Research

Research Highlights

Selected completed projects supported by the Howard-Hopkins Partnership

1.  “Comparative Gene Expression Analysis in African American and Caucasian Breast Cancer” Carolyn W. Broome, Ph.D. (HUCC)
Saraswati Sukumar, Ph.D. (SKCCC)

This collaboration resulted in two publications and an additional grant at completion of the project.  Some of the results of the project were published in a paper entitled “Estrogen receptor/progesterone receptor-negative breast cancers of young African-American women have a higher frequency of methylation of multiple genes than those of Caucasian women.” (Clin Cancer Res. 2004 Mar 15;10(6):2052-7) by J. Mehrotra , MM Ganpat , and Y Kanaan et al.  This paper examined the molecular basis for negative prognostic factors being more prevalent in African-American (AA) than Caucasian (Cau) women.  The investigator examined the frequency of promoter hypermethylation in invasive ductal breast cancers in the two races. HIN-1, Twist, Cyclin D2, RAR-beta, and RASSF1A were analyzed in DNA from 67 AA and 44 Cau invasive ductal breast cancers.  The tumor samples were stratified by age and estrogen receptor/progesterone receptor (ER/PR) status, and by methylation-specific PCR. Hierarchical multiple logistic regression analysis was used to determine estimated probabilities of methylation. There were significant differences between races found in the ER-/PR-, age < 50 subgroup.   AA tumors had higher frequency of methylation in four of five genes compared to Cau tumors.  There was a higher prevalence (80 versus 0%) of three or more methylated genes per tumor.  No differences in gene methylation patterns were observed between the races for ER+/PR+ tumors in all ages and ER-/PR- tumors in age > 50.  ER+/PR+ status was associated with higher frequency of methylation in Cau tumors in all age groups but only with the age > 50 subgroup in AA.  Cyclin D2 methylation was significantly associated with worse survival. The investigators condluded that  ER-/PR-, age < 50 tumors in AAwomen, have a significantly higher frequency of hypermethylation than in those of Cau women. Comparative studies, such as these, provide a molecular basis for differences in tumor biology in the two races.

Lay Abstract: Breast cancer takes a higher toll on African-American women.  It tends to be more deadly in black women than in whites.  Scientists at Johns Hopkins and Howard University found more gene changes in African-American women with breast cancers that do not respond to hormones than white women.  The genetic changes occur in certain genes known to influence breast cancer, in which small methyl groups stick to the “on-off” portion of the gene.  Disrupting a gene’s starter switch can impact a cell’s ability to behave normally and eventually turn cancerous.

2.  “H. Pylori Induced Oxidative DNA Damage” Duane Smoot, M.D. (HUCC) and John Groopman, Ph.D. (SKCCC)

This project has also been continued as an NIH funded grant.  The results of the work were reported in two publications, including one published in Carcinogenesis 11:2091-5, entitled “Influence of Helicobacter pylori on reactive oxygen-induced gastric epithelial cell injury”.  This paper examined risk factors for gastric cancer, given the association of Helicobacter pylori infection with gastric cancer. The study investigated the effects of exposure of gastric cells to H. pylori on oxidant-associated gastric epithelial cell injury. A humangastric epithelial cell line (AGS) was exposed overnight to either live H.pylori (four cagA(+) and four cagA(-)) or broth culture supernatant from an isogenic H.pylori cagA(+) strain with and without vacA activity. Incubation of AGS cells with cagA(+) and cagA(-) H.pylori strains before exposure to reactive oxygen species (ROS) reduced  viability to 73.7% and 39.5% of controls, respectively. The percent viability of cells exposed to ROS after incubation with control broth, vacA(-) broth and vacA(+) broth was 97.7%, 70.5% and 63.5%, respectively. The investigators then examined the effects of H.pylori exposure on the activities of ROS-scavenging enzymes [catalase, glutathione peroxidase and superoxide dismutase (SOD)] and formation of 8-hydroxy-2-deoxyguanosine (8-OH-dG) adducts in AGS cells. Exposure to cagA(-) strains reduced catalase activity by 42%.  Exposure to cagA(+) H.pylori strains increased catalase activity by 51%. Glutathione peroxidase activity increased with exposure to both cagA(-) and cagA(+) strains by 95% and 240%, respectively. Total SOD activity increased 156% after exposure to cagA(+) strains and was increased (52%) with exposure to cagA(-) strains. CuZn-SOD protein levels  were not significantly altered by exposure to H.pylori strains.   Mn-SOD concentrations were significantly increased after exposure to both cagA(-) and cagA(+) H.pylori strains. The increase in catalase, glutathione peroxidase and SOD activity is associated with fewer 8-OH-dG DNA adducts and decreased susceptibility of AGS cells to lethal injury from ROS after exposure to cagA(+) H.pylori strains compared to exposure of cagA(-) H.pylori strains. Alterations in the activity of ROS-scavenging enzymes by the presence of H. pylori may in part be responsible for the increased risk of gastric cancer associated with H.pylori infection.

Lay Abstract: The stomach-dwelling bacterium H. pylori is well-known to cause gastric cancers worldwide.  In the United States, it is more often found lining the stomachs of African-Americans and several other groups.  One reason for its cancer-causing ability may be the bacteria’s penchant for altering enzymes that detoxify reactive oxygen species, according to Hopkins-Howard researchers.  The National Institutes of Health has continued funding these researchers’ efforts to further evaluate H. pylori’s effects on gastric cells.

3. “Genomic Instability and Gene Silencing in Colon Cancer” Hassan Ashktorab, Ph.D. (HUCC) and Francis Giardiello, M.D. (SKCCC)

This project has also resulted in NIH funding to continue the work.  Two publications were produced including “Clinicopathological features and microsatellite instability (MSI) in colorectal cancers from African Americans” by H,.Ashktorab , DT Smoot , HFarzanmehr , et al (Int J Cancer. Oct 10;116(6):914-9, 2005).  This report examined the 1.5 times higher risk of colorectal carcinoma (CRC) found in African Americans (AAs) than in Caucasians. Gene silencing through CpG island hypermethylation has been implicated with the genesis or progression of microsatellite instability (MSI) due to one target for hypermethylation being the DNA mismatch repair gene hMLH1.  There is some evidence of an increased incidence of MSI among AAs. P16 and hMLH1 can be inactivated by hypermethylation of their respective promoter regions.  This prevents the ability of the cell to regulate proliferation and repair processes.   The investigators studied methylation and hMHS2 expression in colorectal cancers from AA patients to determine if MSI is associated with epigenetic silencing. Matched normal and colon cancer tissues from AA patients (n = 51) were examined. Five microsatellite markers (D2S123, D5S346, D17S250, BAT25 and BAT26) were used to evaluate MSI status.   Twenty-two (43%) cancers demonstrated microsatellite instability-high (MSI-H), while 27 were microsatellite stable (MSS) and 2 were microsatellite instability-low (MSH-L). Most of the MSI-H tumors were proximal, well differentiated and highly mucinous. In the MSI-H group, 68% were females.  The p16 promoter was methylated in 19 of 47 (40%) tumors. A total of 7 of these tumors showed MSI-H (33%). The hMLH1promoter was methylated in 29 of 34 (85%) tumors, of which 13 demonstrated MSI-H (87%). hMLH1 and hMSH2 staining was observed in 66% and 38% of MSI-H tumors, respectively. The prevalence of MSI-H colorectal tumor was 2-3-fold higher, while the defect in the percentage expression of mismatch repair (MMR) genes (hMLH1 and hMSH2) was similar in AA patients compared to the U.S. Caucasian population. Similar numbers of AA MSS tumors with p16 and hMLH1 methylation likely indicate hemimethylation of genes that might reflect environmental or genetic influences that might be more common in the AA population.

Lay Abstract: African-American’s are 1.5 times more susceptible to colon cancer than their white counterparts.  Researchers at Johns Hopkins and Howard University are investigating a common DNA mistake in colon cancer cells, called microsatellite instability.  Cells become unstable when genes known to repair DNA change or mutate.  They often make mistakes in areas where the DNA repeats itself by adding or removing a few DNA letters.  Finding these elongated or truncated DNA sections could indicate the presence of mutation in DNA repair genes.  The researchers have received additional funding from the National Institutes of Health to determine ethnic differences in the presence of such gene changes.

Selected Ongoing Projects supported by the Howard-Hopkins Partnership

1. "Synthesis and MAPK Kinase Inhibitory Activities In Vitro and In Vivo in Human Prostate Tumors by Imido-substituted 2-Chloro-1, 4-Naphthoquinones"
Robert L. Copeland, Jr., Ph.D. (HUCC) and
Samuel R. Denmeade, M.D. (SKCCC)

The collaborative relationship between Dr. Denmeade and Dr. Copeland is exactly the type of relationship that the SKCCC/HUCC was designed to facilitate in cancer research.  This proposal provides an opportunity to develop preclinical data that will provide the basis for future translational studies.  These studies by themselves may identify new agents that could represent clinical development candidates.  The prospects are high based on the resource rich environment and exceptional mentorship that is available through the SKCCC/HUCC partnership.

Prostate cancer is a devastating disease that claims the lives of 30,000 American men per year.  The overall goal of this proposal is to develop new therapy for prostate cancer.  African-American men do have the highest incidence of prostate cancer in the world and present with more advanced disease.  Thus, even with current therapies these men have an overall higher death rate from prostate cancer. Therefore, while new therapies for prostate cancer would have significant impact on the health of all men with prostate cancer, African-American men would, in particular, significantly benefit from these therapies.

2. “Novel Bifunctional Anti-Androgens for Prostate Cancer”
Oladapo Bakare, Ph.D. (HUCC) and
John T. Isaacs, Ph.D. (SKCCC)

During the progression of prostatic cancer, malignant cells undergo molecular changes in which AR interacts with partner proteins to generate genomic as well as non-genomic signaling which allows their continuing growth in the presence of low circulating serum T produced by androgen ablation.  Thus, these cells are not eliminated by standard androgen ablation (i.e.,LHRH±casodex) and their continuous growth eventually kills the patient.  Such lethality is highest among our African-American males within the United States.  To address this health disparity, developing effective therapy for such androgen ablation resistant patients is the focus of the present application.  Our working hypothesis is that present androgen ablation therapy is of limited efficacy because the conformation of the AR protein when either unoccupied or bound by low molecular weight partial agonist or antagonist, like Casodex, can be “forced” by the binding of co-activators to displace co-repressors and undergo a change to a full agonist conformation inducing growth stimulation signaling.  Therefore, a novel strategy to block such AR growth signaling in androgen ablation failing patients is to develop “bulky” bifunctional anti-androgens which bind to the ligand binding domain of AR and structurally lock the AF-2 domain of the AR surface in an antagonist conformation not allowing its AF-2 domain to be “forced” into the agonist state.  Therefore Specific Aim 1 is to design and synthesize a series of benzyl or alkyl 11? and 7? side chain-?9-19-nortestosterone analogs and determine their affinity for binding to the ligand binding domain (LBD) of human AR and their in vitro anti-androgen ability against a series of human prostate cancer cell lines. In Specific Aim 2, the best of each of the four classes of analogs will be coupled via their side chain to a synthetic ligand for FK-506 binding protein (i.e., denoted SLF) to produce bifunctional binding analogs which while tethered to the LBD of AR also binds FK-506 producing an adduct sterically bulky enough to prevent any AR co-activator binding.  In Specific Aim 3, the SLF bifunctional analogs will be tested for their efficacy vs. casodex in vitro and in vivo against a series of human prostate cancers in an androgen ablated environment.  To achieve these goals in a timely fashion, a team approach is required involving the collaboration of Dr. Oladapo Bakare of Howard University and Dr. John Isaacs of Johns Hopkins University.  Dr. Bakare’s expertise is in organic chemical synthesis and his laboratory will synthesize all of the proposed analogs.  Dr. Isaacs’ expertise is in tumor biology and chemical therapeutics focused particularly on prostate cancer, and his laboratory will perform all of the biochemical, and in vitro/in vivo evaluations of the newly synthesized compounds.

3. "Tumor-targeted MR Contrast Enhancement Using Molecular Imaging Techniques"
Paul C. Wang, M.D., (HUCC) and
Zaver M. Bhujwalla, Ph.D. (SKCCC)

This partnership between Howard University Cancer Center and the JHU In Vivo Cellular Molecular Imaging Center at Johns Hopkins University/SKCC will enable the faculty and the students to obtain training in molecular imaging through conducting collaborative research and participating in seminars, meetings, laboratory internship, and scientific exchanges.  Through this valuable partnership experience, a modest facility will be established at the Howard Cancer Center to support sustainable long-term molecular imaging research.  For future studies we intend to include making stable clones with transferrin receptor expression under the control of a gene of interest such as NF-kB.  Such a stable clone will allow us to link the imaging of transferrin receptor expression to its related functions such as metabolism, cell proliferation and angiogenesis.  This will be carried out using the molecular biology expertise available within the JHU ICMIC/SKCC Program and will allow these collaborative investigators to relate gene expression to metabolic processes or vascular functions using MRI.

This partnership project will result in the preclinical development of systems for generating targeted contrast specific for cancer cells and potentially for delivery of therapeutic cargo.  The collaboration between the two programs will result in the transfer of preclinical molecular imaging expertise from JHU ICMIC/SKCC to the Howard University Cancer Center.  The imaging technologies to be used have a high probability of clinical translation that may be incorporated into clinical trials at Howard University Cancer Center to improve diagnosis and treatment outcome.

All the research done both at the Howard University and the Johns Hopkins University will be shared by both groups for joint publications.  Based on the initial collaboration, we have successfully obtained a joint training grant from Department of Defense (DOD).  This training grant will prepare the Howard faculty members to pursue molecular imaging research. Additional grant applications to NIH and other funding agencies will be submitted by the end of the project. The cooperation of Dr. Bhujwalla’s and Dr. Wang’s labs will set an important strategic collaboration between JHU/SKCCC and HUH/HUCC in this fascinating field.

Full List of Collaborative Research Studies Between HUCC and SKCCC

Partnership Publications

1. Ashktorab, H., Smoot, D. T., Carethers, J. M., Rahmanian, M., Kittles, R., Vosganian, G., Doura, M., Nidhiry, E., Naab, T., Momen, B., Shakhani, S., and Giardiello, F. M.: High incidence of microsatellite instability in colorectal cancer from African Americans. Clin Cancer Res 9 (3): 1112-7, 2003.
2. Kittles, R. A., Chen, W., Panguluri, R. K., Ahaghotu, C., Jackson, A., Adebamowo, C. A., Griffin, R., Williams, T., Ukoli, F., Adams-Campbell, L., Kwagyan, J., Isaacs, W., Freeman, V., and Dunston, G. M.: CYP3A4-V and prostate cancer in African Americans: causal or confounding association because of population stratification? Hum Genet 110 (6): 553-60, 2002.
3. Kittles, R. A., Panguluri, R. K., Chen, W., Massac, A., Ahaghotu, C., Jackson, A., Ukoli, F., Adams-Campbell, L., Isaacs, W., and Dunston, G. M.: Cyp17 promoter variant associated with prostate cancer aggressiveness in African Americans. Cancer Epidemiol Biomarkers Prev 10 (9): 943-7, 2001.
4. Mehrotra, J., Ganpat, M. M., Kanaan, Y., Fackler, M. J., McVeigh, M., Lahti-Domenici, J., Polyak, K., Argani, P., Naab, T., Garrett, E., Parmigiani, G., Broome, C., and Sukumar, S.: Estrogen receptor/progesterone receptor-negative breast cancers of young African-American women have a higher frequency of methylation of multiple genes than those of Caucasian women. Clin Cancer Res 10 (6): 2052-7, 2004.
5. Panguluri, R. C., Long, L. O., Chen, W., Wang, S., Coulibaly, A., Ukoli, F., Jackson, A., Weinrich, S., Ahaghotu, C., Isaacs, W., and Kittles, R. A.: COX-2 gene promoter haplotypes and prostate cancer risk. Carcinogenesis 25 (6): 961-6, 2004.
6. Smoot, D. T., Elliott, T. B., Verspaget, H. W., Jones, D., Allen, C. R., Vernon, K. G., Bremner, T., Kidd, L. C., Kim, K. S., Groupman, J. D., and Ashktorab, H.: Influence of Helicobacter pylori on reactive oxygen-induced gastric epithelial cell injury. Carcinogenesis 21 (11): 2091-5, 2000.
7. Umbricht, C. B., Conrad, G. T., Clark, D. P., Westra, W. H., Smith, D. C., Zahurak, M., Saji, M., Smallridge, R. C., Goodman, S., and Zeiger, M. A.: Human telomerase reverse transcriptase gene expression and the surgical management of suspicious thyroid tumors. Clin Cancer Res 10 (17): 5762-8, 2004.
8. Adams-Campbell, L. L., Ahaghotu, C., Gaskins, M., Dawkins, F. W., Smoot, D., Polk, O. D., Gooding, R., and DeWitty, R. L.: Enrollment of African Americans onto clinical treatment trials: study design barriers. J Clin Oncol 22 (4): 730-4, 2004.
9. Agurs-Collins, T., Adams-Campbell, L. L., Kim, K. S., and Cullen, K. J.: Insulin-like growth factor-1 and breast cancer risk in postmenopausal African-American women. Cancer Detect Prev 24 (3): 199-206, 2000.
10. Ahaghotu, C., Baffoe-Bonnie, A., Kittles, R., Pettaway, C., Powell, I., Royal, C., Wang, H., Vijayakumar, S., Bennett, J., Hoke, G., Mason, T., Bailey-Wilson, J., Boykin, W., Berg, K., Carpten, J., Weinrich, S., Trent, J., Dunston, G., and Collins, F.: Clinical characteristics of African-American men with hereditary prostate cancer: the AAHPC Study. Prostate Cancer Prostatic Dis 7 (2): 165-9, 2004.
11. Ashktorab, H., Ahmed, A., Littleton, G., Wang, X. W., Allen, C. R., Tackey, R., Walters, C., and Smoot, D. T.: p53 and p14 increase sensitivity of gastric cells to H. pylori-induced apoptosis. Dig Dis Sci 48 (7): 1284-91, 2003.
12. Ford JG, Howerton MW, Bolen S, Gary TL, Lai GY, Tilbert J, Gibbons, MC, Baffi C., Wilson, RF, Feuerstein, CJ, Tanpitukpongse P, Powe NR, Bass EB.  Knowledge and Access to Information on Recruitment of Underrepresented Populations to Cancer Clinical Trials. Evidence Report/Technology Assessment (Summ) 122:1-11, 2005
a. Ford JG and Howerton MW. The science of recruiting minority populations to screening trials. Clinical Trials 2004;1:341-2.
13. Kanaan, Y., Kpenu, E., Utley, K., Adams-Campbell, L., Dunston, G. M., Brody, L. C., and Broome, C.: Inherited BRCA2 mutations in African Americans with breast and/or ovarian cancer: a study of familial and early onset cases. Hum Genet 113 (5): 452-60, 2003.
14. Kittles, R. A., and Weiss, K. M.: Race, ancestry, and genes: implications for defining disease risk. Annu Rev Genomics Hum Genet 4 33-67, 2003. 
15. Kidd, L.C., Paltoo D.N., Wang, S., Chen, W., Akereyeni, F.,Isaacs, W., Ahaghotu, C., and Kittles, R.: Sequence variation within the 5’ regulatory regions of the vitamin D binding protein and receptor genes and prostate cancer risk. Prostate 64 (3):272-82, 2005.
16. Kidd L, Coulibaly A, Templeton T, Chen W, Long L, Mason T, Bonilla C, Akereyeni F, Freeman V, Isaacs W, Ahagotu C, Kittles RA  Germ-line BCL-2 sequence variants and inherited predisposition to prostate cancer. Prostate Cancer and Prostatic Diseases, 2006.
17. Bonilla, C., Mason, T., Long, L., Ahaghotu, C., Chen, W., Zhao, A., Coulibaly, A., Bennett, F., Aiken, W., Tullock, T., Coard , K., Freeman, V., and Kittles, R.A. E-cadherin polymorphisms and haplotypes influence risk for prostate cancer. The Prostate. 66(5):546-56, 2006.
18. Kittles RA, Baffoe-Bonnie A, Moses T, Robbins C, Ahaghotu C, Huusko P, Pettaway C, Vijayakumar S, Bennett J, Hoke G, Mason T, Weinrich S, Trent J, Collins F, Mousses S,  Bailey-Wilson J, Furbert-Harris P, Dunston G,. Powell I, Carpten J. (in press) A common nonsense mutation in EphB2 is associated with prostate cancer risk in African American men with a positive family history. Journal of Medical Genetics.
19. Ashktorab H, Smoot DT, Farzanmehr H, Fidelia-Lambert M, Momen B, Hylind L, Iacosozio-Dononue C, Carethers JM, Goel A, Boland CR, Giardiello FM. Clinicopathological features and microsatellite instability (MSI) in colorectal cancers from African Americans. Int J Cancer. Oct 10;116(6):914-9, 2005.
20. Gordeuk VR, Onojobi G, Schneider MF, Dawkins FW, Delapenha R, Voloshin Y, von Wyl V, Bacon M, Minkoff H, Levine A, Cohen M, Greenblatt RM. The association of serum ferritin and transferrin receptor concentrations with mortality in HIV-infected women. Haematologica 2006, in press.
21. Makambi, K. H., The effect of the heterogeneity variance estimator on some tests of treatment efficacy. Journal of Biopharmaceutical Statistics, 14(2), 439-449, 2004.
22. Reyes, M. O., Archer, J. A., Nunlee-Bland, G., Daniel, G., Morgan, O., Makambi, K. H., Bone mass in physicians: A Howard University Hospital pilot study. Journal of the National Medical Association, 96(3), 299-305, 2004.
23. Makambi, K. H., On positive estimation of the between-study variance in a one-way random effects ANOVA model. Journal of Statistical Research, 38, 33-44, 2004.
24. Carter-Nolan, P. L., Adams-Campbell, L. L., Makambi, K. H., Shantell, L., Palmer, J. R., Rosenberg, L.  Validation of Physical Activity Instruments:  Black Women’s Health Study. To appear: Ethnicity and Disease, 2006.
25. Williams, C. D., Taylor, T.R., Makambi, K.H., Harrell, J., Palmer, J. R., Rosenberg, L., Adams-Campbell, L. L., CES-D four factor structure confirmed but not invariant in a large cohort of African American women. Psychiatry Research, 2006.
26. Bolen S, Tilburt J, Baffi C,. Gary TL , Powe N, Howerton M, Ford J, Lai G, Wilson R, Bass E.  Defining “Success” in Recruitment of Underrepresented Populations to Cancer Clinical Trials: Moving Toward a More Consistent Approach.  Cancer.  2006 Mar 15;106(6):1197-204.
27. Lai GY, Gary TL, Tilburt J, Bolen S, Baffi CR, Wilson RF, Howerton MW, Gibbons MC, Tanpitukpongse TP, Powe NR, Bass EB, Ford JG.  Examination of Efficacy/Effectiveness of Strategies to Recruit Underrepresented Populations into Cancer Clinical Trials.   2006;3(2):133-41. 

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