Lynn Roy

MiR-24 Is Required for Hematopoietic Differentiation of Mouse Embryonic Stem Cells

Overexpression of miRNA, miR-24, in mouse hematopoietic progenitors increases monocytic/ granulocytic differentiation and inhibits B cell development. To determine if endogenous miR-24 is required for hematopoiesis, we antagonized miR-24 in mouse embryonic stem cells (ESCs) and performed in vitro differentiations. Suppression of miR-24 resulted in an inability to produce blood and hematopoietic progenitors (HPCs) from ESCs. The phenotype is not a general defect in mesoderm production since we observe production of nascent mesoderm as well as mesoderm derived cardiac muscle and endothelial cells. Results from blast colony forming cell (BL-CFC) assays demonstrate that miR-24 is not required for generation of the hemangioblast, the mesoderm progenitor that gives rise to blood and endothelial cells. However, expression of the transcription factors Runx1 and Scl is greatly reduced, suggesting an impaired ability of the hemangioblast to differentiate. Lastly, we observed that known miR-24 target, Trib3, is upregulated in the miR-24 antagonized embryoid bodies (EBs). Overexpression of Trib3 alone in ESCs was able to decrease HPC production, though not as great as seen with miR-24 knockdown. These results demonstrate an essential role for miR-24 in the hematopoietic differentiation of ESCs. Although many miRNAs have been implicated in regulation of hematopoiesis, this is the first miRNA observed to be required for the specification of mammalian blood progenitors from early mesoderm.

Differential expression of ARID3B in normal adult tissue and carcinomas

ARID3B is a DNA binding protein that is overexpressed in neuroblastoma and ovarian cancer. To understand the extent that ARID3B participates in tumor development, we assessed protein expression of ARID3B in normal adult and malignant tissues. We found that ARID3B is highly expressed in differentiated layers of squamous epithelium. We also examined expression of an alternative splice form of ARID3B and found that it has similar but not identical expression patterns to the full length ARID3B isoform. ARID3B has two closely related paralogues, ARID3A and ARID3C. Each of these 3 family members exhibits different patterns of expression. Of the ARID3 family members, ARID3B is the most widely expressed and is particularly expressed in epithelium. In addition to examining normal tissue, we investigated ARID3B expression in a variety of tumor types. Most notably we found that ARID3B expression is decreased in esophagus and stomach tumors compared to normal corresponding tissues. Our results indicate that the different patterns of ARID3B in normal tissues translate into different roles for ARID3B in carcinomas.

CD133 Promotes Adhesion to the Ovarian Cancer Metastatic Niche

Cancer stem cells (CSCs) are an attractive therapeutic target due to their predicted role in both metastasis and chemoresistance. One of the most commonly agreed on markers for ovarian CSCs is the cell surface protein CD133. CD133+ ovarian CSCs have increased tumorigenicity, resistance to chemotherapy, and increased metastasis. Therefore, we were interested in defining how CD133 is regulated and whether it has a role in tumor metastasis. Previously we found that overexpression of the transcription factor, ARID3B, increased the expression of PROM1 (CD133 gene) in ovarian cancer cells in vitro and in xenograft tumors. We report that ARID3B directly regulates PROM1 expression. Importantly, in a xenograft mouse model of ovarian cancer, knockdown of PROM1 in cells expressing exogenous ARID3B resulted in increased survival time compared with cells expressing ARID3B and a control short hairpin RNA. This indicated that ARID3B regulation of PROM1 is critical for tumor growth. Moreover, we hypothesized that CD133 may affect metastatic spread. Given that the peritoneal mesothelium is a major site of ovarian cancer metastasis, we explored the role of PROM1 in mesothelial attachment. PROM1 expression increased adhesion to mesothelium in vitro and ex vivo. Collectively, our work demonstrates that ARID3B regulates PROM1 adhesion to the ovarian cancer metastatic niche.

Abstract 256: Elucidating the role of ARID3B in ovarian cancer stem cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Ovarian cancer is the fifth leading cause of cancer death among US women and accounts for more deaths than any other cancer of gynecological origin. In light of the lethality, it is critical to discover and understand the common molecular characteristics in ovarian cancer. Our lab demonstrated that ARID3B, a member of the AT-rich interactive domain family of proteins, is overexpressed in ovarian cancer. Furthermore, ARID3B is essential for embryonic development and is present in embryonic stem cells. Overexpression of ARID3B in ovarian cancer cells leads to increased tumor growth and decreased survival in xenograft studies. Therefore, we hypothesize that ARID3B regulates cancer stem cell production, which enhances tumorigenesis. We have found that stem-cell marker CD133 is upregulated in ovarian cancer human cell line SKOV3IP overexpressing ARID3B. We are currently assessing the mechanism for ARID3B regulation of ovarian cancer stem cells using in vitro models. Determining the role of ARID3B in cancer stem cells may help us better understand the molecular mechanisms that regulate tumor initiation, chemoresistance, and ovarian cancer progression. Citation Format: Victoria E. Deneke, Lynn Roy, Richard Dahl, Karen D. Cowden Dahl. Elucidating the role of ARID3B in ovarian cancer stem cells. [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 256. doi:10.1158/1538-7445.AM2013-256

Abstract A45: An in vivo examination of the effects of ARID3B on ovarian cancer metastasis in nude mice

In 2011, 21,880 women were diagnosed with ovarian cancer and more than 13,850 died from the disease. Unfortunately, the prognosis for most women diagnosed with ovarian cancer is poor due to most patients being diagnosed with local and distant metastases. ARID3B, a DNA binding protein from the AT-rich interactive domain (ARID) family, is overexpressed in late stage neuroblastoma and ovarian cancer. In early development, ARID3B is essential as evidenced by the deaths of Arid3B null embryos by midgestation. The importance of ARID3B in early development and its overexpression in cancer suggest a potential role in cancer progression and metastasis. In addition, our laboratory identified two isoforms of ARID3B a full length (FL) and a short form (SH) that is missing the c-terminal exons 5-9. To determine the effects of the two ARID3B isoforms on metastasis in vivo, human ovarian cancer SKOV3IP cells (selected for their ability to metastasize) overexpressing red fluorescent protein (RFP), ARID3B-FL, or ARID3B-Sh were injected intraperitoneally into nude mice. The mice underwent live optical fluorescence imaging using the Multispectral FX (Kodak) imaging system at frequent intervals to assess tumor burden. The mice were sacrificed when they began to show signs of significant illness. Live imaging of the mice demonstrated that ARID3B-FL expressing SKOV3IP cells formed more and larger tumors more quickly than either SKOV3IP-RFP cells or SKOV3-ARID3B-Sh cells. After sacrificing the mice, tumors and organs were recovered from the abdomen and were imaged ex vivo for tumor burden. Tumors were separated from the organs, weighed, and imaged. The average weights of the tumors were 1.91g for SKOV3IP-RFP derived tumors, 3.22g for SKOV3IP ARID3B-Sh tumors, and 5.46g for SKOV3IP-FL derived tumors. The average tumor burden was greatest for the mice bearing SKOV3IP ARID3B-FL tumors. Gross examination of the tumors post-mortem and post fixation suggested that the tumors from the RFP and ARID3B-Sh expressing cell lines were less vascular or had less vascular leakage than those from the ARID3B-FL mice. Survival analysis showed that the median survival for SKOV3IP ARID3B-FL tumor bearing mice was significantly lower than mice bearing RFP or the ARID3B-Sh expressing tumors (RFP=51 days, ARID3B-Sh=52 days, ARID3B-FL=36 days; P=0.0074). Currently, our lab is addressing potential mechanisms for how ARID3B functions in metastasis. The data presented here suggest that ARID3B plays a significant role in peritoneal metastasis in ovarian cancer. Citation Format: Lynn Roy, Serene Samyesudhas, Martin Carrasco III, Karen Cowden Dahl. An in vivo examination of the effects of ARID3B on ovarian cancer metastasis in nude mice. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A45.

Abstract 3038: Delineating the role of Arid3B in normal and malignant differentiation of esophageal and cervical stratified squamous epithelia

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL ARID3B is DNA binding protein overexpressed in neuroblastoma and ovarian cancer. We have determined that ARID3B expression is also upregulated during squamous epithelial differentiation (cervix, skin, and esophagus). Therefore, we wanted to assess the role of ARID3B in development of squamous cell carcinomas of the esophagus and cervix. In addition to examining the expression of ARID3B, we also wanted to determine the mechanism for ARID3B regulation in cancer and normal squamous cell epithelia. Recent evidence suggests that p63, specifically the ΔNp63α isoform, transcriptionally regulates Arid3B. p63 is a member of the p53 transcription factor family and is essential for the proliferative potential of stem cells in stratified squamous epithelia. p63 is also involved in various cancers including skin cancer and esophageal cancer. In esophageal squamous cell carcinoma, p63 is ubiquitously expressed and low levels are associated with a poor prognosis. We hypothesize that p63 regulates Arid3B in normal and malignant differentiation of stratified squamous epithelium. To evaluate ARID3B expression in cancer we performed immunohistochemistry on tissue microarrays using two independent Arid3B antibodies. Loss of nuclear Arid3B appears to be common in squamous cell carcinoma of cervix and esophagus. We also examined expression of ARID3B in a normal and a squamous cell carcinoma esophageal cell line. We are also examining the coordinate expression of p63 and Arid3B in serial sections of normal and malignant tissue sections of esophagus, cervix, and skin. We are also investigating the regulation of ARID3B in 3D cell culture models of esophagus differentiation. Our data suggests that misregulation of ARID3B may contribute to the development of cancer in stratified epithelia specifically of the cervix and esophagus. 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 3038. doi:1538-7445.AM2012-3038