastatic prostate cancer. Taken together, our analysis of the molecular mechanisms involved in the distinct metastatic potential of these two new mouse models provides us with clinically relevant tools to conduct preclinical studies.

Citation Format: Jingqiang Wang, Alvaro Aytes, Cory Abate-Shen. Combinatorial activation of PI3-kinase and Ras-Raf signaling in mouse models of advanced prostate 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 B45.
Abstract IA14: Functional genomics and cancer vulnerabilities
Authors: Hahn William C.
Published: May 2015

Recent advances in genomics now make it possible to consider enumerating all of the genetic lesions in specific cancers. These approaches will yield critical information regarding the identification, number, and types of alterations found in human tumors and are increasingly being applied prospectively to patient samples in the clinic. At the same time, complementary approach to decipher the molecular basis of malignant transformation depends upon the application of genome scale tools to annotate the function of genes involved in cancer initiation and progression. Over the past several years, we have developed genome scale RNAi libraries and open reading frame expression libraries that permit a systematic evaluation of genes involved in cancer initiation and maintenance. Using these libraries, we have now performed screens in a panel of human cancer cell lines to systematically identify cancer vulnerabilities. By combining these functional approaches with information derived from mapping the structural abnormalities present in cancer genomes, we have identified several new oncogenes that contribute to cancer development. The ability to systematically manipulate gene expression at genome scale now provides opportunities to investigate the function of genes involved in cancer initiation and maintenance, which will provide a framework for therapeutic strategies.

Citation Format: William C. Hahn. Functional genomics and cancer vulnerabilities. [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 IA14.
Abstract IA15: Accelerating translational research using ESC-GEMM models and inducible RNAi
Authors: Lowe Scott W.
Published: May 2015

Cancer arises through an evolutionary process whereby normal cells acquire mutations that erode growth controls, leading to the expansion of aberrantly proliferating cells. Such mutations activate oncogenes or inactivate tumor suppressors, each bestowing new capabilities to developing leukemic cells. Our research is based on the premise that the path of cancer evolution dictates a tumor's subsequent response to therapy and creates unique vulnerabilities that represent therapeutic opportunities. Hence, we in use cancer genomics and functional studies in a coordinated manner to identify cancer drivers and dependencies, the then study their role of key genes in therapy response. To facilitate our research, we frequently employ mouse cancer models based on the genetic manipulation of stem and progenitor cells ex vivo followed by transplantation of the altered cells into the appropriate organ of syngeneic recipient mice. This approach allows us to rapidly study the impact of many genes and gene combinations on tumorigenesis and distinguish driver and passenger events from many candidates. Furthermore, we have developed powerful methods for using RNA interference to suppress gene function in vivo in either a stable and reversible manner, enabling us to study the gene function in established disease. Recently, we have combined these approaches to identify and validate candidate driver genes, and then used the animal models obtained from these studies as preclinical model to test novel therapeutic approaches. In addition, we have used inducible RNAi to study the role of tumor suppressor genes and cancer targets in normal and neoplastic tissues.

Citation Format: Scott W. Lowe. Accelerating translational research using ESC-GEMM models and inducible RNAi. [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 IA15.
Abstract IA18: Pathogenesis of gastric cancer: Lessons and therapeutic targets from mouse models
Authors: Wang Timothy C.
Published: May 2015

Many if not most gastrointestinal cancers arise in the setting of chronic inflammation, and a prime example is H. pylori-associated gastric cancer. We have utilized murine models of both proximal and distal gastric neoplasia to investigate the role of chronic inflammation in the development of malignancy. The development of gastric cancer in the mouse is dependent upon commensal flora and activation of both immature myeloid cells and cancer-associated fibroblasts. Gastric cancer arises in highly innervated portions of the stomach, and can be suppressed by surgical or chemical denervation of the stomach. Neurons and neurotransmitters promote the growth of gastric stem cells in organoid culture in vitro. Carcinogenesis of the stomach is associated with activation and early activation and amplification of epithelial stem or progenitor cells appears to be a requirement for the development of neoplasia. Further, conditional loss in gastric stem cells of tumor suppressor genes in vivo may not be sufficient for tumorigenesis but instead may require inflammatory signals in order to maintain self-renewal. In summary, the early stages of tumor development are often niche-dependent, and targeting the cancer niche represents an attractive strategy for cancer prevention and cancer therapy.

Citation Format: Timothy C. Wang. Pathogenesis of gastric cancer: Lessons and therapeutic targets from 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 IA18.
Abstract IA3: The Co-Clinical Trial Project
Authors: Pandolfi Pier Paolo
Published: May 2015

Herein we report the first application of a “Co-Clinical” approach, and assess the effectiveness of this integrated platform to investigate the genetic basis of resistance to androgen deprivation therapy (ADT) in prostate cancer (CaP). Surprisingly, we find that physical castration counteracts tumour progression in a Pten-loss driven mouse model of CaP through the induction of a potent apoptotic response and proliferation block. We took advantage of these findings to identify “co-clinically” new determinants of ADT resistance. We show that the tumour suppressive response to ADT is completely bypassed as a consequence of concomitant deletion of either Trp53 or Lrf together with Pten, leading to the development of castration resistant prostate cancer (CRPC). Mechanistically, we find that compound loss of Pten with Lrf or Trp53 triggers the down-regulation of the pro-apoptotic factor Xaf1, along with the up-regulation of the steroid 5-alpha-reductase enzyme Srd5a1, hence promoting CaP cell survival in low androgen conditions. Integrated acquisition of data from mouse and human CaP identifies p53/LRF/XAF1/SRD5A1 as a novel predictive signature for castration resistance. Importantly, we further show both in vitro and in vivo that combined inhibition of XAF1/XIAP and AR pathways overcomes castration resistance. Thus these results provide a proof of principle for the “Co-Clinical” approach as a means to facilitate the stratification of human patients and the development of tailored therapeutic treatments.

Citation Format: Pier Paolo Pandolfi. The Co-Clinical Trial Project. [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 IA3.
Abstract IA7: Drosophila cancer models identify complex therapeutics
Authors: Cagan Ross
Published: May 2015

The complex genomic nature of human tumors presents a challenge for cancer research. To capture this genetic complexity, we generated multigenic models of several cancer types in Drosophila based on patient sequencing data, including thyroid, colorectal, and lung tumors. As we increased in genetic and metabolic complexity, the models prove increasingly resistant to targeted cancer therapies. We have mapped this emergent resistance to specific cancer gene combinations, and have found that drug combinations can “break” this resistance. To establish single compound candidate therapeutics, we collaborated with Kevan Shokat's group to combine fly genetics and medicinal chemistry to develop single agents that suppress multiple targets, while rationally avoiding ‘anti-targets’ that promote toxicity. Finally, I will discuss our efforts to bring these Drosophila tools to individual patients through our new Center for Personalized Cancer Therapeutics.

Citation Format: Ross Cagan. Drosophila cancer models identify complex therapeutics. [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 IA7.
Abstract PR13: Using a hypoxia-induced CSC tumor initiation model to improve predictive potential for patients with metastatic breast cancer
Authors: Patel Purvi , Ma Yupo , Zhai Haiyan , Ju Jingfeng , Chen Emily
Published: May 2015

About 10-20% of breast cancers are found to be triple-negative. TNBCs have a relapse pattern that is very different from hormone-positive breast cancers: the risk of relapse is much higher for the first 3-5 years but drops substantially below that of hormone-positive breast cancers after that. Therefore, preventing tumor recurrence and metastasis in high-risk TNBC patients within 3-5 years of diagnosis will dramatically improve the long-term survival of these patients. Currently, most of preclinical animal models have limited predictive clinical potential for advanced metastatic diseases. One possible explanation is that most of the pre-clinical studies focus on the therapeutic benefit of eradicating bulk tumor cells. Using a stem-like subpopulation from triple negative breast cancer (TNBC) cell lines enriched by exposing the cells to cycles of hypoxia and reoxygenation, we showed that mammary tumors initiated from this subpopulation have a high recurrence rate (100%) after the taxol treatment compared to no recurrence from taxol treated tumors initiated from the bulk cancer cells. Our model mimics the progression of recurrence-prone breast tumors with a period of dormancy before the recurrence of local and distal tumors. Molecular analysis of the treatment naïve and recurrent tumors revealed increased expression of chemoresistance-associated miRNAs in the recurred xenograft tumors. In addition, we identified new markers in the recurred xenograft tumors that are also increased in drug resistant human breast tumors compared to the treatment naïve breast tumors. Ultimately, we propose that cancer stem cell based tumor model can be used to improve the predictive potential of pre-clinical models for patients with metastatic breast tumors.

This abstract is also presented as Poster A12.

Citation Format: Purvi Patel, Yupo Ma, Haiyan Zhai, Jingfeng Ju, Emily Chen. Using a hypoxia-induced CSC tumor initiation model to improve predictive potential for patients with metastatic 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 PR13.
Abstract A01: The role of EphA2 in KRAS mutant non-small cell lung cancer
Authors: Amato Katherine , Wang Shan , Hastings Andrew , Chen Haiying , Colvin Daniel , Ye Fei , Chen Jin
Published: May 2015

Lung cancer is the leading cause of cancer-related deaths in the United States accounting for approximately 160,000 deaths per year. Emerging data from sequencing efforts has emphasized the importance of identifying molecular subtypes of non-small cell lung cancer (NSCLC) and matching patients with appropriate targeted therapies for the most effective treatment of this disease. Although significant progress has been made to develop novel, molecularly-targeted therapeutics, challenges still remain in targeting commonly occurring mutations in NSCLC, such as KRAS. Overcoming these challenges will require strategies to identify and inhibit novel targets required for the suppression of cell viability in these subtypes of lung cancer. To this end, we have shown that the EphA2 receptor tyrosine kinase plays a unique role in the maintenance of cell viability in NSCLC, especially those with activating KRAS mutations. EphA2 is generally overexpressed in NSCLC and even further overexpressed in KRAS mutant NSCLC. Preliminary data from our lab has shown that silencing of EphA2 expression dramatically inhibits cell viability in human NSCLC cell lines bearing KRAS mutations. To further understand this phenotype, we are working to define the molecular mechanism downstream of EphA2 that is critical for maintaining cell viability in KRAS mutant NSCLC. Additionally, we have created a transgenic mouse model of KRAS mutant NSCLC with EphA2 depletion to study the role of EphA2 in KRAS driven lung tumorigenesis . Based on our preliminary data, we propose that EphA2 provides a novel target for modulating cell viability in lung cancers with KRAS mutations which are currently associated with limited therapeutic options.

Citation Format: Katherine Amato, Shan Wang, Andrew Hastings, Haiying Chen, Daniel Colvin, Fei Ye, Jin Chen. The role of EphA2 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 A01. doi: 10.1158/1557-3125.RASONC14-A01
Abstract A09: Notch signaling couples with Kras or PI3K to drive EGFR independent pancreatic acinar to ductal metaplasia
Authors: Halbrook Christopher J. , Crawford Howard C.
Published: May 2015

Almost all reported cases of pancreatic ductal adenocarcinoma, the most common form of pancreatic cancer, harbor an oncogenic Kras mutation. Recently, targeting oncogenic Kras to specific cell types in the adult pancreas has shown the acinar compartment to be more susceptible to neoplastic transformation than ductal cells. Acinar to ductal metaplasia (ADM) is a phenomenon where acinar cells undergo a morphological change and begin expressing markers normally found only in ductal cells while losing markers typical of acinar cells. ADM is highly proliferative with the potential to develop into pancreatic intraepithelial neoplasias, a precursor lesion to pancreatic cancer. Acinar cells harvested from mice expressing mutant Kras undergo spontaneous ADM when embedded in 3D collagen cultures, however, we and others have found the epidermal growth factor receptor (EGFR) is required for ADM induced by mutant Kras expression in vitro. Activation of EGFR by treatment of WT collagen embedded acinar cell explants with the EGFR ligand TGF-α has also been shown to induce ADM in a manner requiring Notch pathway activation. Furthermore, it has been demonstrated that expression of Notch2 intracellular domain (N2ICD) is sufficient to induce ADM in WT acinar cells. To directly investigate the relationship of the EGFR, Kras, and Notch signaling pathways in ADM, we examined the effect of Notch signaling in acinar cells harvested from EGFRf/f; Ptf1a+/Cre (EKO) mice, which harbor a pancreas specific deletion of EGFR. Surprisingly, we found that expression of N2ICD failed to induce ADM in acinar cells from EKO mice. We also observed that co-expression of a constitutively active p110α mutant or mutant Kras with N2ICD was able to rescue ADM in acinar cells from EKO mice, though neither was sufficient to drive ADM in the absence of N2ICD. Additionally, treatment of acinar cells from WT mice with either the p110α inhibitor Pik75 or the expression of a dominant negative mutant of the PI3K downstream target Rac1 reduced Erk1/2 phosphorylation, whereas expression of constitutively active p110α increased the level of Erk1/2 phosphorylation. These results demonstrate the importance of EGFR signaling in ADM, and identify the need to determine the mechanism of crosstalk between the PI3K and MAPK pathways in acinar cells.

Citation Format: Christopher J. Halbrook, Howard C. Crawford. Notch signaling couples with Kras or PI3K to drive EGFR independent pancreatic acinar to ductal metaplasia. [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 A09. doi: 10.1158/1557-3125.RASONC14-A09
Abstract A14: Transactivation of the Rho-guanine exchange factor ARHGEF2 by oncogenic K-RAS potentiates invasion-migration in pancreatic ductal adenocarcinoma.
Authors: Kent Oliver A. , Vargas Maria Jose Sandi , Rottapel Robbert
Published: May 2015

Activating mutations in K-RAS are a ubiquitous feature of pancreatic ductal adenocarcinoma (PDAC). Cellular transformation by oncogenic RAS engages the MAPK pathway and involves Arhgef2, the microtubule-associated RhoGEF a potential oncogene activated downstream of oncogenic RAS. Using luciferase reporters under regulation by the human ARHGEF2 promoter, we have found that GEF-H1 is regulated by oncogenic K-RAS in multiple cell lines and is a transcriptional target of the MAPK and Jun N-terminal kinase (JNK) pathways. Treatment of cell lines with small molecule inhibitors revealed that promoter activity is dependent on MEK1/2, JNK, and TGF-beta associated kinase (TAK1). We have mapped the human ARHGEF2 promoter and found it contains three regulatory elements: a Ras responsive element (RRE), a “core” ETS transcription factor binding region and two distal PTTG binding sites. The RRE is negatively regulated by the Ras responsive element binding protein 1 (RREB1), a known oncogene regulated by oncogenic K-RAS downstream of the MAPK pathway. The ETS core region contains conserved consensus elements for AP-1, ELK1 and SP1 transcription factors (TF). The pituitary tumor-derived transforming gene (PTTG) has been implicated in PDAC tumorigenesis but its link to oncogenic K-RAS is unknown. Using RNAi and luciferase reporters with mutations in TF binding sites in the ARHGEF2 promoter, we have found the transactivation of ARHGEF2 is dependent on the ELK1 and SP1 transcription factors. Chromatin immunoprecipitation has confirmed association of these transcription factors with the endogenous ARHGEF2 promoter in multiple PDAC cell lines. Our results suggest transactivation of ARHGEF2 by oncogenic K-RAS may lead to increased RhoA activity and by extension, account for the highly metastatic potential of PDAC.

Citation Format: Oliver A. Kent, Maria Jose Sandi Vargas, Robbert Rottapel. Transactivation of the Rho-guanine exchange factor ARHGEF2 by oncogenic K-RAS potentiates invasion-migration in pancreatic ductal adenocarcinoma.. [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 A14. doi: 10.1158/1557-3125.RASONC14-A14
Abstract A22: An MRAS, SHOC2, and SCRIB complex coordinates ERK pathway activation with polarity and tumorigenic growth
Authors: Young Lucy C. , Hartig Nicole , Munoz-Alegre Marta , Oses-Prieto Juan A. , Durdu Sevi , Bender Sabine , Vijayakumar Vineetha , Rudan Matteo Vietri , Gewinner Christina , Henderson Stephen , Jathoul Amit P. , Ghatrora Rupinder , Lythgoe Mark F. , Burlingame Alma L. , Rodriguez-Viciana Pablo
Published: May 2015

SHOC2 is mutated in Noonan syndrome and plays a key role in the activation of the ERK-MAPK pathway, which is upregulated in the majority of human cancers. SHOC2 functions as a PP1-regulatory protein and as an effector of MRAS. SHOC2 and MRAS form a complex with SCRIB, a polarity protein with tumor suppressor properties. SCRIB functions as a PP1-regulatory protein and antagonizes SHOC2-mediated RAF dephosphorylation through a mechanism involving competition for PP1 molecules within the same macromolecular complex. SHOC2 function is selectively required for the malignant properties of tumor cells with mutant RAS, and both MRAS and SHOC2 play a key role in polarized migration. We propose that MRAS, through its ability to recruit a complex with paradoxical components, coordinates ERK pathway spatiotemporal dynamics with polarity and that this complex plays a key role during tumorigenic growth.

Citation Format: Lucy C. Young, Nicole Hartig, Marta Munoz-Alegre, Juan A. Oses-Prieto, Sevi Durdu, Sabine Bender, Vineetha Vijayakumar, Matteo Vietri Rudan, Christina Gewinner, Stephen Henderson, Amit P. Jathoul, Rupinder Ghatrora, Mark F. Lythgoe, Alma L. Burlingame, Pablo Rodriguez-Viciana. An MRAS, SHOC2, and SCRIB complex coordinates ERK pathway activation with polarity and tumorigenic 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 A22. doi: 10.1158/1557-3125.RASONC14-A22
Abstract A32: Gaps and opportunities for RASopathies: Use of new tools to make the right choice in target and compound selection
Authors: Rosa Salvatore La , Knight Pamela , Pancza Patrice , Scobie Kimberly , Bakker Annette
Published: May 2015

“RASopathies” are a class of developmental disorders caused by germline mutations in genes that encode protein components of the Ras/MAPK pathway. Each syndrome exhibits different phenotypic features, though there are many overlapping features among syndromes, including characteristic facial features, cardiac defects, cutaneous abnormalities, autism, learning disabilities and a predisposition to malignancies. Neurofibromatosis type 1 (NF1) has been more extensively studied than the other syndromes and today 11 clinical trials are ongoing for NF1 versus 3 in all other RASopathies.

In the last couple of years a few studies were published which directly connected the molecular mechanisms of these diseases to each other, creating a new opportunity to cross-validate the effects of modulating the RAS pathway at different stages and with different tools, and providing new overlapping strategies which could also benefit less studied syndromes. We recently acquired in-house capabilities to access MetaCore and Integrity databases to help in the process of data mining and compound selection. We applied these capabilities in a multi-tiered approach to compare NF1 with Noonan Syndrome (NS). First, we analyzed the molecular profiles of these two RASopathies using MetaCore and zeroed in on a well-known common molecular target, MEK. Among the large number of MEK inhibitors now in development, an analysis of these agents has been made with the use of the database to compare the different chemical classes of inhibitors and analyse properties, opportunities and redundancies among these compounds. The same approach will be then applied to try to identify a novel drug target in common in NF1 and NS. For this new target, an analysis of available drugs will be made to try to identify the best candidate/s which could be explored for in vitro or in vivo testing or as a proof-of-concept. Combining the capabilities of bioinformatic analysis and chemical analysis, a more informed drug choice for screening and/or preclinical testing can be done on the basis of detailed information on drugs and interconnections of targets/pathways. This approach, taking into account the various aspects of a particular model or disease, will attempt to accelerate research by allowing the selection of the most suitable compounds for a particular experiment, allowing for more informed choices.

Citation Format: Salvatore La Rosa, Pamela Knight, Patrice Pancza, Kimberly Scobie, Annette Bakker. Gaps and opportunities for RASopathies: Use of new tools to make the right choice in target and compound selection. [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 A32. doi: 10.1158/1557-3125.RASONC14-A32
Abstract A42: H-Ras regulates the dynamics of TRAIL death receptors
Authors: Chen Jun-jie , Di Xu , Zhang Yaqin , Bozza William , Zhang Baolin
Published: May 2015

Multiple clinical trials are ongoing to evaluate the potential antitumor activity of recombinant human TNF-related apoptosis inducing ligand (rhTRAIL) and its agonistic antibodies. These protein therapies act through death receptors (DRs) 4 and/or 5 on the cell surface, followed by activation of a caspase cascade and apoptotic cell death. We have previously shown that DR4 and/or DR5 are mislocalized in nucleus and/or cytoplasm in some cancer cells thereby rendering cellular resistance to the targeted therapies. In this study, we have found a correlation between H-Ras expression and TRAIL resistance in the NCI60 panel of human cancer cell lines. The elevated expression of H-Ras is also generally associated with a deficiency of TRAIL death receptors on cell surface. Blockade of H-Ras activity in TRAIL-resistant cells upregulated the surface expression of both DR4 and DR5 without changing their total protein expressions. Subsequently, the cells were sensitized to apoptosis induction by the targeted therapies. To our knowledge, this is the first evidence that oncogenic H-Ras is involved in the regulation of subcellular localization of TRAIL death receptors. H-Ras, along with other Ras GTPases, is known to stimulate outgrowth of cancer cells. Our data suggest that H-Ras may elicit its oncogenic activity at least partly by blocking cytokine-induced apoptosis of cancer cells. Additional studies are warranted to evaluate the therapeutic potential of combinational therapies to simultaneously target H-Ras and TRAIL receptors.

Citation Format: Jun-jie Chen, Xu Di, Yaqin Zhang, William Bozza, Baolin Zhang. H-Ras regulates the dynamics of TRAIL death receptors. [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 A42. doi: 10.1158/1557-3125.RASONC14-A42
Abstract A44: Identification of new, biologically relevant, G-quadruplexes formed within the critical promoter of kRAS as promising molecular targets
Authors: Hockings Jennifer , Brooks Tracy Ann
Published: May 2015

kRAS is one of, if not the, most prevalent oncogenic aberrations identified to date, and has been well validated as a therapeutic target. It is either upregulated or mutationally activated in ∼30% of all cancers, including 60-90% of pancreatic cancer, and contributes to malignant transformation, tumorigenicity, and a corresponding poor prognosis. Our therapeutic approach to transcriptionally downregulate kRAS is innovate and promising because we are focused on ablating the oncogenic protein, rather than previously unsuccessful attempts focused on modulating its function. Numerous literature reports validate the downregulation of kRAS expression to as an anti-cancer approach, but there has yet to be a concerted effort in this area, primarily due to lack of a molecular target. This is where our findings are exceptionally pioneering, as we focus on globular higher order DNA (G4) formations within the core kRAS promoter as druggable targets with great promise. G4s occur in G/C-rich DNA and are made up of two or more stacked tetrads, formed by the Hoogsteen hydrogen bonding of four guanines, stabilized by monovalent cations. These G/C-rich regions preferentially cluster around the transcriptional start site throughout the genome peaking within 50 bp of the TSS, which is indicative of their regulatory role. Although not universally true, formation of G4s generally functions to silence transcription or translation and thus represents an attractive molecular target for kRAS. The proximal promoter of kRAS contains three separate putative G4-forming regions: near, mid, and far. We have revealed a previously undescribed within the “mid” region of the proximal kRAS promoter, which contains seven contiguous runs three or more continuous guanines. This region forms biologically functional, transcriptionally silencing, equilibrating G4s. This is in contrast to the previously described “near” G4, which we show to have little to no role in transcriptional regulation. These newly described mid-G4 structures have high potential as molecular targets for drug development.

Citation Format: Jennifer Hockings, Tracy Ann Brooks. Identification of new, biologically relevant, G-quadruplexes formed within the critical promoter of kRAS as promising molecular targets. [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 A44. doi: 10.1158/1557-3125.RASONC14-A44
Abstract A48: The K-ras let-7 miRNA binding site variant and K-ras mutations in colon cancer
Authors: Dalay Nejat , Yalniz Zubeyde , Gurbuz Orkun
Published: May 2015

The K-ras gene is one of the most commonly mutated genes in cancer. The ras proteins act as functional switches in the activation of the MAPK pathway in a complex signaling network coupling growth factor receptors to the intracellular kinases. Mutations in the K-ras gene lead to the constitutive activation of the protein and consequently of the MAPK pathway. K-ras mutations are early events in colorectal carcinogenesis and are observed in 30-40 % of the colorectal cancers. Mutations mostly occur at codons 12, 13 and 61 of the gene and result in the deregulation of the protein activity. Several studies have shown that patients with K-ras mutations do not benefit from therapies with anti-EGFR monoclonal antibodies.

miRNAs are global regulators of gene expression by binding to the 3'-untranslated region of the target mRNA. Recently, it has been shown that the K-ras gene is regulated at the translational level by binding of the let-7 miRNA. The let-7 family of miRNAs is known to play an important role in several cancer types. A germline SNP at the 3'-UTR of the K-ras gene (rs: 61764370 T-G) has been shown to disrupt binding of the let-7 molecule to K-ras resulting in overexpression of the gene and was found to confer increased susceptibility for distinct cancer types. The let-7 miRNA variant was found to be associated with poor outcome in lunh and head and neck cancers. However, the frequency and association of this variant with other cancer types has not been investigated widely.

In this study we evaluated the frequency of the K-ras gene variant in 145 patients with colon cancer and 85 healthy individuals and its possible association with the K-ras gene mutations. The K-Ras gene variant was analyzed by polymerase chan reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, K-ras gene mutations were investigated by real time-PCR. The results were evaluated using the Chi-square test.

The variant allele of the K-ras gene was observed in 23 patients (15.9%) in the heterozygote form. A single patient was homozygous for the variant allele. The variant allele was detected in 12 % of the control group. K-ras gene mutations were present in 46 (32 %) of the patients. The frequency of the variant allele was 13 % among the patients harboring a K-ras gene mutation. The distribution of the variant allele is in accordance with a report on the US control population and no significant difference was observed between the patients and the controls.

Citation Format: Nejat Dalay, Zubeyde Yalniz, Orkun Gurbuz. The K-ras let-7 miRNA binding site variant and K-ras mutations in colon 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 A48. doi: 10.1158/1557-3125.RASONC14-A48
Abstract A49: Biological impact of TIMP-1 relative to KRAS status in lung carcinoma
Authors: Gupta Sampa Ghoshal , Kutiyanawalla Ammar , Lee Byung Rho , Rojiani Amyn M. , Rojiani Mumtaz V.
Published: May 2015

Comprehensive sequence analysis of common human cancers has identified several genes that promote tumorigenesis. KRAS is a commonly mutated oncogene in lung adenocarcinoma, most frequently harboring mutations in codon 12 or 13. Therapeutically targeting of KRAS with small molecules has proven ineffective due to difficulties in disrupting protein/protein interactions that are inherent in its activation and function. Recent work has established that many cancers may have KRAS addiction and are considered KRAS dependent while others do not require KRAS for survival. Previously we have shown that TIMP-1 plays an important role in cell survival in lung adenocarcinoma cells. We have overexpressed TIMP-1 in H2009, a KRAS dependent cell line, which rendered these cells resistant to Staurosporine induced apoptosis, whereas knocking down TIMP-1 in A549 (a KRAS independent lung carcinoma cell line) had the opposite effect. We also noted that TIMP-1 overexpression in H2009 cells made them more angiogenic and aggressive in in vitro assays. H2009 and A549 cell lines also showed significant differences in migration, spheroid formation as well as invasiveness. Although H441 cell line is known to be KRAS dependent, it was not as invasive as H2009. These cell lines also showed differential expression of Vimentin and E-cadherin. These results prompted us to review available data regarding these cell lines, which not only harbored different mutations in the cancer promoting genes, but also differed at the regulatory level and functioned through activated ERK1/2, NFkB and Akt pathways. Analysis with the R2 program (http://hgserver1.amc.nl/cgi-bin/r2/) identified a significant correlation between TIMP-1 and KRAS in breast cancers and lung cancer . Interestingly, high TIMP1 levels have been shown to cause chemoresistance by virtue of its anti-apoptotic activity and KRAS has generally been undruggable. Our current study compares KRAS dependent and independent cell lines in relation to TIMP-1 expression. Modulating TIMP-1 renders these cells either more sensitive or resistant to apoptosis. Clinically, patients with elevated TIMP-1 expression have been shown to have shorter overall survival. This study helps to further dissect these pathways and provides a better understanding of the impact of these mutations in lung carcinoma. Supported in part by a Georgia Research Alliance Distinguished Cancer Scientist Award.

Citation Format: Sampa Ghoshal Gupta, Ammar Kutiyanawalla, Byung Rho Lee, Amyn M. Rojiani, Mumtaz V. Rojiani. Biological impact of TIMP-1 relative to KRAS status in lung carcinoma. [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 A49. doi: 10.1158/1557-3125.RASONC14-A49