This document describes a research plan to investigate ROBO1 as a potential biomarker for distinguishing indolent from aggressive prostate cancer phenotypes in African American men. The plan involves 3 aims: 1) Using immunohistochemistry to determine if ROBO1 expression levels correlate with cancer aggressiveness in African American tissue samples, 2) Examining whether ROBO1 hypermethylation predicts localized or metastatic cancer, and 3) Investigating ROBO1 as a therapeutic target using cell line models of treatment-resistant cancer in African American men. Updates are provided on progress made, including developing the necessary techniques and cell lines. The research also involves training an African American student and collaborating with community partners to address health disparities.

1. Novel Biomarker that
Discriminates Indolent from
Aggressive Phenotype in AA
Men
NIH U54MD008620
M. Saleem Bhat, PhD
Asst. Professor
Hormel Institute,
University of Minnesota

2. Prostate cancer (CaP) exhibits the most striking racial
disparity. African-Americans are at 1.4 times higher risk of
being diagnosed of CaP than Caucasians.
Serum-PSA as a disease type discriminating marker is
reported to perform poor in African-American men.
There is no race-specific biomarker that would
provide information at the time of diagnosis if the
tumor is treatable or has tendency to spread to other parts
of body (metastasis) and become resistant to
therapies.
Hypothesis:
ROBO1 could act as a potential biomarker that would
discriminate between primary and metastatic disease in
African-Americans, and possibly a therapeutic target to
treat metastatic CaP in this population.

3. Laboratory Research Plan:
Aim#1: Determine the relevance of ROBO1 as a biomarker for CaP-
disease phenotype-distinction and aggressiveness in
African-Americans using IHC analysis of tissues.
Aim#2: Determine if hypermethylation of ROBO1 would predict the
localized or metastatic phenotype of prostate cancer.
Aim# 3: Investigate the relevance of ROBO1 as a therapeutic
target using race-distinct carcinogenesis model.
To address issue of disparity, we will employ :
1. Laboratory based research
2. Involvement of minority community in understanding the
gravity of the this health problem,
Academic-Community Partnership & Plan:
The PI will impart the health disparity research training to an
African-American student (identified by The 100 Black Men Inc. th
rough its education/fellowship program)

4. Aim#1: Determine the relevance of ROBO1 as a biomarker for CaP-disease
using IHC analysis of tissues.
Update
We obtained >60 AA-pa
tient Specimens.
We perfected the
method;
Performed validation of
several antibodies
against ROBO1
Young
brain
old
brain
150 KD
ROBO1
Cleave
form
Status : Going on

5. NIH U54MD008620
Aim#2: Determine if hypermethylation of ROBO1 would predict the
localized or metastatic phenotype of prostate cancer.
Update
Plan: We would use prostatic tissues and circulatory DNA (proof of the
principle) in serum samples of African-American patients.
We are isolating the DNA from available prostatic tissues.
We also plan to use LCM dissection technique for isolating ROBO1+ve and
ROBO1-ve area of tissues to isolate DNA and perform methylation analysis .
We would perform the methylation analysis of all DNA samples
under similar condition.
We standardized of isolating free DNA from serum samples.

6. Aim# 3: Investigate the relevance of ROBO1 as a therapeutic
target using race-distinct carcinogenesis model.
Using race-distinct in vitro models
we will determine if reactivating R
OBO is an ideal therapeutic strateg
y for treating metastatic-CaP disea
se in African-Americans
Update
2. We are currently generating AA-specific localized and
metastatic cell lines in which the expression of ROBO1
can be switched on and off as per need.
3. We have started experiments to
study the molecular pathways which
could be involved in the regulation of the
differential expression of ROBO1
in the localized and metastatic tumor sta
ges of CaP disease.
1.We have generated cell lines Representing do
cetaxel-resistant and bicalutamide-resistant di
sease in African-American men.

7. NIH U54MD008620
Academic-Community Partnership
1. (With the help of Dr. Mark Alexander ), The 100 Black Men , the
community partner of CHAAMPS program has identified and
nominated one student for a 10-week long training in Health
Disparity research in my laboratory. We anticipate that the student
start the training in the mid-April, 2016.
Update

8. Funding : NIH/NIMHD
Acknowledgment
Collaborators:
1. Dr. Koochekpour (Roswell Cancer Institute, Buffalo, NY).
2. Louisiana-North Carolina Prostate Cancer Consortium
3. Dr. Mark Alexander , 100 Black Men of America, Inc.
4. Dr. Badrinath Konety, Department of Urology, University of
Minnesota
NIH U54MD008620