Growth Regulation of Cancer Program

Overview | Leaders | Members | Research | Publications

 

Research Portfolio

Recent accomplishments of the Growth Regulation of Cancer Program include:

  • Retinoic acid-mediated trans-differentiation of breast cancer cells involves Sox-9, ER81, and VE-cadherin. (Endo et al., PLoS ONE, 2008)
  • Alterations in the TGF-β signaling pathway and disordered regulation by ELF play a powerful role in the transition from a stem cell to a normal differentiated cell such as a gut epithelial cell. (Kim et al., Biochem Biophys Res Commun, 2006; Saha et al., Oncogene, 2006; Varalakshmi et al., Oncogene, 2005)
  • Nitazoxanide, tizoxanide, and other thiazolides are potent inhibitors of hepatitis B virus and hepatitis C virus replication. (Korba et al., Antiviral Res, 2008)

View a full list of accomplishments from 2002 to 2008.

Additionally, there are many ongoing studies:

 

BRCA1 related cancer pathways.
Investigators: Furth, Rosen

One of the GRC Program's productive areas of collaborative publications is in hormonal and growth factor signaling relating to interactions between BRCA1 and hormonal signaling. Investigations in mouse models led by Drs. Furth and Rosen showed that Brca1 loss in mammary epithelial cells results in abnormal growth responses to estrogen, progesterone and tamoxifen which lead to the development of preneoplasia and cancer. Activation of estrogen signaling pathways collaborates with loss of BRCA1 to promote development of ERα negative and ERα positive mammary preneoplasia and cancer (Jones et al., Oncogene, 2008; Ma et al., Mol Endocrinol, 2006; Jones et al., Oncogene, 2005).

Recently, Bae and co-workers found a new function for BRCA1. It is generally agreed that BRCA1 defects cause several abnormalities, each of which can significantly contribute to the genomic instability associated with breast cancer development. BRCA1 was previously known to contribute to cancer development due to affecting alterations in DNA damage repair, specifically, double-strand break repair. The Bae data suggest that BRCA1 protein levels also regulate an even earlier step - the formation of certain kinds of DNA damage that can eventually be converted into point mutations through an effect on the expression of genes encoding proteins that can detoxify genotoxic chemicals. This observation provides a mechanistic explanation for BRCA1 regulation of DNA damage formation (in addition to the already well documented effects of BRCA1 on DNA damage repair). As part of these investigations, a collaboration between Bae and Rosen  showed that BRCA1 modulates xenobiotic stress-inducible gene expression by interacting with aryl hydrocarbon nuclear translocator (ARNT) in human breast cancer cells (Hyo et al., J Biol Chem, 2006).

Top

P75^NTR
Investigators: Djakiew, Brown, Byers, Posey

GRC Member Dr. Djakiew identified the p75^NTR tumor suppressor, a receptor for nerve growth factor, as a druggable target for inhibition of prostate cancer growth (Allen et al., Exp Cell Res, 2005) and this work now includes Dr. Brown, Byers, and Dr. Posey of the Lombardi/University of the District of Columbia U56 partnership. p75^NTR is a member of the tumor necrosis factor receptor super-family of membrane receptors which shares an intracellular sequence of amino acids called the death domain that can induce apoptosis. Normal prostate epithelial cells lose expression of p75^NTR with progression to cancer. However, the gene in these cells remains intact, so that the possibility remains for re-expression of the protein. The Djakiew team is one of the few research laboratories to investigate the role of the p75^NTR tumor suppressor in prostate cancer. This work has led to the discovery of small molecules with potential efficacy in prostate cancer through the induction of p75^NTR. The aryl propionic acid class of non-steroidal anti-inflammatory drugs exhibits differential efficacy for induction of the p75^NTR in prostate cancer cells independent of cyclo-oxygenase inhibition. They also induce p75^NTR mRNA stability, leading to p75^NTR dependent apoptosis of tumor cells. The mechanism of action for this enhanced mRNA stability occurs via aryl propionic acid mediated phosphorylation of the p38 MAPK. Additional classes of compounds are being investigated that can stimulate p75^NTR expression levels to identify more potent compounds. These studies are continuing in collaboration with the laboratory of Milton Brown. In collaboration with Byers, the Djakiew lab showed that p75 NTR mediates a bifurcated signal transduction cascade through the NF kappa B and JNK pathways to inhibit cell survival.

Top

STAT3
Investigators: Banerjee, Albanese

Dr. Banerjee has linked changes in STAT3 signaling to phytoestrogen regulation of prostate cancer growth (Chau et al., Carcinogenesis, 2007). This work was extended as a collaboration with Dr. Albanese. Numerous epidemiological studies have suggested that phytoestrogens in soybeans can reduce the risk for acquisition of hormone-dependent cancers. The Banerjee lab and others demonstrated that incorporation of nM to µM concentrations of dietary genistein can prevent the progression of poorly-differentiated prostate cancer in an animal model if the treatment starts before the development of prostate tumor. However, if treatment starts after the formation of tumor, flow level genistein exposure enhances growth and metastasis of prostate cancer in vivo; this is associated with the activation and/or up-regulation of STAT3, Akt, osteopontin (OPN), and TERT/telomerase activity. Based on these results, the Banerjee lab hypothesizes that genistein has a biphasic effect on prostate cancer cells. This concept represents a paradigm shift in phytoestrogen research in prostate cancer because most published reports have proposed beneficial effects of pharmacologic doses of genistein. With Dr. Albanese, Dr. Banerjee is determining if genistein treatment shows a similar biphasic effect on prostate growth and metastases in PTEN-KO and PTEN-KO-erbB2 mice.

Top

TGF-beta signaling and development of GI malignancies
Investigators: Mishra, Byers, Wellstein, Schlegel, He, Marshall, Pishvaian, Brown, Creswell

This Aim mostly focuses around, but not entirely, the program project grant (PI: Mishra), entitled "Cellular and Molecular Mechanisms of Gastrointestinal Cancer" and investigates the role of altered TGFbeta signaling in gastrointestinal cancer development. The overall hypothesis of the P01 is that disruption of the TGF-beta tumor suppressor pathway (through ELF, Smad and Smad4) leads to a proliferative potential in cells that then acquire secondary events such as activation of pathways that include PRAJA and others, resulting in gastrointestinal cancers. This P01 proposes to 1) use animal models that have predetermined mutations in specific pathways that include wnt, TGF-beta, myc, telomerase (TERT) and CDK4 to determine their role in Beckwith-Wiedemann Syndrome (BWS) and foregut cancer formation; and 2) develop markers and therapeutics targeted to gastrointestinal (GI) cancers.

One of the remarkable features of liver and gastrointestinal stem cells is that despite their large number and rapid rate of cell division, these cells rarely acquire the age-related genetic defects associated with cancer induction or show deterioration in functional competence. This observation suggests that intestinal stem cells have evolved protective mechanisms against genetic damage. Among them is the ability of stem cells to selectively sort the old (parental) and new DNA strands when they divide, retaining only parental DNA strands. This phenomenon is called asymmetric division, and ensures that replication-induced errors are excluded from stem cells. Random errors introduced into parental strands (e.g., after exposure to genotoxic agents) trigger a second protective response with a robust p53-dependent stem cell apoptosis and G1 arrest induced by TGF-b pathways. Recent studies by the Mishra lab and others have shown that there is a powerful role for the TGF-β signaling pathway in the transition from a stem cell to a normal differentiated GI epithelial cell. Conceivably, alteration of either mechanism by stem cell/cell cycle activators such as b-catenin, PRAJA, CDK4, h-TERT or, c-MYC could expedite tumorigenesis and conversely tumorigenesis can be prevented by targeting these activators. It has been shown that CDK4, PRAJA, b-catenin, h-TERT and c-Myc represent key proteins that are dramatically increased in human and mouse foregut cancers when TGF-β signaling is inactivated (Gallicano, Schlegel, Mishra, Kallakury, Pishvaian, Albanese). Also, disruption of TGF-beta signaling through beta-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation (Kitisin et al., Oncogene, 2007). The adaptor protein ELF is emerging as a molecule that plays a role in gut stem cell homeostasis and is a potent regulator of GI tumorigenesis through its ability to affect the activity of TGF-β mediated tumor suppression- specifically Smad3 and Smad4 signaling. ELF and Smad3 are CDK4 and PRAJA substrates, and are also activated in GI progenitor cells. The hypothesis is that the inactivation of this pathway by CDK4, PRAJA, b-catenin, h-TERT and c-Myc leads to uncontrolled activation of oncogenic pathways that can be utilized as a therapeutic approach at these lethal cancers. In addition, proteins such as ELF, TBRII, Smad4, PRAJA, CDK4, c-Myc, nuclear b-catenin, h-TERT, as a group, could potentially be utilized as functional markers for the early detection of these cancers. ELF and Smad 4 expression is observed in intestinal crypt bases consistent with the hypothesis that these proteins contribute to normal gut stem cell homeostasis. In fact, mouse model studies demonstrate that when ELF function is lost or reduced, both developmental abnormalities and a spectrum of gastrointestinal cancers appear. Total deletion of ELF results in the generation of liver and gastrointestinal cancers; ELF haploinsufficiency in combination with Smad4 haploinsufficiency results in gastric cancers and colonic adenomas; and ELF haploinsufficiency (alone or in combination with Smad3 haploinsufficiency) results in visceromegaly as well as multiple GI cancers, including metastatic pancreatic, hepatocellular, bowel adenocarcinomas and other cancers in mouse models. This last mouse model provides a compelling phenocopy of human Beckwith-Wiedemann syndrome (BWS), a hereditary cancer syndrome. In humans, ELF expression is lost in gastric cancers as well as Duke B1 colon carcinoma, consistent with a role for ELF in suppression of early human gastrointestinal cancer.

The Mishra lab work focuses on the ELF/Smad3/Smad4 in vivo tumor models to analyze the suppressor role of TGF-β signaling in the initiation and progression of intestinal cancer. This basic science work in mouse models has led to a series of preclinical and translational experiments that are now supported by a P01 awarded to Dr. Mishra in 2008. First, Dr. Mishra discovered that CDK4 was highly activated in the tumors from elf/smad3/4 mutant mice. This finding led to collaboration with Dr. Prem Reddy (Fels Institute for Cancer Research, Temple University) to develop CDK4 specific inhibitors for the GI cancers that the elf/smad3/4 mutant mice develop. In related experiments Dr. Mishra found that b-catenin is markedly activated in the tumors from the elf/smad3/4 mutant mice. This led to a collaboration with Dr. Byers to develop Vitamin D analogues specifically for foregut cancers such as hepatocellular cancer (HCC) and an interprogrammatic collaboration with Dr. Wellstein to take a similar approach for pancreatic cancer. The translational work is being spear-headed by Drs. He and J. Marshall  for the CDK4 inhibitors, and Pishvaian  for the Vitamin D analogs. Finally, Dr. Mishra observed a marked activation of PRAJA, c-MYC and h-TERT in the murine cancer models. Significantly, h-TERT was also found to be increased in the human BW syndrome. Also, part of this P01, Dr. Schlegel is analyzing the role of the TGF-β pathway with respect to TERT suppression. In an interprogrammatic collaboration, Dr. Brown is developing new therapeutics targeted allosterically at the c-MYC and h-TERT sites, as well as the drug discovery efforts focused around the TGF-b pathway. In an intra- and interprogrammatic collaboration with Drs. Mishra  and Pishvaian, the Byers lab carried out a series of studies aimed at determining the role of TGFbeta and wnt signal transduction (Mishra et al., Oncogene, 2005).

Top

TGF-beta signaling, BRCA1 and development of breast cancer
Investigators: Furth, Shields

In a separate research project under Aim 2, Drs. Furth and Shields have an ongoing interprogrammatic study funded by the DOD Synergy Award Mechanism on the role of the TGFbeta pathway in BRCA1 mutation related breast cancer. While the Furth lab executes experiments using mouse models to study the role in pathophysiology, the Shields lab is investigating if loss of BRCA1 expression in primary human mammary epithelial cells modulates TGFbeta family member expression. In an earlier study, Bae and Avantaggiati with Rosen found that BRCA1 is required for orderly cell cycle progression (Bae et al., Cell Cycle, 2005).

Top

Expressing the papillomavirus L1 capsid protein in native conformation to spontaneously assemble into virus-like particles (VLPs)
Investigators: Schlegel

Utilizing a newly developed canine model for evaluating mucosal infection, the Schlegel laboratory demonstrated that these VLPs were strongly immunogenic and that they induced neutralizing antibodies that completely protected against viral challenge. It was clear that this approach could therefore be used to generate a similar vaccine for humans. Thus, this technology was first licensed to MedImmune and later sublicensed and cross-licensed to GlaxoSmithKline and Merck for the current HPV vaccines that are commercially available. In an effort to make more cost-effective and stable HPV vaccines, the laboratory (in collaboration with Bob Garcea at the University of Colorado) is supported by a RAPID Award from the NIH to develop a second-generation HPV vaccine. This capsomeric vaccine is produced as a fusion protein in bacteria, and is readily purified using an attached GST protein. Not only is this inexpensive to produce, the vaccine is highly stable and can be converted to a powder by precipitation or lyophilization, making it more accessible to populations around the world. This vaccine is currently being produced by Shanta Biotechnics in India where it will also be tested soon (within a year) in Phase 1 trials. The intent of this vaccine is to make it for approximately $2-5, such that its widespread use can be afforded by developing countries.

A third-generation HPV vaccine is also being developed by Schlegel with the assistance of a grant from the Bill and Melinda Gates Foundation. This vaccine utilizes the platform of the second-generation vaccine, but it also includes epitopes of the HPV E7 protein that is always expressed in HPV-induced tumors. Thus, this vaccine is intended primarily to be therapeutic, although we anticipate that it may be prophylactic at the same time. This project is an international collaboration (Bob Garcea from the University of Colorado; Lutz Gissmann from the German Cancer Research Center, DKFZ, in Heidelberg; and Luisa Villa at the Ludwig Cancer Center in Sao Paulo, Brazil). Currently the vaccine is in the early phases of production in Buenos Aires, Brazil, by the Biosidus biotechnology company and the expectation is that Phase 1 trials will commence in approximately 1 year. Again, this vaccine is being produced in bacteria and should be low enough in cost to be available to developing countries.

Top

Improving the efficacy of human papillomavirus vaccines
Investigators: Schlegel

In established papillomavirus infections, cellular immune responses, particularly by antigen-specific CTLs, are important defense mechanisms against progression of the disease. During the HPV life cycle, late functions including structural protein synthesis and virus assembly occur exclusively in outermost layers of the infected epithelium, which are less accessible to immune surveillance. In addition, in cervical carcinomas the high risk HPV genomes integrate into the cellular chromosome often silencing transcription of the structural protein genes. As a consequence, the cellular immune system has minimal exposure to viral structural proteins; therefore, vaccines based solely on papillomavirus structural proteins would not be expected to be effective against established infections. Nevertheless, proliferating cells transformed by the entire HPV16 genome can express sufficient levels of L1 to serve as targets for CTLs.

To improve the therapeutic potential of HPV vaccines, Dr. Schlegel's lab has focused on the E7 viral oncoprotein. E7 is a 10-14kDa phosphorylated protein expressed early in the viral life cycle. E7 of high risk subtypes binds retinoblastoma-susceptibility protein thereby modulating cell cycle control. Expression of E7 is required for the maintenance of the proliferative state of HPV-infected cells and E7 can be detected in all layers of the infected epithelium of patients with cervical dysplasia and carcinomas. Memory CTLs specific for E7 epitopes are produced in cervical cancer patients. However, E7-induced cellular responses are not strong enough to influence disease development. Schlegel aims to enhance this E7-specific CTL response by developing vaccines that can efficiently deliver E7 to the MHC class I pathway resulting in the induction of specific CTLs that can then eliminate E7-expressing epithelial cells. Ideally, this vaccine would also incorporate the proven prophylactic qualities of L1 VLPs or L1 pentamers to make a vaccine that is both prophylactic and therapeutic.

Top

Wnt Pathway in Normal Development and Malignant Progression
Investigators: Dym, Üren, Gallicano

There are established links between the gene expression patterns found in normal stem cells and in cancer precursor cells (termed cancer stem cells by some investigators) and these are being explored as part of Aim 2. The GRC Program includes Dr. Martin Dym, who is an experienced spermatogonial stem cell biologist. Dr. Dym works with Dr. Üren to explore the role of the Wnt pathway in normal development to help Üren understand its role in cervical cancer generation. Spermatogonial stem cells (SSCs) constitute one of the most important stem cell systems in the body, not only because they produce sperm that transmit genetic information from generation to generation, but also because of recent studies from several laboratories showing that in mice they can be reprogrammed to pluripotent embryonic stem (ES)-like cells without the necessity of exogenously added genes. The Dym lab focuses on the biology of the SSCs and in the past few years isolated and characterized SSCs in mice and then examined signaling pathways regulating renewal and differentiation. Immortalized SSC lines have been developed and used for biological studies by Dr. Üren and others to examine the role of Wnt signaling in SSCs. A manuscript on this topic has recently been submitted for publication. In man, very little is known about SSCs, but the Dym lab demonstrated that GPR125 may be a marker for human SSCs. Putative human SSCs can also be reprogrammed to pluripotency. The Dym lab was able to achieve this result without the addition of genes, suggesting that human SSCs have great potential for cell-based, autologous organ regeneration therapy for various diseases. At the same time, it will be critical to be sure that cells that possess great regenerative potential do not also possess the capacity to generate a cancer. A team of developmental, reproductive and cancer biologists who are able to investigate and understand the full impact of a pluripotent cell is best suited to carry out this type of work. The work demonstrating reprogrammed to pluripotency is ongoing, in collaboration with fellow GRC member Ian Gallicano. A manuscript has recently been submitted for publication. This line of studies demonstrates the value of GRC to align the research interests of cancer-focused scientists with fundamental researchers who might not otherwise employ cancer model systems.

Top

HPV and Wnt/beta-catenin signaling
Investigators: Üren, Toretsky, Schlegel

HPV exposure appears to be necessary but not sufficient for cervical cancer development. Dr. Üren discovered that Wnt/beta-catenin signaling activation is sufficient to induce malignant transformation in cells expressing HPV oncogenes. Drs. Üren, Toretsky, and Schlegel showed that malignancy-associated HPVs immortalize, but do not fully transform, human keratinocytes (Üren et al., Cancer Res, 2005). Furthermore, malignant transformation of these keratinocytes by SV-40 small-t antigen was also found to be through activation of the beta-catenin pathway. Additionally, immunohistochemical examination of human cervical cancer samples proved the existence of an activated Wnt/beta-catenin pathway. This work identifies potential molecular targets for millions of women previously exposed to HPV. Development of a secondary screen to identify activation of Wnt/beta-catenin pathway in HPV positive individuals may predict infected women at risk of malignant progression. Furthermore, targeting Wnt/beta-catenin pathway may also provide therapeutic opportunities in advanced stage tumors. The HTSR was essential to the success of this research.

Top

HPV and BRCA1
Investigators: Schlegel, Rosen

BRCA1 is able to repress the transcriptional activity of estrogen receptor-alpha (ER-alpha) in human breast and prostate cancer cells but only weakly inhibits ER-alpha in cervical cancer cells. Drs. Schlegel and Rosen  found that human papillomavirus E7 or E6 oncogenes placed into human papillomavirus-negative cells rescued the BRCA1 repression of ER-alpha activity and that the E7 and E6 oncoproteins interact directly with BRCA1 in vitro and associate with BRCA1 in vivo in cultured cells. E6 and E7 also antagonized the ability of BRCA1 to inhibit c-Myc E-box-mediated transactivation and human telomerase reverse transcriptase promoter activity, in a manner dependent upon the zinc finger domains. These findings suggest functional interactions of BRCA1 with E7 and E6 (Zhang et al., J Biol Chem, 2005).

Top

Development of anti-viral hepatitis therapies
Investigators: Casey, Korba

In a small controlled study, Drs. Casey and Korba found that clevudine, a potent inhibitor of hepadnaviruses, including hepatitis B virus and woodchuck hepatitis virus, suppressed hepatitis delta virus (HDV) viremia in chronically infected woodchucks. Suppression was correlated with the marked reduction of woodchuck hepatitis virus surface antigen in individual animals, consistent with the concept that repression of surface antigen expression may be a useful antiviral strategy for HDV (Casey et al., Antimicrob Agents Chemother, 2005). This work is relevant to the GRC Program and to the mission of Lombardi because suppression of infectious hepatitis and its complications has the potential to reduce the risk of hepatocellular carcinoma in infected individuals.