Erika Pond

Gemcitabine resistant pancreatic cancer cell lines acquire an invasive phenotype with collateral hypersensitivity to histone deacetylase inhibitors

Gemcitabine based treatment is currently a standard first line treatment for patients with advanced pancreatic cancer, however overall survival remains poor, and few options are available for patients that fail gemcitabine based therapy. To identify potential molecular targets in gemcitabine refractory pancreatic cancer, we developed a series of gemcitabine resistant (GR) cell lines. Initial drug exposure selected for an early resistant phenotype that was independent of drug metabolic pathways. Prolonged drug selection pressure after 16 weeks, led to an induction of cytidine deaminase (CDA) and enhanced drug detoxification. Cross resistance profiles demonstrate approximately 100-fold cross resistance to the pyrimidine nucleoside cytarabine, but no resistance to the same in class agents, azacytidine and decitabine. GR cell lines demonstrated a dose dependent collateral hypersensitivity to class I and II histone deacetylase (HDAC) inhibitors and decreased expression of 3 different global heterochromatin marks, as detected by H4K20me3, H3K9me3 and H3K27me3. Cell morphology of the drug resistant cell lines demonstrated a fibroblastic type appearance with loss of cell-cell junctions and an altered microarray expression pattern, using Gene Ontology (GO) annotation, consistent with progression to an invasive phenotype. Of particular note, the gemcitabine resistant cell lines displayed up to a 15 fold increase in invasive potential that directly correlates with the level of gemcitabine resistance. These findings suggest a mechanistic relationship between chemoresistance and metastatic potential in pancreatic carcinoma and provide evidence for molecular pathways that may be exploited to develop therapeutic strategies for refractory pancreatic cancer.

Targeting integrin α6 stimulates curative-type bone metastasis lesions in a xenograft model.

Laminin-binding integrin receptors are key mediators of epithelial cell migration and tumor metastasis. Recent studies have demonstrated a role for the α6 integrin (ITGA6/CD49f) in maintaining stem cell compartments within normal bone marrow and in residency of tumors metastatic to bone. In this study, we tested a function-blocking antibody specific for ITGA6, called J8H, to determine if preexisting cancer lesions in bone could be slowed and/or animal survival improved. Human prostate tumors were established by intracardiac injection into male SCID mice and treatment with J8H antibody was initiated after 1 week. Tumor progression was monitored by micro-computed tomography (CT) imaging of skeletal lesions. Animals that received weekly injections of the anti-ITGA6 antibody showed radiographic progression in only 40% of osseous tumors (femur or tibia), compared with control animals, where 80% of the lesions (femur or tibia) showed progression at 5 weeks. Kaplan–Meier survival analysis demonstrated a significant survival advantage for J8H-treated animals. Unexpectedly, CT image analysis revealed an increased proportion of bone lesions displaying a sclerotic rim of new bone formation, encapsulating the arrested lytic lesions in animals that received the anti-ITGA6 antibody treatment. Histopathology of the sclerotic lesions demonstrated well-circumscribed tumor within bone, surrounded by fibrosis. These data suggest that systemic targeting of the ITGA6-dependent function of established tumors in bone may offer a noncytotoxic approach to arrest the osteolytic progression of metastatic prostate cancer, thereby providing a new therapeutic strategy for advanced disease. Mol Cancer Ther; 13(6); 1558–66. ©2014 AACR.

Implementation of educational video improves patient understanding of basic breast cancer concepts in an undereducated county hospital population

The purpose of this study was to evaluate the effect of a video on patient understanding of basic breast cancer concepts.

Abstract 09: Laminin binding integrin copy number variations in distinct types of epithelial cancers

Background: The laminin binding integrin (LBI) family are adhesion molecules that are essential for invasion and metastasis of human epithelial cancers and cell adhesion mediated drug resistance. Our objective was to investigate whether copy number variation (CNV) of a five gene signature (ITGB4, ITGA3, LAMB3, PLEC and SYNE3) comprised of essential genes for laminin adhesion would correlate with the reported clinical outcome in human epithelial tumors. Methods: We investigated the relative alteration frequency of the CNV of integrin B4 (ITGB4), integrin A3 (ITGA3), laminin beta 3 chain (LAMB3), plectin (PLEC) and nesprin 3 (SYNE3) as a five gene signature independent of the epithelial cancer type using the publically available and published data. Provisional data was excluded from the analysis. We rank ordered the results using a 20% alteration frequency cut off and limited the analysis to studies containing at least 100 samples. The Kaplan Meier survival curves were analyzed to determine any alterations in patient survival in terms of months. Results: Eight different cancer types representing 1,985 samples, contained at least a 20% alteration frequency of the five gene signature. The frequency of alteration ranged from 32.2% to 22.9% with the rank order (highest to lowest) as skin cutaneous melanoma, ovarian serous adenocarcinoma, stomach adenocarcinoma, lung adenocarcinoma, bladder urothelial carcinoma, breast invasive carcinoma and uterine corpus endometrial carcinoma. Within the gene signature, PLEC was the most commonly altered followed by LAMB3, ITGB4, ITGA3 and SYNE3 across all eight cancer types. Although specific mutations occurred in PLEC, amplification was the most common feature. Within a cancer type, there was little overlap of the individual amplified genes with the specific case, suggesting different specific amplicons may alter the LBI adhesion structures. Of the eight cancer types, a significant alteration in survival was indicated in bladder urothelial carcinoma (p=0.052714) with a median survival change of 19.02 to 24.28 months. Conclusions: A five gene signature was created representing the essential and genetically verified functional components of laminin binding integrins and their adhesion structures. The CNV signature of the laminin binding integrins may have prognostic and predictive significance depending on the type of epithelial cancer. These may be promising biomarkers but also rational targets for personalized therapy in selected epithelial tumors for preventing metastatic spread. (Supported in part by NIH grants CA 23074 and CA 159406) Citation Format: Erika Pond, William L. Harryman, Anne E. Cress. Laminin binding integrin copy number variations in distinct types of epithelial cancers. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 09.

Abstract B42: Adaptive drug resistance is associated with phenotypic reprogramming and hypersensitivity to HDAC inhibitors

Pancreatic cancer is the fourth leading cause of cancer death in the United States. One of the major reasons for the poor prognosis of pancreatic cancer is its high resistance to gemcitabine and other currently available chemotherapeutic agents. In the present study, we have developed gemcitabine-resistant cell lines to investigate the mechanisms by which adaptive resistance occurs during pancreatic tumor progression. Gemcitabine-resistant (GR) cell lines were established from the MiaPaCa2 cell line. Increasing drug selection pressure resulted in the establishment of populations that proliferate in the continuous presence of 300 nm (GR300), 800 nM (GR800), or 2µM (GR2000) gemcitabine. Using a SCID mouse xenograft mouse model, we demonstrate that the gemcitabine resistant phenotype is maintained in vivo. Growth inhibition profiles demonstrate cross resistance to Ara-c (cytarabine), and 2-chlorodeoxyadenosine (2CDA/cladribine), but no resistance to DNA damaging agents or microtubule inhibitors. Most strikingly, the gemcitabine resistant subclones demonstrated a dose dependent hypersensitivity to the Class I histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) (IC50: MP 1.6uM; GR300 1.0uM; GR800 0.2uM; GR2000 0.08uM). This collateral hypersensitivity extended to additional Class I HDAC inhibitors including MS-275 (Entinostat) and Trichostatin A, but not to the Class IIa selective agent MC1568. There was no significant difference in basal levels of histone acetylation, but the gemcitabine resistant clones demonstrated a greater increase in histone acetylation in response to HDAC treatment. Cell morphology of the gemcitabine resistant cells demonstrate an EMT-like phenotype, however evaluation of classic EMT markersidentified no significant changes in expression compared to the parental cell line. Invasive potential was measured using matrigel invasion assays, and found to be increased in a dose dependent manner. These data demonstrate that selection for drug resistance co-selects for an invasive phenotype with EMT-like characteristics. In addition, the resistance phenotype that emerges under drug pressure is associated with a phenotypic reprogramming that can be exploited to devise new therapeutic options for patients with advanced pancreatic adenocarcinoma. Citation Format: Betty k. Samulitis, Erika Pond, Kelvin Pond, Anne E. Cress, Robert T. Dorr, Terry H. Landowski. Adaptive drug resistance is associated with phenotypic reprogramming and hypersensitivity to HDAC inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B42.

Abstract 4345: Inadequate repair of ionizing radiation damage: A consequence of the invasive tumor phenotype

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL A major characteristic of the transition of prostatic intraepithelial neoplasia (PIN) to invasive prostate cancer is the progressive loss of laminin 322 in the basal lamina and loss of A6B4 integrin, a laminin 322 adhesion receptor in the basal cells. Previous work from our group reported that the loss of interaction with laminin 322 decreased the ability of normal prostate cells to respond to DNA damage. The defect was manifested as an alteration of an ionizing radiation (IR) induced G2 progression block. In this study, the goal was to determine if an invasive phenotype of human tumor cells, expressing A6B1 integrin, a laminin 511 adhesion receptor, influenced a DNA damage response (DDR) to IR. DU145 tumor cells were grown on laminin 511 and DDR was determined under conditions of stimulated invasion via HGF. The DDR endpoint chosen was the production, resolution and persistence of nuclear H2AX phosphorylation foci as an indicator of damage, repair and inadequate repair, respectively. Similar to a recent report, we observed a fast and transient phosphorylation of H2AX in response to IR within the basal cells of the normal human prostate epithelium that was both dose and time dependent. Utilizing tissue culture conditions, we determined that under optimal growth conditions on laminin 511 there was a dose and time response of H2AX phosphorylation in response to IR treatment. Under conditions of HGF stimulation, the persistence of H2AX foci 24 hours after IR was two to four fold higher as compared to unstimulated cells. The amplified persistence of H2AX foci was observed at 1, 2, 4, 6 and 8 Gy at both 24 and 36 hours after IR treatment. Current studies are determining if the inadequate repair response to IR under conditions of stimulated invasion results in an increased tumor cell killing or an increased genomic instability of surviving tumor cells. (Supported in part by NIH Cancer Center Core Grant CA 23074 and DOD grant W81XWH-10-1-0496) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4345. doi:1538-7445.AM2012-4345

Abstract 1534: Laminin binding integrin functions in human bone metastatic cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Laminin binding integrins are major determinants of cancer cell migration, invasion and metastasis. In human prostate cancer, both A6B1 and A3B1 laminin-binding integrins are expressed on tumor cell surfaces as observed in either prostatectomy or needle biopsy specimens. In human breast cancer, the A6 integrin is expressed in both primary tumors and in metastatic lesions. Integrin A6B1 is post-translationally modified on the tumor cell surface by urokinase-type plasminogen activator (uPA), producing a structural variant, A6pB1. While the A6pB1 form is devoid of the ligand binding domain, A3B1 is fully functional and not subject to cleavage. In this study, we observed the continued expression of the A6B1 integrin on tumor cell surfaces within human bone metastatic lesions as well as in human tumor xenografts growing within bone. The cleavage function of the A6 integrin was found to be constitutively expressed on nine different cancer cell lines derived from breast, prostate, lung, pancreatic tumors. Interestingly, five different normal cell types expressing the adhesion active A6B1 integrin did not have the constitutive cleavage function. However, the cleavage function in normal cells was inducible by the exogenous addition of uPA. Selection of successive bone resident cancer cells selects for constitutive cleavage of A6 integrin and increasing expression of A3B1 integrin. Taken together, these data suggest that the cleavage function is constitutively activated in cancer specimens and opens the possibility cleavage mediated release from laminin binding may be coordinated with A3 dependent adhesion. (Supported in part by CA23752 and the TACMASS core service of the Arizona Cancer Center). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1534. doi:10.1158/1538-7445.AM2011-1534

Abstract A80: Parity and obesity in Mexican and Mexican-American women: Findings from the Ella Binational Breast Cancer Study

Background: Obesity rates are increasing rapidly around the globe. Obesity is associated with negative health consequences including increased risk of all-cause mortality and increased breast cancer morbidity. In the U.S., some of the highest rates of obesity are found among Hispanic women. Additionally, factors associated with low socioeconomic status have also been shown to be positively associated with obesity in the U.S., independent of race or ethnicity; higher parity (i.e., three or more full-term births) has also been implicated as a contributing factor. The aim of this study was to evaluate the independent contribution of parity on obesity in a highly parous population of Mexican and Mexican-American women diagnosed with breast cancer. Methods: Participants are from the Ella Binational Breast Cancer Study, a study of women of Mexican descent who were 18 years of age and older at time of enrollment and who were diagnosed with invasive breast cancer within 24 months of recruitment. Each participant completed an interviewer-administered risk factor questionnaire, provided a blood or saliva sample, and consented to provide tumor tissue and access to medical record data. Obesity was defined as having a body mass index (BMI) ≥30 kg/m2, which was calculated from self-reported height and weight. Parity was defined as the number of full-term live births. Age at interview, age at first full-term pregnancy, level of education, employment, smoking status, menopausal status, age at menarche, and breastfeeding were assessed as potential confounding factors. Among U.S. participants, level of acculturation, interview language and nativity were also evaluated as confounders. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using multinomial logistic regression. Results: Mean age was higher for U.S. compared to Mexican participants (54.4 ± 12.6 vs. 50.1 ± 12.0). Approximately 51% (n=608) of participants resided in Mexico and 49% (n=579) in the U.S. Among U.S. participants, 54% were born in Mexico and of these, 42% have lived in the U.S. Conclusions: Our results show a significant, positive association between parity and obesity that was not independent of education. Results of our work suggest that both pre and postnatal periods are important adult life periods for health care providers to implement obesity prevention strategies and interventions. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):A80.

Abstract PR-5: Risk factor distribution among women of Mexican descent by level of acculturation: Findings from the ELLA Binational Breast Cancer Study

Purpose of Study: Studies have shown that breast cancer (BC) risk is higher in U.S.-born Hispanics than foreign-born Hispanics and is modified by age at migration, duration of residence in the U.S., and level of acculturation. Furthermore, epidemiological data support the notion of distinct reproductive factor associations for specific BC tumor subtypes. The aim of this study is to assess the association between established BC risk factors and level of acculturation and country of residence among Mexican American (MA) and Mexican BC cases. Experimental Procedures: This case-only study examined the risk factor profile in 606 Mexican and 488 MA (20% US-born and 80% foreign-born) women, with a confirmed diagnosis of invasive BC, who were recruited into the ELLA Binational Breast Cancer Study between March 1,2007, and May 31,2010. Participants completed an interviewer-administered risk factor questionnaire, consented to a medical record review, and provided tissue and saliva or blood specimens. An eight-item language-based Bidimensional Acculturation Scale (BAS) was used to classify MA women as English-dominant (n=88), bilingual (n=221), or Spanish-dominant (n=179). The BAS is highly correlated with nativity and time in the US, however language use/exposure was focal for this study since it is the single strongest predictor of acculturation. Chi-square tests, ANOVA, and regression models to test for trend were used to assess variation in the risk factor profile by level of acculturation. Summary of Results: Age at diagnosis was generally low; the lowest was among MA-bilingual women (48.9 = 12.0 years) and highest among Mexican women (53.7 = 12.7 years). Considering a gradient of increasing acculturation from Mexican (lowest) to MA-English dominant (highest) women, there were clear trends for decreasing rates of breastfeeding (80.2 to 35.2%; p-trend Conclusion: Our results show that heterogeneity in BC risk factor patterns by level of acculturation is present. Given the recent data supporting distinct correlations between specific risk factors and BC subtypes, it will be essential to consider level of acculturation and country of residence when assessing the prevalence of these subtypes. The trends observed in risk factor profiles by level of acculturation in the ELLA Study could provide important explanations for differences in disease patterns between groups. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):PR-5.