Roseman University of Health Sciences
From left: Ronald R. Fiscus, PhD, VP of Research and Professor of Pharmaceutical Sciences, Ben Costantino, PhD, Postdoctoral Fellow, Mary Johlfs, MS, Director of Research Operations, Priyatham Gorjala, PhD, Postdoctoral Fellow, Aurelio Lorico, MD, PhD, Co-Director, Cancer Research Center, Germana Rappa, MD, PhD, Co-Director, Cancer Research Center, Olivia Chao, PhD, Postdoctoral Fellow, Kasey Zanolli, MHA, Administrative Grants Officer, Fabio Anzanello, Research Technician, Toni Green, PhD, Postdoctoral Fellow
Cancer Research Projects
Role of cell-cell fusion in breast cancer progression (Rappa and Lorico Labs)
More than a million new breast cancer cases are diagnosed every year worldwide. More effective, targeted therapies are needed to ameliorate the prognosis of patients with advanced and metastatic breast cancer. We have recently published seminal work on breast cancer stem cells and on the formation of hybrids between mesenchymal stem cells and breast cancer cells as cause of breast cancer metastases (Rappa G, and Lorico A. Phenotypic characterization of mammosphere-forming cells from the human MA-11 breast carcinoma cell line. Exp Cell Res. 2010; Rappa G., and Lorico, A. Phenotypic Heterogeneity of Breast Cancer Stem Cells J. of Oncology 2011; Rappa, G., Mercapide, J., and Lorico, A. Spontaneous formation of tumor-mesenchymal stem cell hybrids results in breast cancer heterogeneity and progression. Am. J. Pathol., 2012). To address the key issue of whether cell fusion plays a significant role in tumor progression, we propose to (i) Establish the frequency of fusion in vivo in an appropriate animal model; (ii) Demonstrate the clinical occurrence of heterotypic cell fusion in secondary breast cancers of patients which have previously received an allogenic bone marrow transplantation; (iii) Discover inhibitors of breast cancer cell fusion and evaluate their anti-cancer and anti-metastatic activity. In addition to advancing our knowledge on the mechanism of breast cancer progression and formation of metastasis, the outcomes from the proposed research plan will dramatically change the way that research is conducted on these issues: in particular, novel therapeutic strategies and novel compounds targeting fusion hybrids and/or capable of preventing dangerous fusion events will be developed, and strategies to distinguish which patients carry an advanced form of breast carcinoma as a result of heterotypic fusion will be devised to select the appropriate therapeutic treatment.
Spontaneous formation by breast carcinoma cells of heterotypic (with MSC) and homotypic proliferating hybrids. (FromSPONTANEOUS FORMATION OF TUMORIGENIC HYBRIDS BETWEEN BREAST CANCER AND MULTIPOTENT STROMAL CELLS IS A SOURCE OF TUMOR HETEROGENEITY. Rappa G., Mercapide J. and Lorico A. – American Journal of Pathology 2012)
A: Fluorescent micrographs of a co-culture of MDA-DsRed (red) and human MSC-GFP (green) that, within 24 hr, and in the absence of selective pressure, results in formation of hybrids (yellow). B: MSC-GFP/MA-11-DsRed hybrid (green + red), as visualized by Z-stack confocal microscopy. C: Picture of a co-culture of MSC-GFP (green) and MDA-DsRed (red), showing proliferating hybrid cells (arrows) that display the fibroblastic shape of MSC. D: Left: Spontaneous hybridization between MA-11-GFP (green) and MDA-DsRed (red) cells results in yellow cells in the area where the colonies of both cell lines establish contact. Right: MA-11/MDA hybrid cells isolated by cloning cylinders. E: Colony composed mainly by proliferating MA-11-GFP/MA-11-DsRed hybrids (yellow) after hybrid isolation by cloning cylinders from a coculture of MA-11 cells expressing either GFP or DsRed. Scale bars: 50 mm in A and D, 25 mm in B, 100 mm in C and E.
Mechanisms of metastasis in malignant melanoma (Rappa Lab)
Development of metastasis is by far the main cause of death for patients with melanoma, a disease largely refractory to all currently available systemic therapies. Unfortunately, the great majority of drugs currently in use for metastatic melanoma have been discovered and validated for their potential to inhibit local melanoma growth in mouse models. In 2008, we published in Stem Cells a seminal paper, showing for the first time that CD133 (Prominin-1), the main cancer stem cell marker identified to date, contributes to melanoma cells pro-metastatic properties and is therefore an excellent target to prevent melanoma dissemination. We have recently found that CD133 interacts with the Wnt pathway and that CD133 is a component of microvesicles that are released by melanoma cells in the extra-cellular space and signal stem cell expansion to the surrounding tissues (A. Lorico, J. Mercapide and G. Rappa, Prominin-1 (CD133) and Metastatic Melanoma: Current Knowledge and Therapeutic Perspectives in Prominin-1 (CD133): Advances in Experimental Medicine and Biology; G. Rappa, J. Mercapide, F. Anzanello, T. Le, M. G. Johlfs, R. R. Fiscus, M. Wilsch-Bräuninger, D. Corbeil and A. Lorico. Wnt Interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells, Exp. Cell Res., 2013):
We intend to expand these studies, and to develop anti-melanoma agents that target CD133, its downstream effectors, and/or the release of CD133-containing membrane vesicles. In a subsequent phase, the new therapeutic approaches discovered will be tested in pre-clinical models. Due to the expression of CD133 in many types of cancer, our findings will also have a potential impact on the treatment of different types of malignancies, and at the same time will improve our understanding of both cancer and stem cell biology.
Study of cancer exosomes (Lorico Lab)
The field of microvesicles, and in particular of exosomes, is rapidly expanding, with accumulating evidence of their function as mediators of intercellular communication, due to their capacity to merge with and transfer a cargo of bioactive molecules to recipient cells. We have now for the first time observed release of exosomes expressing CD133 in the extracellular medium of cancer cells, specifically of FEMX-I melanoma. We intend to elucidate the significance of prominin-1-expressing exosomes in cancer, and in particular their composition and function.
While compelling evidence supports the significance of microvesicle release in a broad range of physiological and pathological processes, their classification, protocols of their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. We have recently characterized the proteome, lipidome and the microRNA composition of CD133 exosomes (Rappa et al., Molecular Cancer, 2013). Our hypothesis is that cancer cell-derived exosomes are critical for the malignant properties of cancer cells and that their composition is different from exosomes derived from somatic stem cells. The final aim is to identify cancer-specific exosomal targets and develop specific anti-cancer strategies.
Neutral endopeptidase (NEP/CD10) and CYP3A5 in prostate (Goodman Lab)
NEP is a transmembrane cell surface peptidase normally expressed on epithelial, endothelial and stromal cells of numerous tissues. It catalytically inactivates a variety of small peptide substrates; loss of NEP expression has been implicated in a number of cancers, such as prostate cancer, lung cancer and pancreatic cancer. In prostate cancer the loss of NEP is perpetuated by androgen deprivation therapy, the mainstay therapy for advanced or recurrent prostate cancer. The lab is focused on developing therapeutic strategies aimed at restoring NEP expression or targeting its loss. As NEP is a angiogenesis inhibitor the lab is also focused on understanding the interaction between NEP and PTEN a AKT inhibitor in endothelial cells.
Detection and culturing of circulating tumor cells to identify and develop biomarkers for better prognosis (Goodman Lab)
Circulating tumor cells (CTC) are an important indicator of metastasis and associated with a poor prognosis. Detection sensitivity and specificity of CTC in the peripheral blood of metastatic cancer patient remains a technical challenge. In collaboration with Dr. Thuc T. Le we use Coherent anti-Stokes Raman scattering (CARS) microscopy to examine the lipid content of CTC. Intracellular lipid could serve as a biomarker for prostate CTC which could be sensitively detected with CARS microscopy in a label-free manner. Strong affinity for lipid by metastatic prostate cancer cells could be used to improve detection sensitivity and therapeutic targeting of prostate CTC.
Comparison of lipid uptake between cancer cells (LNCaP) and peripheral blood mononuclear cells (PBMC). A. Bodipy lipid probe has been used to differentiate lipid intake and storage between cancer cells and PBMCs. B.CARS microscopy showing increased lipid intake by LNCaP cells as compared to PBMCs. CK-cytokeratin.
CYP3A5 as modulator of androgen receptor signaling- Influence of polypharmacy on the therapeutic efficacy of androgen deprivation therapy (Goodman Lab)
Androgen-targeted therapy plays a central role in the treatment of advanced metastatic prostate cancer. Prostate cancer is generally a disease of the elderly, polypharmacy is a major concern in the context of treatment. Our data indicatethat CYP3A5, which metabolizes 30% of the commonly used drugs via the hepatic route, modifies Androgen receptor (AR) expression in prostate cancer cell lines. As many of the commonly used drugs are substrates, inducers or inhibitors of CYP3A5, these drugs have the capacity to modulate AR signaling and therefore impact, either positively or adversely, therapeutic efficacy of AR-directed therapies. Understanding the regulation of downstream signaling by CYP3A will facilitate the selection of the appropriate concomitant medications for men with advanced prostate cancer.
Targeting DNA repair to enhance potency of chemotherapeutics in prostate cancer treatment (Goodman Lab)
Defects in DNA repair mechanism lead to genomic instability and transformation to malignancy. Yet, for cancer cells to maintain replication and prevent cell death, they in turn become critically reliant on alternative DNA repair processes. This reliance on alternate DNA repair pathways can be therapeutically exploited based on the concept of synthetic lethality, where the simultaneous perturbation of two genes is toxic to the cells. Our lab is currently working on identifying synthetically lethal relationship in the deficient DNA repair of prostate cancer cells and to develop strategies to target these pathways through rationale combination of inhibitors and DNA damaging agents.
γ-H2AX foci staining indicative of double-stranded DNA breaks (DSBs) in DU145 prostate cancer cells treated with DNA-damaging agent, etoposide.
Therapeutic targeting of cancer stem cells (CSCs), “the seeds of cancer development and recurrence”, in lung cancer, mesothelioma and ovarian cancer (Fiscus Lab)
A special type of adult stem cells, called cancer stem cells or CSCs, have recently been identified in both blood cancers and solid tumor cancers. The CSCs, which make up only a small percentage (e.g. 0.1% - 10%) of the total cell population in tumors, have characteristics of stem cells (e.g. growing slowly, and thus resistant to most anti-cancer therapies) but are capable of differentiating into rapidly-growing tumor cells. The Fiscus Lab, in collaboration with Dr. Maria Wong’s Lab at the University of Hong Kong, has found the presence of CSCs in the tumors of lung cancer patients and in many commonly-used lung cancer cell lines and has further identified useful biomarkers for lung cancer CSCs that can be used as potential diagnostic and therapeutic tools for identifying and targeting lung cancer stem cells (Leung et al., 2010a).
Almost all of the current therapies for treating the multiple types of cancers work by targeting the rapidly-growing tumor cells, leaving the CSCs (the seeds) untouched and thus able to re-constitute the original tumor. This often results in a recurrence of the original cancer - a common problem with most cancers. One therapeutic agent that specifically targets CSCs is metformin, a pharmaceutical agent commonly used for treating type-2 diabetes. The Fiscus lab is studying the molecular mechanisms of metformin, including its ability to target CSCs, with the goal of developing newer and better agents that target and selectively eliminate the CSCs in the multiple forms of cancer. The new findings from these studies will be used to develop novel therapies that specifically target the CSCs, thus effectively stopping the recurrence of the cancers.
Identifying CREB and Inhibitor of Apoptosis Proteins (IAPs), e.g. cIAP-1, cIAP-2, Livin and Survivin, as key mediators of the exaggerated proliferation and chemoresistance in blood cancers (leukemia and lymphoma), lung cancer, mesothelioma and ovarian cancer (Fiscus Lab):
The Fiscus Lab at Roseman University is a leading lab in identifying new, previously unrecognized proteins and molecular mechanisms that mediate cell survival (e.g. PKG-Ia, Livin, Survivin) in both normal cells (Fiscus, 2002; Fiscus et al., 2002; Wong and Fiscus, 2010; Wong and Fiscus, 2011; Fiscus and Johlfs, 2012) and cancerous cells (Johlfs and Fiscus, 2010; Leung et al., 2010a,b; Wong et al, 2012; Fiscus et al., 2012; Wong and Fiscus, 2013).
Recent studies by Janica Wong, a Postdoctoral Research Associate in the Fiscus Lab, have identified the importance of the NO/cGMP/PKG-Ia signaling pathways in regulating DNA synthesis/cell proliferation, cell migration and resistance to apoptosis (promoting cell survival) in both normal non-cancerous cells [e.g. OP9 bone marrow-dervied mesenchymal (stromal) stem cells, vascular endothelial cells/smooth muscle cells and pancreatic islet cells] and cancer cells [e.g. ovarian cancer and non-small cell lung cancer (NSCLC) cells]. Janica has discovered that the kinase (catalytic) activity of PKG-Ia has a key role in maintaining high levels of expression of the anti-apoptotic (pro-cell-survival) proteins cIAP-1, Livin, Mcl-1 and Survivin, via its ability to phosphorylate the transcription factor CREB (shown in cellular model). The cellular model also illustrates our recent findings that PKG-Ia directly phosphorylates the oncogenic protein c-Src, enhancing its tyrosine kinase and oncogenic activity (Fiscus et al., 2012; Fiscus and Johlfs, 2012). Via this mechanism, the NO/ cGMP/PKG-Ia signaling pathway plays a central role in promoting c-Src activation and the exaggerated DNA synthesis/cell proliferation of cancer cells, as well as the resistance to chemotherapeutic agents like cisplatin (i.e. chemoresistance). The exaggerated proliferation and chemoresistance can result in a relapse of tumor growth following chemotherapy. Also illustrated above in our cellular model is the key role of the NO/cGMP/PKG-Ia signaling pathway in vascular endothelial cells, promoting angiogenesis (new blood vessel growth) that is essential for the growth of the tumor.
Cellular model illustrating our data identifying the role of nitric oxide (NO) and downstream activation of PKG-Ia in cancer cells of epithelial origin (including breast, lung, ovarian and prostate cancers as well as mesothel-ioma). The NO / cGMP / PKG-Ia signaling pathway plays a central role in promoting c-Src activation & the exaggerated DNA synthesis / cell prolifer-ation of cancer cells as well as resistance to chemotherapeutic agents like cisplatin (chemoresistance) in certain resistant populations of cancer cells. The exaggerated proliferation & chemoresistance can result in a relapse of tumor growth following chemotherapy. Also illustrated above is the key role of the NO / cGMP / PKG-Ia signaling pathway in vascular endothelial cells, promoting angiogenesis (new blood vessel growth).
We are using state-of-the-art microscopy, including TIRF (total internal reflection fluorescence) microscopy coupled with high-speed confocal microscopy (shown above), to identify subcellular localization of key cancer-associated proteins (e.g. the IAPs cIAP-1, cIAP-2, Livin, Survivin and XIAP, and the Bcl-2 family members, especially Mcl-1) in select types of cancers, blood cancers (leukemia and lymphoma), lung cancer, mesothelioma and ovarian cancer. Our goal is to develop new anti-cancer therapies that target specific subcellular fractions of these proteins in order to minimize the toxic side effects often associated with cancer treatments.
Molecular mechanisms mediating the anti-cancer/anti-tumor-angiogenesis actions of resveratrol, Geum japonicum (rose family) extracts and other natural products in foods, drinks and nutraceuticals (Fiscus Lab).
Earlier studies from the Fiscus Lab had shown that extracts and purified tannins from the rose family member Geum japonicum have anti-hypertensive effects (and thus therapeutic potential) via increased production of nitric oxide (NO) (Xie et al., 2007). Similar tannins from related plants, as well as resveratrol (a polyphenol from grapes, red wine, peanuts and berries) have been found to have anti-cancer effects. The Fiscus Lab at Roseman University is studying the molecular mechanisms of these natural products, particularly their direct anti-cancer effects on cancer cells and the indirect anti-cancer effects via inhibition of tumor angiogenesis. We have found that resveratrol directly inhibits the proliferation of cancer cells, which is associated with a dramatic down-regulation of PKG-I gene expression (needed for proliferation in cancer cells). Our studies have also shown that resveratrol decreases multiple components of angiogenesis, including endothelial cell proliferation and tube formation, via its ability to suppress the kinase activity of PKG-I. This resveratrol-induced suppression of PKG-I activity in vascular endothelial cells results in a dramatic decrease in the expression of survival proteins, such as cIAP-1, cIAP-2, Livin and XIAP (Wong and Fiscus, 2013, manuscript submitted for publication). We will be conducting similar experiments with the tannins of Geum japonicum and other natural products from foods, drinks and nutraceuticals to find better ways of preventing cancer.