Narayanan, Ramaswamy

Pancreatic cancer is an abhorrent malignancy with limited diagnostics and
response to drug therapy. It is believed that noncoding RNAs (ncRNAs) will further the
understanding behind the mechanisms of pancreatic cancer development and progression,
providing a novel approach for drug development and biomarker discovery. Therefore, a
database of pancreatic cancer ncRNAs was established using bioinformatics and text
mining approaches. These ncRNAs were characterized for RNA expression, copy number
variation, disease association, single nucleotide polymorphisms, secretome analysis, and
identification of protein targets. Exosomal proteins and ncRNA identified through this
study provide the basis for noninvasive diagnostic potential. Additionally, a secreted
microRNA, MIR3620, emerged from this study as a potential prognostic and diagnostic
biomarker for pancreatic cancer. By analyzing MIR3620 and its protein targets, a
mechanism of regulation for these genes in contributing to the progression and
development of pancreatic cancer was established.

A Down's Syndrome related Single Minded 2 gene (SIM2), previously known to be
associated with Trisomy 21 was predicted by bioinformatics to be colon cancer specific.
In previous work from the laboratory using a patient tissue repository, an isoform of this
gene, short form (SIM2-s) was shown to be colon cancer specific. Inhibition of SIM2-s
expression by antisense technology resulted in cancer-cell specific apoptosis within 24
hours. Microarray-based gene expression profiling of the antisense-treated colon cancer
cells provided a fingerprint of genes involving key cell cycle, apoptosis, DNA damage
and differentiation genes. Taking hints from the microarray database, experiments were
initiated to decipher the molecular mechanism underlying the cancer specific function of
the SIM2-s gene. Using an isogenic cell system, apoptosis was found to be dependent
on DNA damage and repair gene, GADD45-a. Further, key pathways including p38 MAP
kinase (MAPK) and specific caspases were essential for apoptosis. Programmed cell
death was not dependant on cell cycle and was preceded by the induction of terminal
differentiation. To clarify whether SIM2-s function is a critical determinant of differentiation, stable transfectants of SIM2-s were established in a murine adipocytic
cell line (3T3-L 1 ). SIM2-s overexpression caused a pronounced block of differentiation
of the pre-adipocytes into mature adipocytes. A study of the differentiation pathway in
3T3-L 1 cells suggested that this block occurs early on in the cascade. These results
supported the starting premise that SIM2-s is a critical mediator of cell differentiation. To
clarify whether the SIM2-s gene has transforming potential, the SIM2-s gene was
overexpressed in the NIH3T3 murine fibroblast cell line. The cells expressing the human
SIM2-s gene exhibited shorter doubling time, abrogation of growth serum requirement,
greater cell number at saturation density and focus formation. In vivo tumorigenicity
assays showed tumor formation with long latency. These results provide strong evidence
for the role of SIM2-s gene in tumor cell growth and differentiation, and validate drug
therapy use for the gene.

Understanding the host response immediately following mucosal HIV-1
infection will be pivotal in determining whether the immune response induced by
a vaccine will successfully sense and control viral replication. In order for
effective vaccine strategies and modalities to be developed, these earliest
immunological events must be fully assessed in a non-biased manner.
Nonhuman primates (NHP), specifically Rhesus macaques (RM), serve as a
model to investigate the immunological landscape immediately post-challenge
and to define the spatiotemporal path of simian immunodeficiency virus (SIV).
SIV infection of RM serves as a model of human HIV infection as it recapitulates
many of the virological, immunological, and pathological features of HIV infection in the human host. In this thesis I will test the hypothesis whether
transcriptional analysis will allow a sensitive measure of the early innate immune
responses that accompany detection of the SIV virus in the periphery. I have
determined that an early inflammatory profile arises early in tissues proximal to
the challenge site that precedes widespread immune activation and the systemic
antiviral interferon response. This study defines in detail the spatiotemporal
relationship between virus and host immune response and may be a valuable
resource in guiding future vaccine design strategies.

Mining the human genome for therapeutic target(s) discovery promises novel outcome. Over half of the proteins in the human genome however, remain uncharacterized. These proteins offer a potential for new target(s) discovery for diverse diseases. Additional targets for cancer diagnosis and therapy are urgently needed to help move away from the cytotoxic era to a targeted therapy approach. Bioinformatics and proteomics approaches can be used to characterize novel sequences in the genome database to infer putative function. The hypothesis that the amino acid motifs and proteins domains of the uncharacterized proteins can be used as a starting point to predict putative function of these proteins provided the framework for the research discussed in this dissertation.

Harnessing the human genome using bioinformatics lead to the discovery of a highly cancer-selective gene, Single Minded 2 gene (SIM2). An isoform of the SIM2 gene, the short-form (SIM2-s), was shown to be specific to colon, pancreas, and prostate tumors. Antisense inhibition of SIM2-s in a colon carcinoma derived cell line (RKO) caused inhibition of gene expression, growth inhibition and apoptosis in vitro and in nude mice tumorigenicity models. To understand the mechanism of Sim2-s antisense, the antisense treated RKO colon cancer cells were monitored for genome wide expression using Affymetrix GeneChipRTM technology. A list of apoptosis related genes was generated using GeneSpringRTM software. Select GeneChip RTM output was validated by Quantitative RT-PCR. Relevance of a key gene, Growth arrest and DNA damage inducible (GADD45a), in the SIM2-s pathway was established. These results will provide a basis for the future experiments to understand the mechanism underlying Sim2-s activation in specific tumors.

Recently Dr. Narayanan's laboratory, utilizing bioinformatics approaches, identified a novel gene which may play a role in colon cancer. This gene in view of its expression specificity was termed Colon Carcinoma Related Gene (CCRG). The CCRG belongs to a novel class of secreted molecules with a unique cysteine rich motif. The function of CCRG however, remains unknown. The basis of this project revolved around establishing the putative function (functional genomics) of CCRG. The rationale for the project was to test a hypothesis that CCRG may offer a growth advantage to cancer cells. The availability of diverse tumor-derived cell lines, which were CCRG negative offered a possibility to study the consequence of enforced expression of CCRG. A breast carcinoma cell line was transfected with an exogenous CCRG expression vector and the stable clones were characterized. The stable transfectants of CCRG showed enhanced growth and a partial abrogation of serum growth factor(s) requirement. These results provide a framework for future experiments to further elucidate the function of CCRG.

The Cancer Genome Anatomy Project (CGAP) database of the National Cancer Institute contains thousands of expressed sequences, both known and novel, derived from diverse sets of normal, precancerous, and tumor cDNA libraries. This offers the possibility of using this database as a rational starting point for bioinformatics-based cancer gene discovery. Using the Digital Differential Display tool of the CGAP database, a hypothesis-driven gene discovery approach was undertaken to analyze differential expression of various solid tumor types. Two hundred known genes and five hundred novel sequences were discovered to be differentially expressed, and a comprehensive database was established to facilitate identification of cancer diagnostic and therapeutic targets. To validate the use of bioinformatics in discovering genes with organ- and tumor-selectivity, novel ESTs predicted to be colon tumor-specific were analyzed further for expression specificity. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis using matched sets of colon normal- and tumor-derived cDNAs identified one EST to be specifically expressed in the majority of colon tumors and normal small intestine. Due to this apparent specificity, the gene was termed Colon Carcinoma Related Gene (CCRG). Based on protein sequence analysis, CCRG belongs to a novel class of secreted factors. Another gene identified in this study showed homology to Single Minded 2 gene (SIM2). Involvement between SIM2 and cancer has not yet been reported. Isoform-specific expression of SIM2 short-form (SIM2-s) was seen in colon, pancreas, and prostate carcinomas but not in most normal tissues. Using a large collection of paraffin sections from colon, pancreas, and prostate tumor and normal tissues, elevated protein expression was seen in tumors compared to normal tissue specimens, demonstrating the diagnostic potential of SIM2-s. Antisense inhibition of SIM2-s expression in colon and pancreatic cancer cell lines caused inhibition of gene expression, growth inhibition, and apoptosis. Administration of SIM2-s antisense in nude mice caused inhibition of colon tumor growth without pronounced gross toxicity. Using GeneChipRTM technology, a gene expression profile indicative of apoptosis was observed in the colon cancer model. CCRG and SIM2-s offer both a diagnostic and therapeutic potential in select cancers and validate the use of bioinformatics approaches in the gene discovery paradigm.