Srikanth Santhanam

Therapeutic targeting of inflammation and tryptophan metabolism in colon and gastrointestinal cancer

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer death in the United States. Cytotoxic therapies cause significant adverse effects for most patients and do not offer cure in many advanced cases of CRC. Immunotherapy is a promising new approach to harness the bodys own immune system and inflammatory response to attack and clear the cancer. Tryptophan metabolism along the kynurenine pathway (KP) is a particularly promising target for immunotherapy. Indoleamine 2,3-dioxygenase 1 (IDO1) is the most well studied of the enzymes that initiate this pathway and it is commonly overexpressed in CRC. Herein, we provide an in-depth review of how tryptophan metabolism and KP metabolites shape factors important to CRC pathogenesis including the host mucosal immune system, pivotal transcriptional pathways of neoplastic growth, and luminal microbiota. This pathways role in other gastrointestinal (GI) malignancies such as gastric, pancreatic, esophageal, and GI stromal tumors is also discussed. Finally, we highlight how currently available small molecule inhibitors and emerging methods for therapeutic targeting of IDO1 might be applied to colon, rectal, and colitis-associated cancer.

Blood Group O-Dependent cellular responses to cholera toxin: Parallel clinical and epidemiological links to severe cholera

Because O blood group has been associated with more severe cholera infections, it has been hypothesized that cholera toxin (CT) may bind non-O blood group antigens of the intestinal mucosae, thereby preventing efficient interaction with target GM1 gangliosides required for uptake of the toxin and activation of cyclic adenosine monophosphate (cAMP) signaling in target epithelia. Herein, we show that after exposure to CT, human enteroids expressing O blood group exhibited marked increase in cAMP relative to cells derived from blood group A individuals. Likewise, using CRISPR/Cas9 engineering, a functional group O line (HT-29-A(-/-)) was generated from a parent group A HT-29 line. CT stimulated robust cAMP responses in HT-29-A(-/-) cells relative to HT-29 cells. These findings provide a direct molecular link between blood group O expression and differential cellular responses to CT, recapitulating clinical and epidemiologic observations.

Enterotoxigenic Escherichia coli blood group A interactions intensify diarrheal severity

Enterotoxigenic Escherichia coli (ETEC) infections are highly prevalent in developing countries, where clinical presentations range from asymptomatic colonization to severe cholera-like illness. The molecular basis for these varied presentations, which may involve strain-specific virulence features as well as host factors, has not been elucidated. We demonstrate that, when challenged with ETEC strain H10407, originally isolated from a case of cholera-like illness, blood group A human volunteers developed severe diarrhea more frequently than individuals from other blood groups. Interestingly, a diverse population of ETEC strains, including H10407, secrete the EtpA adhesin molecule. As many bacterial adhesins also agglutinate red blood cells, we combined the use of glycan arrays, biolayer inferometry, and noncanonical amino acid labeling with hemagglutination studies to demonstrate that EtpA is a dominant ETEC blood group A–specific lectin/hemagglutinin. Importantly, we have also shown that EtpA interacts specifically with glycans expressed on intestinal epithelial cells from blood group A individuals and that EtpA-mediated bacterial-host interactions accelerate bacterial adhesion and effective delivery of both the heat-labile and heat-stable toxins of ETEC. Collectively, these data provide additional insight into the complex molecular basis of severe ETEC diarrheal illness that may inform rational design of vaccines to protect those at highest risk.

Role of Kynurenine Pathway in Gastrointestinal Diseases

In the course of obtaining nutrients from the environment to sustain life, the gastrointestinal (GI) tract encounters a myriad diversity of foreign antigens, microbiota, and environmental toxins. To maintain homeostasis in this setting, the GI mucosal immune system employs a multitude of techniques to distinguish the helpful from harmful. Recent studies reveal amino acid metabolism pathways as an integral mechanism to maintaining homeostasis at the mucosal interface. Tryptophan metabolism along the kynurenine pathway in particular is now recognized as an important feature of both GI inflammation and malignancy. Indoleamine 2,3 dioxygenase (IDO1), the most prevalent of the tryptophan catabolizing enzymes, is expressed widely across tissues and cell types within the GI tract. This chapter will address the current knowledge of kynurenine pathway functions in the diseased GI tract and highlight the potential benefits of exploiting this pathway for therapeutic purposes.

Sa1977 Kynurenine Metabolic Pathway As a Rational Target for Colorectal Cancer: Defining Its Role in β-Catenin Signaling

Background: Enzymes that initiate tryptophan metabolism along the kynurenine pathway (KP) are commonly overexpressed in colorectal cancer (CRC). One of these enzymes, IDO1, has recently been shown to promote tumorigenesis in a mouse model of colitis associated cancer and promote nuclear translocation of β-catenin and proliferation in CRC cells. The current investigation is aimed at identifying the signaling pathways activated by various KP metabolites and determining if a similar effect exists in non-neoplastic primary colonic epithelial cells (CEC). Methods: In vitro models of human CRC using various CRC cell lines (HT29, HCT116 and SW480) as well as human colon primary CECs cultured in growth factor (Wnt3a, Noggin, R-Spondin) enriched media were used. All experiments were performed in serum and growth factor free culture media with or without the addition of KP metabolites such as Kynurenine (Kyn), Quinolinic Acid (QA) and Picolinic Acid (PA). Expression of mRNA and protein as well as their phosphorylation status was quantified at multiple time points, and cell proliferation was measured by MTT assay. Results: Addition of Kyn, QA and PA (100 μM) rapidly induced (5 min) phosphorylation of AKT (Ser473) and LRP (Ser1490), a required co-receptor for activating Wnt/β-catenin signaling. The KP metabolites also rapidly inactivated GSK-3β as indicated by phosphorylation at Ser9 and induced expression of nuclear β-catenin (P-Ser552). These changes were followed by an induction of CyclinD1 expression at 12-36 hours along with an increase in CRC cell proliferation after Kyn and QA application. However, with PA, no functional changes in proliferation were observed. In contrast to CRC cells, KP metabolites treatment of primary human CECs led to comparatively moderate and delayed changes in AKT, GSK-3β and βcatenin phosphorylation. Additionally, no change in LRP phosphorylation or effect on primary CEC proliferation was observed. Conclusions: KP metabolites induce rapid and reversible changes in the activation state of cellular machinery linked to CRC cell proliferation and neoplastic growth. While similarities exist between initial signaling events of KP metabolites, important distinctions were observed in their ultimate effects on proliferation of CRC and primary colonic epithelial cells. Together these observations highlight the role of the KP metabolites in β-catenin signaling and form the basis of future interrogation of this pathway for therapeutic targeting of colorectal cancer that may spare the normal epithelium.