The program in this area specifically focuses on tumor progression, invasion and metastasis and UV carcinogenesis. The investigators in this program are Dr. Ramesh Ganju (chemokines, inflammation and metastasis), Dr. Tatiana Oberyszyn (UV carcinogenesis), Dr. Traci Wilgus (inflammation and angiogenesis), Dr. Sujit Basu (neuro-immune interaction in cancer progression and metastasis), Dr. Xianghong Zou (cell cycle and cancer), Dr. Rolf Barth (treatment for gliomas), Dr. Nancy Lill (regulation of EGF receptor signaling), and Dr. Kalpana Ghoshal (Role of DNA methylation and microRNAs in liver disease)
Chemokines/Inflammation and Metastasis
Dr. Ganju’s laboratory is analyzing the molecular mechanisms of breast cancer progression and metastasis. In addition, they are developing novel therapies to inhibit breast and lung cancer growth and metastasis. Project 1: They are analyzing the role of chemokine receptor CXCR4 and its ligand CXCL12 in breast cancer growth, angiogenesis and metastasis. Dr. Ganju’s laboratory has elucidated novel signaling pathways mediated by CXCR4 that regulate the CXCL12-induced chemotaxis, chemoinvasion, and adhesion of breast cancer cells. They are further analyzing the role of CXCL12/CXCR4 pathway by using CXCL12 conditional knockout mouse models which they have recently generated. Project 2: Dr. Ganju’s laboratory has recently shown that a novel molecule Slit2, which binds to Robo receptor, inhibits breast tumor growth. In addition, they have shown that Slit/Robot complex inhibits tumor growth by modulating the beta-catenin pathway. They are further characterizing the role of Slit2/Robo comples in modulating breast cancer progression and metastasis and are also performing structural and functional studies to determine the domain on Slit2 that possesses anti-tumorigenic activity. Project 3: They are analyzing the role of S100A7 in breast cancer progression and metastasis. S100A7 is among the most highly expressed genes in ER-α-negative breast carcinoma and in high grade pre-invasive ductal carcinoma in situ. Furthermore, a poor prognosis is associated with the persistent expression of S100A7 in invasive carcinoma. Dr. Ganju’s laboratory has shown that S100A7 may modulate breast cancer growth through modulation of inflammatory pathways using a novel inducible bi-transgenic MMTV-rtTA; mS100a7a15 mouse model. They have shown that mS100a7a15 overexpression in mammary glands enhances ductal hyperplasia. They have shown that orthotopic implantation of highly metastatic MVT-1 cells in mammary glands of inducible transgenic mS100a7a15 mice resulted in enhanced tumor growth and metastasis. These effects were mediated by tumor-associated macrophages (TAMs). In addition, soluble S100A7 or mS100a7a15 enhanced chemotaxis of macrophages through activation of RAGE receptors. These studies revealed that S100A7 may enhance breast tumor growth and metastasis through activation of proinflammatory and pro-metastatic pathways. Dr. Ganju’s laboratory has also shown that S100A7-overexpression in ERα+ breast cancer cells exhibits decreased proliferation in vitro and in vivo. Further mechanistic studies revealed that S100A7 mediates the tumor-suppressive effects via a coordinated regulation of β-catenin/TCF4 pathway and an enhanced interaction of β-catenin and E-cadherin in S100A7-overexpressing ERα+ breast cancer cells. We are further analyzing the role of S100A7 in ERα+ and ERα-, especially triple-negative, breast cancer using various breast cancer mouse model systems. Project 4: Dr. Ganju’s laboratory is also identifying small molecular weight molecules that have potential to be used as drugs to block metastasis. In this regard they have shown that synthetic cannabinoids, which are small molecular weight molecules and do not possess psychoactive activity, inhibit breast and lung cancer growth and metastasis in vitro and in vivo using nude mice and transgenic mouse models. Dr. Ganju’s laboratory is further elucidating the mechanism by which synthetic cannabinoids inhibit tumor growth and metastasis. In this regard, they have shown that synthetic cannabinoids that bind to cannabinoid receptors CB1 and CB2 may modulate the activity of epidermal growth factor receptor and chemokine receptor CXCR4. Dr. Ganju’s laboratory is further analyzing the CB1/CB2-mediated molecular mechanism that leads to inhibition of growth and metastasis in lung and breast cancers using various transgenic and knockout mouse models.
Non-melanoma skin cancer (NMSC) is the most common human malignancy with over 1 million new cases being reported annually in the immunocompetent population the United States. Research in the Oberyszyn laboratory in the Department of Pathology centers on using murine models to understand the role of the inflammatory response and determining the function of biological mediators including prostaglandins, cytokines and reactive oxygen intermediates during ultraviolet light B (UVB) mediated skin cancer development. Currently there are 3 main projects ongoing in the lab. Project 1: Epidemiological studies have shown that there are gender differences in the development of NMSC, with men being twice as likely to develop basal cell carcinoma and three times as likely to develop SCC as women. This disparity has been attributed to lifestyle choices, since males historically have had professions requiring more time out in the sun and are less likely to use sun protection. Studies from our laboratory have found that when exposed to equal amounts of UVB male mice exhibited less UVB-induced inflammation compared to female mice, but they displayed higher levels of epidermal oxidative DNA damage and lower baseline total antioxidant activity levels. The male mice also developed tumors earlier, in greater number and with a more advanced grade than female mice. We are currently working on identifying the underlying mechanistic differences in the response between male and female skin to UVB and on identifying differences between the genders in initiation, promotion and progression of UV-induced SCC. We have also found that male mice have higher levels of an immunosuppressive cell population called myeloid derived suppressor cell (MDSC), which may contribute to the higher cancer rates seen in male mice. We have identified a link between the higher levels of this cell type and lower levels of skin catalase levels. We are continuing to explore differences in male and female skin as well as their immune response following UVB exposure, to ultimately allow for the development of more appropriately targeted prevention and treatment strategies. Project 2: Americans live in a culture that glorifies youth. According to market researcher FIND/SVP, the anti-aging products market is expected to hit $56 billion by 2007. Studies in post-menopausal women have found that hormone replacement therapy is effective at reversing the dryness and wrinkling that affects aging skin. Based on these studies, there is increasing interest in the use of topical creams containing hormones such as estrogen to prevent or reverse some of the normal cutaneous aging processes in younger pre-menopausal women. While exposure to these creams may be beneficial cosmetically, the effect of applying estrogen to sun exposed sites for prolonged periods of time, on skin cancer development is not known. Our preliminary studies using female Skh-1 hairless mice found a significant increase in the number of tumors in mice treated topically with estrogen immediately following UVB exposure compared to mice treated with vehicle control. These data indicate that increased levels of estrogen in the skin combined with UV exposure may act to enhance initiation and promotion of UV-induced skin cancers. These findings also suggest that the use of lotions and creams containing estrogenic compounds on sun exposed sites by younger women may be contributing to the increase in the number of skin tumors being diagnosed in women under the age of 40. Our recently completed murine studies found that topical estrogen treatment of previously UV exposed skin increased tumor burden in a model of both pre and post-menopausal mice. Ongoing analysis will determine whether topical estrogen differentially enhances the progression of benign UVB-induced tumors to malignant SCC in pre-menopausal and/or post-menopausal mice. Project 3: The obesity rate in the United States is reaching epidemic proportions. Nearly one third of adults are now classified as obese. According to the National Cancer Institute, studies have linked obesity with increases in the development of endometrial, colon, kidney and esophageal cancers. Initial studies have reported a link between obesity and an increased UV-induced inflammatory response in the skin. However, to data there is no clear link between the increases in NMSC cases seen in younger patients with increases in obesity. We have begun to use our murine model to explore the contribution of selected fat depots to both UV induced inflammation and tumor development.
Inflammation and Angiogenesis
Research in Dr. Wilgus’ laboratory focuses on skin biology and cutaneous pathology. Several ongoing projects aim at determining how inflammation and angiogenesis contribute to skin cancer development, wound healing and fibrosis.
One project, funded by NIH-NCI, will explore the role of vascular endothelial growth factor-1 (VEGFR-1) in the development and growth of skin cancer. Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis, or new blood vessel growth, which is an important step in skin carcinogenesis. VEGF induces angiogenesis by signaling through its receptors (VEGFR-1 and VEGFR-2) on endothelial cells. Until recently, expression of the VEGF receptors was thought to be restricted to endothelial cells; however, there is mounting evidence that many non-endothelial cell types express one or more VEGF receptor enabling them to respond to VEGF. They have found that VEGFR-1 is expressed by keratinocytes, the epithelial cells of the skin. Studies will be performed in conditional VEGFR-1 knockout mice, which have VEGFR-1 specifically deleted in keratinocytes, to determine whether VEGF can directly influence keratinocytes through VEGFR-1, and whether this interaction stimulates skin carcinogenesis.
The Wilgus laboratory also has several ongoing projects in the area of fetal wound healing. Early in development (within the first and second trimesters of development) fetal skin can regenerate and heal wounds without a scar. However, the fetus does not retain the ability to heal wounds without a scar. In the third trimester, fetal skin heals in a similar pattern to adult skin, loses the ability to regenerate and begins to heal with a scar. Currently, they are studying the role of mast cells, fibroblasts and various inflammatory mediators in the transition from scarless to fibrotic healing using a mouse model of fetal wound healing. The overall goal of these studies is to identify putative anti-scarring molecules and to determine mechanisms of skin regeneration.
Neuro-immune Interaction in Cancer Progression and Metastasis
Endogenous molecules play important roles in the growth and metastasis of cancer. Dr. Basu’s research has demonstrated that the endogenous tissue dopamine is lost in gastrointestinal (GI) tract cancers. Because endogenous GI tract dopamine regulates several important physiological functions of the GI tract, his interest is to elucidate the role of dopamine in GI tract cancers.
Angiogenesis and vasculogenesis (bone marrow-derived progenitor cell-mediated) are essential for several physiological and pathological processes. Dr. Basu’s interest is to identify and explore the role of regulators (such as the neurotransmitter dopamine) of angiogenesis and vasculogenesis and to develop anti-angiogenic therapy utilizing small endogenous molecules.
Neuro-immune interaction is now a well established phenomenon. Another interest of their research is to investigate the role of neuro-immune interactions in different physiological and pathological conditions.
Cell Cycle and Cancer
Dr. Zou’s research focuses on dissecting the novel function of cell cycle factors Cdks and Cdc25A in cell development and tumorigenesis. Dr. Zou’s laboratory has recently shown that phosphatase Cdc25A plays an important role in breast cancer metastasis. They further analyzed the mechanisms and showed that Cdc25A modulates metalloportease (MMP1) through Foxo1 and thereby regulates breast cancer metastasis (Mol Cell Biol, 2011). Indole-3-Carbinol, which is derived from broccoli, has been shown to inhibit growth of various tumors. His laboratory has recently shown that indole-3-Carbinol may inhibit growth and metastasis of breast cancer through a novel mechanism by inducing Cdc25A degradation (Cancer Pre Research, 2010). His group has also revealed that Cdk1 is required for the self-renewal of mouse embryonic stem cells (J Cell Biochem, 2011). Dr. Zou’s laboratory is further analyzing the novel role of Cdk4 in B-lymphomagenesis and tumor survival using knockout/transgenic mouse model systems. Furthermore, they are also characterizing the role of Cdc25A in breast cancer progression and metastasis and are developing indole-3-carbinol as a novel therapeutic strategy against breast cancer.
Novel Strategies for the Treatment of Brain Tumors
Dr. Barth’s laboratory is undertaking several projects for developing novel treatments for brain tumors. Project 1. Chemoradiotherapy of brain tumors using carboplatin or cisplatin containing nanovehicles in combination with photon irradiation. They have evaluated the therapeutic efficacy of i.c. delivery of carboplatin in combination with radiation therapy for treatment of the F98 rat glioma. Their studies have demonstrated the superiority of i.c. chemoradiotherapy with carboplatin and X irradiation compared to mono¬therapy. Dr. Barth’s lab is further opti¬mizing the therapeutic regimen by administering carboplatin by osmotic pumps and escalating the radia¬tion doses. In addition, neurotoxicologic studies will be carried out in non-tumor bearing rats. Project 2. Carboranylporphyrins as boron delivery agents for neutron capture therapy of brain tumors. This project and the following one both focus on boron neutron capture therapy (BNCT) as a way to treat brain tumors. BNCT is based on the nuclear capture and fission reactions that occur when non-radioactive 10B is irradiated with low energy thermal neutrons to produce α-particles (10B[n,α]7Li). Carboranyl¬porphyrins are a class of substituted porphyrins containing multiple car¬borane clusters. Three of these compounds, designated H2TBP, H2TCP, and H2DCP, have been evaluated in the present study. The goals were two-fold. First, to determine their biodistribution following intracerebral (i.c.) administration by short term (30 min) convection enhanced delivery (CED) or sustained delivery over 24 h by Alzetä osmotic pumps to F98 glioma bearing rats. Second, to determine the efficacy of H2TCP and H2TBP as boron delivery agents for BNCT in F98 glioma bearing rats. Their data is the first to show that carboranylporphyrins can be used as delivery agents for BNCT of an experimental brain tumor. Based on these results, they now are in the process of synthesizing and evaluating carboranylporphyrins that could have enhanced cellular uptake and improved therapeutic efficacy. Project 3. Boronated Unnatural Amino Acids as New Boron Carriers for BNCT. The development of new and more efficient boron delivery agents to tumor cells is an area of intense research in boron neutron capture therapy (BNCT) of cancer. This study focuses on the evaluation of boron delivery characteristics of cis and trans isomers of a boronated unnatural amino acid, 1-amino-3-boronocyclopentanecarboxylic acid (ABCPC), which were synthesized by our collaborator, Dr. George Kabalka, Department of Chemistry, University of Tennessee, Knoxville. Boron delivery characteristics of cis and trans isomers of ABCPC were tested in B16 mouse model for human melanoma and in B16 cells growing in cultures. Boron content of blood, tumor, liver, and kidney tissue samples was determined using inductively coupled plasma-optical emission spec¬troscopy (ICP-OES). Secondary ion mass spectrometry (SIMS) was carried out by our collaborator, Dr. Subhash Chandra, Department of Biomedical Engineering, Cornell University, Ithaca, New York. SIMS was used for imaging the distribution of boron at 500 nm spatial resolution of cis and trans ABCPC in B16 cells grown in vitro. Both enantiomers of ABCPC delivered comparable amounts of boron to B16 melanoma tumor cells, and these were similar to values obtained with BPA. SIMS imaging data revealed the presence of boron throughout the tumor with heterogeneous distribution for all compounds. The single cell SIMS imaging studies of B16 cells in cultures revealed that both cis and trans isomers of ABCPC delivered boron to the cytoplasm as well to the nucleus with the cell interior to the nutrient medium partitioning of approximately 4/1 at 2.5 hr. exposure. The water solubility of ABCPC compounds is an important advantage for BNCT. These encouraging observa¬tions provide support for continued studies of boronated unnatural amino acids as new and efficient boron carriers for BNCT. Project 4. In Vivo Studies on the Thrombopoietic Activity of Irgenal 3, 20 Dibenzoate (IDB), a Novel PKC Agonist. These studies were carried out in collaboration with Dr. Fred Racke, Division of Hemotopathology. Dr. Racke has carried out extensive in vitro studies with IDB. In vitro, IDB is a potent stimulator of megakaryopoiesis and promoted megakaryocyte production at the expense of erythroblast produc¬tion in the presence of erythropoietin. The purpose of the studies carried out in Dr. Barth’s laboratory was to determine if IDB could stimulate megakaryopoiesis in mice that had been exposed to varying doses, ranging from 2 to 10 Gy, of whole body X-irradiation using a Siemens linear accelerator (LINAC) as the radiation source. Mice were treated with varying doses of IDB, administered at varying times prior to or following X-irradiation. A single intraperitoneal (i.p.) injection of IDB mitigated radiation-induced thrombocytopenia, even when administered at 24 hours after X-irradiation. This suggests that IDB may have use as a mitigator of radiation induced thrombo¬cytopenia as a con¬sequence of either radiation therapy or a terrorist-perpetrated radiation attack.
Regulation of EGF Receptor Signaling by Cbl
Research in the Lill laboratory focuses on negative regulation of receptor-mediated signaling. One member of the ErbB receptor family, the epidermal growth factor receptor (EGFR), is negatively regulated by its recruitment of the signaling protein Cbl. Cbl associates with activated EGFR, undergoes tyrosine phosphorylation, and colocalizes with EGFR in tubulovesicular structures of the endocytic pathway. They and others have shown that overexpression of Cbl enhances EGFR downregulation from the cell surface, recruitment into the endocytic trafficking pathway, and degradation. Although Cbl functions as a component of the ubiquitylation machinery that targets proteins for proteasome-mediated degradation, the bulk of EGFR degradation occurs in the lysosome. Studies in their laboratory are aimed at defining the mechanism for Cbl-mediated enhancement of EGFR lysosomal degradation.
Ongoing work addresses the following questions: (1) which Cbl structural domains or motifs are critical for enhanced EGFR downregulation and degradation?; (2) can these domains be used as targeting motifs to degrade other oncogenic receptor tyrosine kinases?; and (3) which protein-protein interactions are required to route activated EGFR to the lysosome, and how are they affected by Cbl overexpression, mutation, or knock-down? The laboratory utilizes a variety of genetic, biochemical, and cell biological techniques to address these questions. Exciting ongoing work involves live cell imaging of endocytic fusion events.
Role of DNA methylation and microRNAs in liver disease
Dr. Ghoshal’s laboratory is involved with two major research projects 1) MicroRNA-122 is a master regulator of liver homeostasis and 2) Liposomal nanoparticles (LNPs) mediated delivery of microRNA-122 mimic inhibits growth of hepatocellular carcinoma in xenograft model.
Project 1): MicroRNA-122, is the most abundant liver specific microRNA, positively regulates cholesterol metabolism and promotes hepatitis C virus replication in the liver. miR-122 expression is dramatically suppressed in primary hepatocellular carcinoma (HCC) of both rodent and human origin and its down regulation correlates with poor prognosis and metastasis. Furthermore, both in vitro and xenograft studies demonstrated that miR-122 functions as a tumor suppressor by targeting several oncogenic targets. To understand its biological function, we have generated miR-122 conditional knockout (floxed) mice using Cre-lox system and crossed these mice to Alb-Cre and E2-Cre mice to generate both liver specific knockout (LKO) and germ-line knockout (KO) mice respectively, and monitored their phenotype. These mice were born normal and fertile but exhibited lower serum cholesterol, glucose and higher alkaline phosphatase level in early adult life, steatohepatitis by 6 month of age and spontaneous hepatocellular carcinoma by 12 month. Incorporation of 3H1-glycerol in the hepatic triglyceride (TG) in vivo showed increase in de novo TG synthesis in LKO mice. In contrast, TG secretion measured by monitoring serum triglyceride levels after injecting lipoprotein lipase inhibitor was significantly reduced in LKO mice. Thus, increase in hepatic steatosis in LKO mice probably caused due to increased TG synthesis and reduced TG secretion. Flow analysis showed that hepatic inflammation in knockout mice is likely due to increase in granulocyte neutrophil population in the livers. Ingenuity Pathway Analysis of the microarray data revealed that genes regulating lipid metabolism, such as TG synthesis and storage, are significantly upregulated whereas those involved in cholesterol metabolism were downregulated in the livers of the knockout mice. In addition, a large number of genes involved in cell survival, proliferation and migration are also significantly altered in the livers of knockout mice. Sylamer analysis identified many of the upregulated genes harbor miR-122 seed sequence in their 3’-UTRs. Upregulation of several oncogenes probably plays a causal role in the development of spontaneous HCC in knockout mice, whereas dramatic induction of fetal genes e.g. a-fetoprotein (Afp), the imprinted genes (Igf2 and H19), EpCAM explain their hepatoblast-like phenotype. Interestingly, HCC incidence in male LKO mice was significantly higher than in female mice, which correlated with fold elevated serum IL-6. Microarray analysis revealed that the genes involved in inflammation, cholestasis, steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma were dysregulated in the tumors. Taken together, these data suggested that miR-122 plays critical role in liver function, loss of which predisposes mice to spontaneous HCC. This project is supported by a RO1 grant from NIDDK.
Project 2): Hepatocellular carcinoma (HCC) is the third-leading cause of death from cancer and the fifth most common malignancy worldwide. In the United States, there has been a striking increase in the incidence and mortality from hepatocellular carcinoma in recent years because of late stage of detection when most of the conventional chemotherapeutic agents are ineffective. Alternative approaches are therefore warranted. Therefore, We have focused on developing microRNA based therapeutics for HCC. Recently, our collaborators Drs James Lee and Robert Lee’s group at OSU synthesized several novel LNPs for delivery of miR mimics, anti-miRs or si-RNA in mice. Using miR-122 mimic loaded in a specific LNP formulation we treated nude mice that developed tumors after injecting human HCC (SK-Hep-1) cells. Tumor growth was significantly reduced in mice treated with miR-122-LNPs compared to those treated with scrambled RNA-LNPs. These results indicate that miR-122-LNPs alone or in combination with anti-cancer drugs may be an effective therapeutic regimen in human HCC patients. We are planning to extend these studies in primate models of liver cancer. This project is funded by a R21 grant from NCI.