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Department of Biological Sciences


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Franklin G. Berger

Director, Center for Colon Cancer Research
Department of Biology
University of South Carolina

Office: PSC 614
Phone Number: Office: 803-777-1171; Lab: 803-777-7216; Office: 803-777-1231
Email: fgberger@mailbox.sc.edu
Website: Berger Lab/ Center for Colon Cancer Research
Vitae: Download PDF

Background: Colorectal cancer is the fourth-highest cancer in terms of incidence, and second-leading cause of cancer death.  In the US, over 140,000 new cases of colorectal cancer are diagnosed each year, and nearly 50,000 patients die from the disease.  Like most cancers, less-than-optimal efficacy of anti-neoplastic agents is a major barrier to the successful treatment of advanced colorectal cancer.  The high genetic plasticity of neoplastic cells generally leads to rapid emergence of resistance to chemotherapeutic agents, reducing therapeutic effectiveness.  For several years, we have been examining an important class of chemotherapeutic agents that target the pyrimidine biosynthetic enzyme thymidylate synthase (TS).  TS catalyzes the  reductive methylation of dUMP to form dTMP, which is indispensible for DNA synthesis during cell proliferation and DNA repair. Treatment of cells with fluoropyrimidine analogs (e.g., 5-fluorouracil and 5-fluoro-2'- deoxyuridine), as well as antifolates (e.g., tomudex, AG337, and BW1843), results in the generation of metabolites that inhibit TS, causing depletion of thymidylate pools, cessation of cell growth, and eventually, cell death. Thus, TS inhibitors have been useful in the treatment of a variety of neoplasms.  However, a large fraction of patients do not respond to these drugs; furthermore, those that do respond typically develop resistance.

 Mechanisms of intracellular TS degradation: It is well-established that TS-directed drugs induce the concentration of TS protein, a phenomenon that occurs via stabilization of the polypeptide and that may contribute to reduced drug efficacy.  To pursue this observation, we have studied the mechanisms underlying TS degradation and its alteration by inhibitory ligands (see references below).  We have found that TS is degraded by the 26S proteasome; however, unlike most proteins, such degradation is ubiquitin-independent.  A 42-residue region, which is located at the N-terminal end of the polypeptide, regulates TS degradation.  The region functions as a degron capable of destabilizing a heterologous protein to which it is fused.  The TS degron is composed of an intrinsically disordered region (residues 1-27) followed by an amphipathic alpha-helix (residues 31-42) that cooperate to promote degradation of the polypeptide.  Using targeted in vitro mutagenesis, we have defined specific elements within the degron that regulate its activity.  A free, unblocked N-terminal amino group is required for degron activity.  In addition, an Arg-Arg motif at residues 10-11 within the disordered domain is required.  The alpha-helical segment of the degron does not function simply as an extension or scaffold for the disordered domain; rather, it provides a specific structural component that is necessary for degradation and that requires its helical conformation.  Small domains from heterologous proteins can substitute for both the disordered and helical regions, indicating that the degradation-promoting function of these regions is not sequence-specific. Thus, there appears to be little sequence constraint on the ability of these regions to function as degron constituents; rather, it is the overall conformation (or lack thereof) that is critical.

Oxidative stress and cell death in response to TS inhibition:  We have recently shown that apoptosis in response to TS inhibitors is due to oxidative stress brought on by increased concentrations of reactive oxygen species (ROS) resulting from activation of the enzyme NADPH oxidase (NOX).  NOX, which occurs as a membrane-bound multi-subunit complex, catalyzes transfer of an electron from NADPH to molecular oxygen to form superoxide, which is essential for maximal response to TS inhibitors.  The mechanism of NOX activation by TS inhibitors is not completely understood, but likely involves induction of expression of the NCF2 gene, which encode the NOX regulatory subunit p67phox .

Role of the anti-oxidant genes in response to TS inhibitors: Using cDNA microarrays, we have recently determined that cells mount a protective response to TS inhibitors, involving activation of genes involved in amelioration of oxidative and electrophilic stress.  Many of these genes are regulated by transcription factor Nrf2, which is well-known to attenuate the concentration and impact of ROS.  We have postulated that the expression and activation of Nrf2 is a constraining factor in cell death following TS inhibition.  Indeed, we have observed that down-regulation of Nrf2 by siRNAs sensitizes cells to the cytotoxic effects of TS inhibitors.  Our current efforts, summarized in the figure below, are focused on understanding the role of Nrf2 in response toTS-directed drugs, and using the information gained to increase the efficacy of this important class of therpaeutic agents.




SELECTED REFERENCES

Forsthoefel, A.M., M. Peña, Y.Y. Xing, Z. Rafique, and F.G. Berger. 2004.  Structural determinants for the intracellular degradation of human thymidylate synthase.  Biochemistry 43:1972-1979.

Berger, F.G.  2004.  Interleukin-6: a susceptibility factor that may be involved in racial and ethnic disparities in breast cancer mortality.  Breast Cancer Res. Treat. 88:281-285. [This review article was highlighted in the December 10 issue of BreastCancer Net News.]

Murphy, J.T.,  J.M. Tucker, C. Davis, and F.G. Berger.  2004.  Raltitrexed  increases tumorigenesis as a single agent yet exhibits anti-tumor synergy with 5-fluorouracil in ApcMin/+ mice.  Cancer Biol. Therapy 3:1169-1176.

Peña, M., Y.Y. Xing, S. Koli, and F.G. Berger.  2006.  Role of N-terminal residues inubiquitin-independent degradation of human thymidylate synthase.  Biochem. J. 394:355-363.

Berger, F.G. and S.H. Berger. 2006.  Thymidylate synthase as a chemotherapeutic drug target: Where are we after fifty years?  Cancer Biol. Therapy 5:1238-1241.

Barbour, K.W. and F.G. Berger.  2008.  Cell death in response to antimetabolites directed at thymidylate synthase.  Cancer Chemother. Pharmacol. 61:189-201.

Peña, M.M.,  S. Melo, Y.-Y. Xing, K. White, K.W. Barbour, and F.G. Berger. 2009.  The intrinsically disordered N-terminal domain of thymidylate synthase targets the enzyme to the ubiquitin-independent proteasomal degradation pathway.  J. Biol. Chem.  284:31597-31607.

Huang, X., L.M. Gibson, B.J. Bell, L.L. Lovelace, M.M.O. Peña, F.G. Berger, S.H. Berger, and L. Lebioda. 2010.  Replacement of Val3 in human thymidylate synthase affects its kinetic properties and intracellular stability.  Biochemistry 29:2475-2482.

Melo, S.P., A. Yoshida, and F.G. Berger.  2010.  Functional dissection of the N-terminal degron of human thymidylate synthase.  Biochem. J. 432:217-226.

Davis, C., R. Price, G. Acharya, T. Baudino, T. Borg, F.G. Berger, and M.M. Peña.  2011.  Hematopoietic derived cell infiltration of the intestinal tumor microenvironment in ApcMin/+ mice.  Microsc. Microanal. 17:528-539.

Melo, S.P., K.W. Barbour, and F.G. Berger.  2011.  Cooperation between an intrinsically disordered region and a helical segment is required for ubiquitin-independent degradation by the proteasome.  J. Biol. Chem. 286:36559–36567.

Brandt, H.M., H.L. Dolinger, P.A. Sharpe, J.W. Hardin, and F.G. Berger. 2012. The relationship between colorectal cancer awareness and knowledge with colorectal cancer screening.  Colorectal Cancer 1:383-396.

Barbour, K.W., Y.-Y. Xing, E.A. Peña, and F.G. Berger.  2013.  Characterization of the bipartite degron that regulates ubiquitin-independent degradation of thymidylate synthase.  Bioscience Rep.  33:165-173.

Wallace, K., E.G. Hill, D.N. Lewin, G. Williamson, S. Oppenheimer, M.E. Ford, M.J. Wargovich, F.G. Berger, S.W. Bolick, M.B. Thomas, and A.J. Alberg.  2013.  Racial disparities in advanced stage colorectal cancer survival.  Cancer Causes Control.  24:463-471.

Ozer, U., K.W. Barbour, and F.G. Berger.  Oxidative stress and response to thymidylate synthase inhibitors in colon cancer cells.  Submitted.