Dr. Godwin has been involved in both basic and translational research for many years, filling several important niches within Fox Chase Cancer Center (FCCC) and the translational research community as a whole (e.g., translational research initiatives, molecular pathology, biosample ascertainment). The central theme of his work centers on the idea of obtaining a molecular definition of a tumor to define its treatment-sensitive elements. As such, he has a long-standing interest in the field of cancer genetics and recent efforts in the study of molecular targeted therapies. His studies primarily involve three different types of cancer: breast, ovarian, and gastrointestinal stromal tumor (GIST). He uses these models to investigate how to improve patient care.

Schematic representation of a
dualistic model depicting the
development of ovarian cancer

The incidence of epithelial ovarian cancer has remained stable over the last 20 years, with approximately 20,200 cases estimated to occur annually, and a lifetime risk in the general population of 1.4%. Aggressive debulking surgery to remove the primary tumor and its metastatic implants has long been the accepted initial step in the management of ovarian cancer. The achievement of optimum cytoreduction at the end of the initial surgery has been shown as a favorable prognostic factor for the disease course. It has been recently demonstrated that clinical response may not correlate with survival due to the preferential survival of chemotherapy resistant tumor-initiating cells (also referred to as cancer stem cells, cancer progenitor cells, and cancer initiating cells) that eventually grow and repopulate the tumor. Our group studies the origins of ovarian cancer, the role of the extracellular environment (stromal/epithelial/ECM interactions) in regulating tumor cell growth and the use of molecular targeted therapies for the treatment of this disease. Below are examples of ongoing studies in these areas.

Molecular characterization of tubal intraepithelial carcinoma: application to prevention and early detection strategies

Q. Cai & A.K. Godwin, in collaboration with R. Burger, M. Daly, L. Martin

p53 and MIB1 expression in fallopian tube and ovary

The majority of advanced epithelial ovarian, primary peritoneal and fallopian tube cancers are invasive serous adenocarcinomas; recently classified as Type II epithelial ovarian cancers. Identification of morphologic or molecular precursors for Type II ovarian cancer would be paramount to the development of effective prevention strategies. Preliminary studies in hereditary breast-ovarian cancer (HBOC) kindred suggest that Type II ovarian cancer may arise from dysplastic tubal epithelium (tubal intraepithelial carcinoma, TIC) and are related to early mutational loss of TP53. Our preliminary data show strongly stained p53 in both areas of TIC and the concurrent ovarian carcinoma. We hypothesize that TIC represents a precursor of invasive disease for Type II tumors in HBOC (and perhaps sporadic cases). In order to test this hypothesis, we are using molecular genetic approaches to evaluate concurrent TIC and ovarian adenocarcinoma to determine additional molecular alterations that lead to disease development and to determine whether sporadic cases of ovarian cancer share these molecular features. If our data support the model of TIC as an origin of Type II serous tumors, this would justify development of in situ imaging technologies specific to morphologic or molecular changes associated with TIC in the prevention of Type II serous adenocarcinomas. Such technologies are currently undergoing development at Fox Chase using molecular imaging probes.Top

Therapeutic targeting of the tumor microenvironment in ovarian cancer

Y. Kwon, C. Slater, L. Vanderveer, & A.K. Godwin, in collaboration with E. Cukierman.

Development of in vivo-like 3D culture system to study
ovarian tumorigenesis

The tumor microenvironment is complex and contains non-cancerous stromal cells, such as fibroblasts, immune cells, and endothelial cells and products secreted from these cells that intermingle with the tumor cells. Since the trigger for tumor growth and spread may actually come from the stroma, these studies explore how to best use therapies that target the stromal components. We are utilizing 3 dimensional (3D) models to study how the tumor microenvironment supports ovarian tumor cell growth. We have reported that the basement membrane (a thin layer of extracellular proteins that anchors epithelial tissue to underlying connective tissue and provides mechanical structure, separates different cell types, and signals for cell differentiation, migration, and survival) is disturbed very early in the initiation of ovarian cancer. Using this 3D model we have shown that different ovarian cancer cell lines migrate and invade in different ways and these cancer cells change their phenotype by acquiring tumor stem cell characteristics depending on signals provided by their immediate surroundings. Although it is believed that a normal microenvironment has a negative effect on tumor growth, the tumor-associated microenvironment actively contributes to tumor development and progression. Our research is focusing on ways to alter how ovarian tumor cells interact with their microenvironment as a means to interfere with tumor development.Top

RNA interference screens to identify genes essential for ovarian tumor growth and maintenance

G. Sethi, H. Pathak, H. Zhang, & A.K. Godwin, in collaboration with J. Weaver, Y. Zhou

Primary ovarian cancer cells in vitro

We have utilized recent technological advancements in the field of RNAi and high-throughput screening to perform several types of RNAi screens to identify genes essential for ovarian tumor growth and to identify genes that help sensitize tumor cells to molecular targeted therapies. In the former screen, we employed a library of 24,800 siRNAs targeting ~6,200 genes. From this screen we identified 300 unique human genes that were considered potential targets for drug development and represent essential genes for tumor growth and survival. A custom siRNA library was derived and this library has been screened across a panel of ovarian cancer and non-tumorigenic human ovarian surface epithelium cell lines. For each tumor and non-tumor cell line a gene signature was established. A total of 45 common genes across the 10 ovarian cancer cell lines were discovered and found to induce varying degrees of cytotoxic effects as assessed by a cell viability assay. These genes are being further validated using deconvoluted siRNAs and quantitative RT-PCR. We will also evaluate the functional significance of these genes in primary cultures obtained by processing ascites from ovarian cancer patients as a basic step towards establishing personalized medicine. As siRNA therapy holds a promising future, we believe that these studies lay the foundation to develop novel molecular approaches and targets that may ultimately improve the treatment of patients with ovarian cancer.Top

Exploiting biological networks to improve clinical treatment of ovarian cancer

H. Pathak, H. Zhang, A. Kouadio, & A.K. Godwin, in collaboration with E. Golemis, Y. Zhou

High-throughput siRNA synthetic-lethality screening

At the time of presentation most epithelial ovarian cancers are no longer dependent on single genetic determinants for growth and/or survival. The overall response to oncogene-targeted therapies used as single agents is not highly significant in the general population, and therefore, treatment of ovarian cancer poses particularly formidable challenges. We hypothesize that targeted agents will become potent when used in combinations that simultaneously block multiple oncogenic pathways, where such simultaneous blockade will reduce the ability of cancer cells to select resistance mutations. Therefore, we have used second-site siRNA lethality screens to help identify critical pathways to target in combination with novel biologics. Our studies have used bioinformatics to devise a tryrosine kinase-centered network, and probed a corresponding siRNA library targeting 638 genes in synthetic lethality screens. Using this library, we have identified a number of genes that synergize with anti-cancer drugs targeting KIT, PDGFR, EGFR and SRC to identify critical pathways which sensitize ovarian cancer cells to these novel biologics. We will complete the initial validation process and explore the expression patterns of proteins and transcripts for genes identified through our initial screens in patient samples to assess their clinical relevance. These sensitizing genes represent potential targets for multi-drug therapies with biologics in clinical trials. We view this work as ultimately enabling the next-generation approach for cancer therapies: combining targeted drugs in coherently designed multi-agent combinations for use in the clinic.Top

Evaluation of therapeutic agents in treating human ovarian cancer

A. Kouadio, C. Slater, H. Zhang & A.K. Godwin, in collaboration with H. Hensley

Bioluminescence imaging of ovarian tumor cells

Our group uses various in vitro and in vivo models to assess the activity of novel anti-tumor drugs alone or in combination for the treatment of ovarian cancer, including 2D- and 3D-cell culture models, subcutaneous/intraperitoneal/intrabursal tumor xenograft models, and bioluminescence-based animal imaging. We are examining many biologics including cetuximab, panitubimab, gefitinib (EGFR inhibitors), dasatinib (SRC family kinases inhibitor), and AMG706 (VEGFR inhibitor) in vitro and in vivo. In addition to drug evaluations, our studies are also focused on understanding the mechanisms of action at the cellular level. The goals of these therapeutic studies are to identify important drug targets, promising agents, and treatment combinations that can ultimately help improve the clinical outcome of human ovarian cancer treatment.Top

Women who carry mutations in the BRCA1 and BRCA2 (BRCA1/2) genes have a substantially increased risk of developing breast and/or ovarian cancer as compared to the general population. However, risk estimates for breast cancer range from 20-80% suggesting the presence of genetic and/or environmental effect modifiers. We have recently reported that loss of allele specific expression (also referred to as allelic imbalance or AI) of BRCA1 is associated with increased breast cancer risk and demonstrated that these expression patterns can be transmitted by Mendelian inheritance, suggesting that alternate mechanisms, other than deleterious coding mutations, may contribute to breast cancer. We are also participating in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA), an international consortium that is evaluating large cohorts (>15,000) of BRCA1 and BRCA2 mutation carriers for genetic modifiers of breast cancer risk. Our group continues to explore additional high-penetrant breast cancer susceptibility genes. We have shown that BRCC36 is aberrantly expressed in breast cancer, is mutated in cancer prone kindreds, and plays an important role in the regulation of the ubiquitin E3 ligase activities of the BRCA1 containing complex (BRCC). We have also recently initiated studies to identify proteomic markers of benign breast disease (BBD) that can be used to better predict a woman’s risk of developing invasive breast cancer. Below are examples of ongoing studies in these areas.Top

Loss of BRCA1 allelic expression in breast cancer

X. Chen & A.K. Godwin, in collaboration with M. Daly

Allelic imbalance in MRCA1 expression

The contribution of BRCA1 to familial and non-familial forms of breast cancer has been difficult to accurately estimate because the myriad of potential genetic and epigenetic mechanisms that can ultimately influence their expression and cellular activities. Therefore, we investigated if allele specific loss of BRCA1 expression was associated with an increased risk of developing breast cancer. By designing allele specific real-time PCR to target a common polymorphism, BRCA1-c.4308T/C, we were able to evaluate quantitatively the allelic imbalance (AI) in BRCA1 gene expression. By using mRNA from BRCA1 frameshift mutation carriers, we first demonstrated that the transcript ratios of wild-type alleles to the corresponding mutant alleles (BRCA1-c.3671ins4) were significantly increased (Chen et al, 2008). The observed AI is the result of nonsense mediated mRNA decay and leads to subsequent loss of protein expression. We next measured AI for BRCA1 in heterozygous lymphocytes from three groups, familial breast cancer patients, non-familial breast cancer patients, and age-matched cancer-free females. The AI ratios of BRCA1 in the lymphocytes from familial breast cancer patients were found to be significantly increased as compared to cancer-free women [BRCA1: 0.424 ± 0.157 (n=32) vs. 0.211 ± 0.169 (n=40), p< 0.001]. Similarly, the AI ratios were greater for BRCA1 in the lymphocytes of non-familial breast cancer cases versus controls [BRCA1: 0.353 ± 0.209 (n=21), p=0.002]. Furthermore, we demonstrated that the AI patterns for BRCA1 expression could be transmitted by Mendelian inheritance (Chen et al, 2008). These findings indicate that AI in BRCA1 appears to be a common event in breast cancer development and point towards the importance of identifying other mechanisms that regulate/suppress BRCA1 activity as contributors to breast tumorigenesis.Top

BRCA1-associated protein BRCC36, a novel target for breast cancer therapy

X. Chen & A.K. Godwin, in collaboration with G. Adams & M. Robinson

siRNA targeting mediated by diabody-protamine

Since tumor cells in general are genomically unstable and have defects in DNA damage responses, it has been proposed that targeting DNA repair pathways may lead to a therapeutic index in tumor cells over “normal” cells. Our previous studies have demonstrated that BRCC36 is over-expressed in the vast majority of invasive breast cancers and that depletion of BRCC36 sensitizes breast cancer cells to ionizing radiation via the BRCA1 DNA repair pathway (Chen et al, 2006). Therefore, we hypothesize that abrogation of BRCC36 will sensitize breast tumors to DNA-damage based therapies. To test this hypothesis, we will utilize a novel diabody (anti-HER2-protamine) based siRNA delivery system to selectively deplete BRCC36 in breast tumor xenografts. The diabody (C6.5) used in the proposed studies displays approximately 40-fold greater affinity for HER2 and significantly prolongs association with HER2 as compared with scFv. Therefore, this C6.5 diabody provides an in vivo targeting advantage and has been effectively used as a delivery vehicle for therapeutic radioisotopes.Top

Early detection of preneoplastic lesions

D. Pape-Zambito & A.K. Godwin, in collaboration with M. Daly, A. Patchesfky, H. Wu, A. Yeung

Laser capture microdissection of mammary epithelium with
ductal hyperplasia

Although breast cancer rates have been declining in recent years, the number of women receiving benign breast diagnoses is increasing. Some benign lesions have a higher relative risk of subsequent breast cancer development than others, however it is unknown which lesions are most likely to progress to invasive breast cancer. As a proof of principle study, laser capture microdissection was employed to isolate histologically identified regions of BBD (i.e, ductal hyperplasia; DH) and normal breast epithelium from mastectomy specimens. Using proteomic approaches, we identified differentially expressed proteins and demonstrated that a set of these proteins could identify a subpopulation of DH cells exhibiting columnar cell changes (CCC). This is clinically relevant given that cells with atypical CCC have characteristics similar to those observed in atypical DH (ADH) and well-differentiated in situ and invasive carcinomas; thus atypical CCC may represent nonobligate precursors to neoplasia. We have expanded our studies using more sensitive proteomic technologies to compare ADH from women who remain disease free or subsequently develop invasive disease. Together, these markers may prove to be clinically relevant in identifying regions of BBD and in assigning prognostic significance in biopsy samples. We are collaborating with Dr. M. Daly to access clinical specimens from the Benign Registry to Assess Valid Endpoints (BeBRAVE) to test the value of our markers in predicting which lesions have the greatest risk of invasive breast cancer development.Top

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract, with an estimated annual occurrence of 3,300-6,000 in the United States. GISTs are believed to arise from the Interstitial Cells of Cajal, the pacemaker cells of the gut, or from interstitial mesenchymal precursor stem cells. GISTs express and are clinically diagnosed by immunohistochemical staining of CD117, the 145 kDa transmembrane glycoprotein KIT. The most common primary sites for these neoplasms are the stomach (60-70%), followed by the small intestine (25-35%). The majority (~80%) of GISTs possess gain-of-function mutations in KIT in either exons 9, 11, 13 or 17, causing constitutive activation of the kinase receptor, whereas smaller subsets of GISTs possess either gain-of-function mutations in PDGFRA (exons 12, 14, or 18) (~5-8%) or no mutations in either KIT or PDGFRA (~12-15%). Imatinib mesylate (IM), an oral drug that inhibits the KIT/PDGFRA is very effective at controlling metastatic disease and preventing recurrence after initial surgery. Unfortunately, in patients with advanced disease, IM stops working after approximately two years. Our group is studying what happens to GIST cells when they are treated with imatinib and what leads to clinical resistance. Our initial studies identified genetic markers that could predict the response of patients with metastatic/recurrent GIST to imatinib and current studies are focusing on genomic and proteomic changes associated with the pathogenesis of GIST and response to molecular targeted therapies. We have also shown an important role for IGF signaling in adult and pediatric GISTs that lack activating kinase mutations. Based on these studies clinical trials are being developed with the goal of ultimately eradicating this disease. Below are examples of ongoing studies in these areas.Top

Insulin-like growth factor 1 receptor is a potential therapeutic target for gastrointestinal stromal tumors

L. Rink, M. Belinsky, & A.K. Godwin, in collaboration with B.L Eisenberg (Dartmouth-Hitchcock Medical Center), D. Flieder, C. Tarn (McNeil Laboratories), J.R. Testa, M. von Mehren

The insulin-like growth factor system and its role
in tumorigenesis

A subset of GISTs lack gain-of-function mutations in KIT and PDGFRA. These GISTs tend to be less responsive to imatinib-based therapies and have a worse prognosis than GISTs harboring mutations in KIT or PDGFRA. We have shown that the aberrant expression of IGF-1R, as opposed to oncogenic RTK-mutations, may be the driving event in these GISTs. This observation suggests an alternative and/or complementary therapeutic regimen in the clinical management of all GISTs, especially in the subset of tumors that respond less favorably to imatinib. Immunoblotting of GIST specimens demonstrated that IGF-1R was present in all tumors but was markedly overexpressed (10- to 30- fold) in GISTs lacking RTK-mutations as compared to mutant GISTs. IGF-1R overexpression in these tumors was confirmed by IHC analysis using an extended set of GIST specimens. In addition, qPCR analysis demonstrated that IGF-1R mRNA is overexpressed ~17- to 19-fold in GISTs lacking RTK-mutations versus mutant GISTs (P = 0.0013). Interestingly, IGF-1R is constitutively activated in all GISTs irrespective of KIT/PDGFRA mutational status. Using a genomic-based qPCR assay, we found that the IGF-1R gene copy number was increased in 70% of GISTs lacking mutation in KIT and PDGFRA (copy number range, 2.5 to 4 copies/tumor cell), as compared with 28% of mutant GISTs (P = 0.04). Fluorescent in situ hybridization analysis confirmed the IGF-1R amplification (3 to 10 copies/tumor cell). Inhibition of IGF-1R signaling with NVP-AEW541 (Novartis, Basel, Switzerland), a specific inhibitor of IGF-1R or down-regulation of expression of IGF-1R with siRNA led to cytotoxicity and induced apoptosis in GIST cells. Combination of NVP-AEW541 and imatinib in GIST cells induced a strong cytotoxicity response. These findings are particularly exciting given the number of agents targeting IGF-1R that are currently being tested in clinical trials. We are currently testing neutralizing monoclonal antibodies specific for IGF-1R alone and in combination with imatinib, with the hope of improving current therapeutic modalities in GIST patients. Clinical trials using IGF-1R-targeted therapies for imatinib-refractory GIST patients, initially focusing on adult and pediatric GIST patients lacking KIT or PDGFRA mutations are in development within our sarcoma program.Top

Our group is actively involved in evaluating clinical trial samples for predictive markers of drug response. As such, Dr. Godwin is the Translational Science Co-chair or the lead translational researcher for several Gynecologic Oncology Group (GOG), Eastern Cooperative Oncology Group (ECOG), and Southwestern Oncology Group (SWOG) clinical trials evaluating molecularly targeted agents, such as AMG 102, AMG 386, AMG 479, AMG 706, cetuximab, dasatinib, everolimus, enzastaurin, gefitinib, lapatinib, RAD001, sorafenib, temsirolimus, and VEGF-TRAP. Through the Clinical Molecular Genetics Laboratory (CMGL, Director, Godwin) we are evaluating clinical trial samples for predictive markers of response to therapy. For example, our group demonstrated in a phase II clinical trial that certain serologic markers were predictive of disease control when women with recurrent epithelial ovarian carcinoma were treated with cetuximab alone. We have also recently evaluated a series of tumor vascular proteins identified through gene profiling studies as serum prognostic and diagnostic markers and are assessing the predictive value in large cohorts of patient samples. In a collaborative phase II clinical trial of patients with colorectal cancer we demonstrated that tumors with high gene expression levels of epiregulin, amphiregulin, and/or wild-type K-RAS were more likely to have disease control with cetuximab treatment. This study led to numerous other confirmatory studies and a recent Provisional Clinical Opinion statement by the American Society of Clinical Oncology that all patients with metastatic colorectal cancer who are candidates for anti-EFGR therapy (i.e., cetuximab and panitumumab) have their tumors tested for K-RAS mutations. In general, our translational correlatives are aimed at extracting proteomic and genomic information from patients’ blood and/or tumor tissue samples prior to and during the course of treatment to identify potential biomarkers associated with clinical activity and/or predictive of outcome. The ultimate goal of these efforts is to better identify patients more likely to benefit from a particular therapy. A targeted approach to patient selection could potentially improve survival, spare patients needless toxicity, and reduce expenses associated with futile therapy. Our clinical research efforts will continue to explore and expand the use of molecular pathology in personalizing patient care.Top