Karla Esbona

Titin diversity--alternative splicing gone wild.

Titin is an extremely large protein found in highest concentrations in heart and skeletal muscle. The single mammalian gene is expressed in multiple isoforms as a result of alternative splicing. Although titin isoform expression is controlled developmentally and in a tissue specific manner, the vast number of potential splicing pathways far exceeds those described in any other alternatively spliced gene. Over 1 million human splice pathways for a single individual can be potentially derived from the PEVK region alone. A new splicing pattern for the human cardiac N2BA isoform type has been found in which the PEVK region includes only the N2B type exons. The alterations in splicing and titin isoform expression in human heart disease provide impetus for future detailed study of the splicing mechanisms for this giant protein.

Mutation that dramatically alters rat titin isoform expression and cardiomyocyte passive tension

Titin is a very large alternatively spliced protein that performs multiple functions in heart and skeletal muscles. A rat strain is described with an autosomal dominant mutation that alters the isoform expression of titin. While wild type animals go through a developmental program where the 3.0 MDa N2B becomes the major isoform expressed by two to three weeks after birth (approximately 85%), the appearance of the N2B is markedly delayed in heterozygotes and never reaches more than 50% of the titin in the adult. Homozygote mutants express a giant titin of the N2BA isoform type (3.9 MDa) that persists as the primary titin species through ages of more than one and a half years. The mutation does not affect the isoform switching of troponin T, a protein that is also alternatively spliced with developmental changes. The basis for the apparently greater size of the giant titin in homozygous mutants was not determined, but the additional length was not due to inclusion of sequence from larger numbers of PEVK exons or the Novex III exon. Passive tension measurements using isolated cardiomyocytes from homozygous mutants showed that cells could be stretched to sarcomere lengths greater than 4 mum without breakage. This novel rat model should be useful for exploring the potential role of titin in the Frank-Starling relationship and mechano-sensing/signaling mechanisms.

COX-2 modulates mammary tumor progression in response to collagen density

BackgroundHigh breast density is linked to an increased risk of breast cancer, and correlates with changes in collagen. In a mouse model of mammary carcinoma in the context of increased collagen deposition, the MMTV-PyMT/Col1a1tm1jae, there is accelerated mammary tumor formation and progression. Previous gene expression analysis suggests that increased collagen density elevates expression of PTGS2 (prostaglandin-endoperoxide synthase 2), the gene for cyclooxygenase-2 (COX-2).MethodsTo understand the role of COX-2 in tumor progression within a collagen-dense microenvironment, we treated MMTV-PyMT or MMTV-PyMT/Col1a1tm1jae tumors prior to and after tumor formation. Animals received treatment with celecoxib, a specific COX-2 inhibitor, or placebo. Mammary tumors were examined for COX-2, inflammatory and stromal cell components, and collagen deposition through immunohistochemical analysis, immunofluorescence, multiplex cytokine ELISA and tissue imaging techniques.ResultsPyMT/Col1a1tm1jae tumors were larger, more proliferative, and expressed higher levels of COX-2 and PGE2 than PyMT tumors in wild type (WT) mice. Treatment with celecoxib significantly decreased the induced tumor size and metastasis of the PyMT/Col1a1 tumors, such that their size was not different from the smaller PyMT tumors. Celecoxib had minimal effect on the PyMT tumors. Celecoxib decreased expression levels of COX-2, PGE2, and Ki-67. Several cytokines were over-expressed in PyMT/Col1a1 compared to PyMT, and celecoxib treatment prevented their over-expression. Furthermore, macrophage and neutrophil recruitment were enhanced in PyMT/Col1a1 tumors, and this effect was inhibited by celecoxib. Notably, COX-2 inhibition reduced overall collagen deposition. Finally, when celecoxib was used prior to tumor formation, PyMT/Col1a1 tumors were fewer and smaller than in untreated animals.ConclusionThese findings suggest that COX-2 has a direct role in modulating tumor progression in tumors arising within collagen-dense microenvironments, and suggest that COX-2 may be an effective therapeutic target for women with dense breast tissue and early-stage breast cancer.

The Presence of Cyclooxygenase 2, Tumor-Associated Macrophages, and Collagen Alignment as Prognostic Markers for Invasive Breast Carcinoma Patients

Inflammation, and the organization of collagen in the breast tumor microenvironment, is an important mediator of breast tumor progression. However, a direct link between markers of inflammation, collagen organization, and patient outcome has yet to be established. A tumor microarray of 371 invasive breast carcinoma biopsy specimens was analyzed for expression of inflammatory markers, including cyclooxygenase 2 (COX-2), macrophages, and several collagen features in the tumor nest (TN) or the tumor-associated stroma (TS). The tumor microarray cohort included females, aged 18 to 80 years, with a median follow-up of 8.4 years. High expression of COX-2 (TN), CD68 (TS), and CD163 (TN and TS) predicted worse patient overall survival (OS). This notion was strengthened by the finding from the multivariate analysis that high numbers of CD163+ macrophages in the TS is an independent prognostic factor. Overall collagen deposition was associated with high stromal expression of COX-2 and CD163; however, total collagen deposition was not a predictor for OS. Conversely, local collagen density, alignment and perpendicular alignment to the tumor boundary (tumor-associated collagen signature-3) were predictors of OS. These results suggest that in invasive carcinoma, the localization of inflammatory cells and aligned collagen orientation predict poor patient survival. Additional clinical studies may help validate whether therapy with selective COX-2 inhibitors alters expression of CD68 and CD163 inflammatory markers.

Abstract 4960: COX-2 inhibition with celecoxib delays the progression of invasive mammary carcinoma in a murine model of collagen dense stroma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Breast cancer is the most common invasive cancer in women causing over 40,000 deaths yearly in the United States. Increased mammographic density correlates with over four-fold greater risk for breast cancer, making it one of the most important risk factors known for this disease. High breast density is correlated to increased collagen in the breast tissue, and we have found that increased collagen in the Col1a1tm1jae mouse model promotes mammary tumor formation and progression. Elevated collagen density in vitro increases expression of PTGS2 (prostaglandin-endoperoxide synthase 2), the gene for COX-2, by over four fold. COX-2 over-expression is observed in 40% of invasive breast carcinoma cases and correlates with poor prognosis. Based on these findings, we hypothesized that inhibition of COX-2 may be an effective therapy in the context of mammary tumors arising in dense tissue. Celecoxib is a selective non-steroidal anti-inflammatory drug (NSAID) that specifically inhibits COX-2. To understand the mechanism by which COX-2 affects cell signaling and response to collagen matrix density, we utilized our previously characterized model of MMTV-PyVT tumors on a wild type (wt) or Col1a1tm1jae background. Col1a1tm1jae heterozygote and wt littermates were randomly assigned at 11 weeks of age to treatment with placebo or celecoxib at 400mg per kilogram body weight. Oral treatment was given daily for 21 days. We found that MMTV-PyVT tumors responded to celecoxib in a manner that is regulated by matrix density (p-value <.0001). Tumors that arose on the dense Col1a1tm1jae background were larger and more invasive (p-value <.0001). Endpoint tumor size was diminished in mice treated with a daily dose of celecoxib, which delayed the development of tumors and changed the structure of the dense matrix. The effect of celecoxib was not as prominent for masses which developed on the low density wt background. Immunohistochemistry of relevant markers in these tissues is ongoing and will further our understanding of the effects of COX-2 inhibition in dense matrices. These findings suggest that COX-2 has a direct role in modulating the configuration of dense matrices which promote a more invasive cancer effect. Moreover, these findings suggest that COX-2 may be an effective therapeutic target for women with dense breast tissue. Citation Format: Karla Esbona, David R. Inman, Kevin W. Eliceiri, Lee G. Wilke, Patricia J. Keely. COX-2 inhibition with celecoxib delays the progression of invasive mammary carcinoma in a murine model of collagen dense stroma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4960. doi:10.1158/1538-7445.AM2013-4960

AKR1B10 expression by immunohistochemistry in surgical resections and fine needle aspiration cytology material in patients with cystic pancreatic lesions; potential for improved nonoperative diagnosis

Cystic pancreatic tumors account for 10% of cystic lesions in the pancreas. Evaluation focuses on identifying lesions that require surgical resection due to actual or potential malignancy. Cystic tumors with malignant potential include mucinous cystic neoplasms (MCNs), intraductal papillary mucinous neoplasms (IPMNs), and cystic neuroendocrine tumors (NETs). The sensitivity of endoscopic fine needle aspiration (FNA) to diagnose such lesions is low, and a more accurate marker of malignant potential is needed. Aldo-keto reductase 1B10 (AKR1B10) was originally found in human hepatocellular carcinoma. Since then, it has been identified in pancreatic adenocarcinoma and pancreatic intraepithelial neoplasia. Because there is difficulty in determining the malignant potential of cystic pancreatic tumors, we set out to examine the expression of AKR1B10 in these lesions as a potential biomarker of malignancy. AKR1B10 expression was analyzed in cell blocks from FNAs and surgical resection specimens using immunohistochemistry. We examined MCN (n=28), IPMN (n=18), and cystic NET (n=20) as well as nonmucinous cysts including pseudocysts (n=13) and serous cystadenomas (n=16). AKR1B10 expression was seen in 45 of 46 (98%) mucinous lesions evaluated. Strong staining (2+-3+/60%-100% staining) was seen in 16 of 18 (89%) IPMNs and 25 of 28 (90%) MCNs. No staining was seen in the nonmucinous lesions (n=49). In conclusion, AKR1B10 is upregulated in mucinous cystic pancreatic tumors, and this staining can be accomplished in cytology FNA material, making AKR1B10 a promising biomarker of malignant potential. Most importantly, this application could impact the clinical management of these patients by determining the best candidates for surgical resection.

Abstract 3927: The presence of COX-2 and tumor-associated macrophages as a prognostic marker for invasive breast carcinoma patients

Inflammation is an important mediator of tumor progression. The objective of this study was to assess whether the tissue localization of COX-2 and tumor-associated macrophages were associated with clinicopathological features of invasive carcinoma, including collagen deposition and patient survival outcome. A tumor microarray (TMA) of 371 biopsy specimens from patients with invasive breast carcinoma was analyzed for expression of high levels of COX-2, and the macrophage markers CD68 and CD163 in either the tumor nest (TN) or the tumor-associated stroma (TS). The study population for this TMA was female patients, 18 to 80 years of age with a median follow up of 8.4 years. Survival tables were calculated according to the Kaplan-Meier method. We found that elevated collagen deposition was associated with high stromal expression of COX-2 (P Citation Format: Karla Esbona, Sandeep Saha, Yanyao Yi, Menggang Yu, Kari Wisinski, Lee G. Wilke, Patricia J. Keely. The presence of COX-2 and tumor-associated macrophages as a prognostic marker for invasive breast carcinoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3927.

Abstract 1116: Response to cyclooxygenase-2 inhibition is regulated by collagen dense stroma

Increased mammographic density correlates with over four-fold increase risk for breast cancer, making it one of the greatest risk factors known for this disease. High breast density is correlated to increased collagen in the breast tissue, and we have found that increased collagen in the Col1a1 tm1jae mouse model promotes mammary tumor formation and progression. Increased collagen density in vitro elevates expression of PTGS2 (prostaglandin-endoperoxide synthase 2), the gene for cyclooxygenase-2 (COX-2), by over four fold. Because COX-2 over-expression is observed in 40% of invasive breast carcinoma cases and correlates with poor prognosis, we hypothesized that inhibition of COX-2 may be an effective therapeutic in the context of mammary tumors arising in dense tissue. Celecoxib is a selective non-steroidal anti-inflammatory drug (NSAID) that specifically inhibits COX-2. To understand how COX-2 affects response to collagen matrix density, we made use of our previously characterized mouse model of MMTV-PyVT tumors in a wild type (wt) or Col1a1 tm1jae background (HD). Col1a1 tm1jae /+ or wild-type (+/+) littermates were randomly assigned at 11 weeks of age to treatment with vehicle or celecoxib at 0.2mg per mouse per day. Oral treatment was given daily for 21 days. We found a link between matrix density and the role of COX-2. Tumors that arose on the dense Col1a1 tm1jae background (HD) were larger and more invasive (p tm1jae /+ animals. Other features were affected by celecoxib in a non-density manner, as the total number of macrophages (wt = p = 0.0052, HD = p tm1jae /+ and +/+ animals. Ongoing studies are aimed at identifying different macrophage populations, and which stromal cell populations are expressing COX-2 and PGE2. These findings suggest that COX-2 has a direct role in modulating tumor progression in dense matrices, which in turn promote more aggressive tumors. As dense breast tissue is a common occurrence, these findings suggest that COX-2 may be an effective therapeutic target for women with dense breast tissue. Citation Format: Karla Esbona, David Inman, Sandeep Saha, Kevin Eliceiri, Lee G. Wilke, Patricia J. Keely. Response to cyclooxygenase-2 inhibition is regulated by collagen dense stroma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1116. doi:10.1158/1538-7445.AM2014-1116

Abstract P1-06-02: Inflammatory stromal cell response induced by collagen dense stroma is regulated by cyclooxygenase-2 in a mouse breast cancer model

Breast cancer is the most common invasive cancer in women, causing 40,000 deaths yearly in the United States. Increased mammographic density correlates with over four-fold increase risk for breast cancer, making it one of the greatest risk factors known for this disease. High breast density is mainly attributable to elevated collagen matrix deposition in the breast tissue, and we have found that increased collagen in the Col1a1tm1jae mouse model promotes mammary tumor formation and progression. Increased collagen density in vitro increases expression of PTGS2 (prostaglandin-endoperoxide synthase 2), the gene for cyclooxygenase-2 (COX-2), by over four fold. COX-2 over-expression is observed in 40% of invasive breast carcinoma cases and correlates with poor prognosis. Based on these findings, we hypothesized that inhibition of COX-2 may be an effective therapeutic in the context of mammary tumors arising in dense tissue. Celecoxib is a selective non-steroidal anti-inflammatory drug (NSAID) that specifically inhibits COX-2. To understand how COX-2 affects response to collagen matrix density, we utilized our previously characterized mouse model of MMTV-PyVT tumors in a wild type (wt) or Col1a1 tm1jae background (HD, High Density). Col1a1tm1jae heterozygote or wt littermates were randomly assigned at 11 weeks of age to treatment with vehicle or celecoxib at 0.2mg per mouse per day. Oral treatment was given daily for 21 days. We found that MMTV-PyVT tumors responded to celecoxib in a manner that is regulated by matrix density. Tumors that arose on the dense Col1a1 tm1jae background (HD) were larger and more invasive (p Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-02.