Jayshree Mishra

Prospective of colon cancer treatments and scope for combinatorial approach to enhanced cancer cell apoptosis

Colorectal cancer is the leading cause of cancer-related mortality in the western world. It is also the third most common cancer diagnosed in both men and women in the United States with a recent estimate for new cases of colorectal cancer in the year 2012 being around 103,170. Various risk factors for colorectal cancer include life-style, diet, age, personal and family history, and racial and ethnic background. While a few cancers are certainly preventable but this does not hold true for colon cancer as it is often detected in its advanced stage and generally not diagnosed until symptoms become apparent. Despite the fact that several options are available for treating this cancer through surgery, chemotherapy, radiation therapy, immunotherapy, and nutritional-supplement therapy, but the success rates are not very encouraging when used alone where secondary complications appear in almost all these therapies. To maximize the therapeutic-effects in patients, combinatorial approaches are essential. In this review we have discussed the therapies previously and currently available to patients diagnosed with colorectal-cancer, focus on some recent developments in basic research that has shaded lights on new therapeutic-concepts utilizing macrophages/dendritic cells, natural killer cells, gene delivery, siRNA-, and microRNA-technology, and specific-targeting of tyrosine kinases that are either mutated or over-expressed in the cancerous cell to treat these cancer. Potential strategies are discussed where these concepts could be applied to the existing therapies under a comprehensive approach to enhance the therapeutic effects.

Molecular mechanism of interleukin-2-induced mucosal homeostasis.

Sustained damage to the mucosal lining in patients with inflammatory bowel disease (IBD) facilitates translocation of intestinal microbes to submucosal immune cells leading to chronic inflammation. Previously, we demonstrated the role of Jak3 in IL-2-induced intestinal epithelial cell (IEC) migration, one of the early events during intestinal wound repair. In this study, we demonstrate that IL-2 also plays a role in IEC homeostasis through concentration-dependent regulation of IEC proliferation and cell death. At lower concentrations (≤50 U/ml), IL-2 promoted proliferation, while at higher concentrations (100 U/ml), it promoted apoptosis. Activation by IL-2 led to tyrosine phosphorylation-dependent interactions between Jak3 and p52ShcA only at lower concentrations. Phosphatase SHP1 dephosphorylated IL-2-induced phosphorylated p52ShcA. Higher concentrations of IL-2 decreased the phosphorylation of Jak3 and p52ShcA, disrupted their interactions, redistributed Jak3 to the nucleus, and induced apoptosis in IEC. IL-2 also induced dose-dependent upregulation of p52shcA and downregulation of jak3-mRNA. Constitutive overexpression and mir-shRNA-mediated knockdown studies showed that expression of both Jak3 and p52ShcA were necessary for IL-2-induced proliferation of IEC. Doxycycline-regulated sh-RNA expression demonstrated that IL-2-induced downregulation of jak3-mRNA was responsible for higher IL-2-induced apoptosis in IEC. Collectively, these data demonstrate a novel mechanism of IL-2-induced mucosal homeostasis through posttranslational and transcriptional regulation of Jak3 and p52ShcA.

Role of Janus Kinase 3 in Mucosal Differentiation and Predisposition to Colitis

Background: Jak3 is a tyrosine kinase, and its role in mucosal differentiation is not known. Results: Jak3, expressed in colonic mucosa, was essential for differentiation, goblet cell surface localization, muc2 expression, and epithelial barrier functions. Conclusion: Jak3 plays a protective role against predisposition to colitis by promoting mucosal differentiation. Significance: Understanding mucosal functions of Jak3 has important implications for patients with inflammatory bowel disease. Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase expressed in both hematopoietic and nonhematopoietic cells. Previously, we characterized the functions of Jak3 in cytoskeletal remodeling, epithelial wound healing, and mucosal homeostasis. However, the role of Jak3 in mucosal differentiation and inflammatory bowel disease was not known. In this report, we characterize the role of Jak3 in mucosal differentiation, basal colonic inflammation, and predisposition toward colitis. Using the Jak3 knock-out (KO) mouse model, we show that Jak3 is expressed in colonic mucosa of mice, and the loss of mucosal expression of Jak3 resulted in reduced expression of differentiation markers for the cells of both enterocytic and secretory lineages. Jak3 KO mice showed reduced expression of colonic villin, carbonic anhydrase, secretory mucin muc2, and increased basal colonic inflammation reflected by increased levels of pro-inflammatory cytokines IL-6 and IL-17A in colon along with increased colonic myeloperoxidase activity. The inflammations in KO mice were associated with shortening of colon length, reduced cecum length, decreased crypt heights, and increased severity toward dextran sulfate sodium-induced colitis. In differentiated human colonic epithelial cells, Jak3 redistributed to basolateral surfaces and interacted with adherens junction (AJ) protein β-catenin. Jak3 expression in these cells was essential for AJ localization of β-catenin and maintenance of epithelial barrier functions. Collectively, these results demonstrate the essential role of Jak3 in the colon where it facilitated mucosal differentiation by promoting the expression of differentiation markers and enhanced colonic barrier functions through AJ localization of β-catenin.

Identification of Molecular Switch Regulating Interactions of Janus Kinase 3 with Cytoskeletal Proteins

Background: Jak3 is a tyrosine kinase, and the mechanism of Jak3 interactions with cytoskeletal proteins is not known. Results: Tyrosine autophosphorylation of SH2 domain of Jak3 facilitated the interactions between the Jak3-FERM domain and cytoskeletal proteins. Conclusion: Results demonstrate the molecular mechanism of interactions between Jak3 and cytoskeletal proteins. Significance: Understanding of Jak3 functions has important implications in transplant biology, epithelial wound repair, cancer metastasis, and immune cell migration. Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase expressed in both hematopoietic and nonhematopoietic cells. Although mutations that abrogate Jak3 functions cause different immunological disorders, its constitutive activation leads to various types of cancer. Previously, we demonstrated that Jak3 interacted with actin-binding protein villin, thereby facilitating cytoskeletal remodeling and wound repair. In this study, we characterize the structural determinants that regulate the interactions between Jak3 and cytoskeletal proteins of the villin/gelsolin family. Functional reconstitution of kinase activity by recombinant full-length (wt) Jak3 using Jak3-wt or villin/gelsolin-wt as substrate showed that Jak3 autophosphorylation was the rate-limiting step during interactions between Jak3 and cytoskeletal proteins. Determination of kinetic parameters showed that phosphorylated (P) Jak3-wt binds to P-villin-wt with a dissociation constant (Kd) of 23 nm and a Hills coefficient of 3.7. Pairwise binding between Jak3 mutants and P-villin-wt showed that the FERM domain of Jak3 was sufficient for binding to P-villin-wt with a Kd of 40.0 nm. However, the SH2 domain of Jak3 prevented P-villin-wt from binding to the FERM domain of nonphosphorylated protein. We demonstrate that the intramolecular interaction between the FERM and SH2 domains of nonphosphorylated Jak3 prevented Jak3 from binding to villin and that tyrosine autophosphorylation of Jak3 at the SH2 domain decreased these intramolecular interactions and facilitated binding of the FERM domain to villin. Thus we demonstrate the molecular mechanism of interactions between Jak3 and cytoskeletal proteins where tyrosine phosphorylation of the SH2 domain acted as an intramolecular switch for the interactions between Jak3 and cytoskeletal proteins.

Role of Janus Kinase 3 in Predisposition to Obesity-associated Metabolic Syndrome

Background: Jak3 is a tyrosine kinase and its role in metabolic syndrome is not known. Results: Jak3 was essential for intestinal mucosal tolerance through suppressed expression and activation of TLRs. Conclusion: Jak3 expression prevented development of obesity and metabolic syndrome. Significance: Understanding mucosal functions of Jak3 have important implications for patients with diabetes. Obesity, a worldwide epidemic, is a major risk factor for the development of metabolic syndrome (MetS) including diabetes and associated health complications. Recent studies indicate that chronic low-grade inflammation (CLGI) plays a key role in metabolic deterioration in the obese population. Previously, we reported that Jak3 was essential for mucosal differentiation and enhanced colonic barrier functions and its loss in mice resulted in basal CLGI and predisposition to DSS induced colitis. Since CLGI is associated with diabetes, obesity, and metabolic syndrome, present studies determined the role of Jak3 in development of such conditions. Our data show that loss of Jak3 resulted in increased body weight, basal systemic CLGI, compromised glycemic homeostasis, hyperinsulinemia, and early symptoms of liver steatosis. Lack of Jak3 also resulted in exaggerated symptoms of metabolic syndrome by western high-fat diet. Mechanistically, Jak3 was essential for reduced expression and activation of Toll-like receptors (TLRs) in murine intestinal mucosa and human intestinal epithelial cells where Jak3 interacted with and activated p85, the regulatory subunit of the PI3K, through tyrosine phosphorylation of adapter protein insulin receptor substrate (IRS1). These interactions resulted in activation of PI3K-Akt axis, which was essential for reduced TLR expression and TLR associated NFκB activation. Collectively, these results demonstrate the essential role of Jak3 in promoting mucosal tolerance through suppressed expression and limiting activation of TLRs thereby preventing intestinal and systemic CLGI and associated obesity and MetS.

Adapter Protein Shc Regulates Janus Kinase 3 Phosphorylation

Background: The trans-molecular mechanism of Jak3 regulation is not known. Results: p52ShcA regulated Jak3 dephosphorylation through direct interactions with both Jak3 and tyrosine phosphatases. Conclusion: Results demonstrate the molecular mechanism of deregulation of Jak3 activation. Significance: Understanding the regulation of Jak3 activation has implications in mucosal homeostasis, EMT, cancer metastasis, and immune cell migration. Although constitutive activation of Janus kinase 3 (Jak3) leads to different cancers, the mechanism of trans-molecular regulation of Jak3 activation is not known. Previously we reported that Jak3 interactions with adapter protein p52ShcA (Shc) facilitate mucosal homeostasis. In this study, we characterize the structural determinants that regulate the interactions between Jak3 and Shc and demonstrate the trans-molecular mechanism of regulation of Jak3 activation by Shc. We show that Jak3 autophosphorylation was the rate-limiting step during Jak3 trans-phosphorylation of Shc where Jak3 directly phosphorylated two tyrosine residues in Src homology 2 (SH2) domain and one tyrosine residue each in calponin homology 1 (CH1) domain and phosphotyrosine interaction domain (PID) of Shc. Direct interactions between mutants of Jak3 and Shc showed that although FERM domain of Jak3 was sufficient for binding to Shc, CH1 and PID domains of Shc were responsible for binding to Jak3. Functionally Jak3 was autophosphorylated under IL-2 stimulation in epithelial cells. However, Shc recruited tyrosine phosphatases SHP2 and PTP1B to Jak3 and thereby dephosphorylated Jak3. Thus we not only characterize Jak3 interaction with Shc, but also demonstrate the molecular mechanism of intracellular regulation of Jak3 activation where Jak3 interactions with Shc acted as regulators of Jak3 dephosphorylation through direct interactions of Shc with both Jak3 and tyrosine phosphatases.

Janus kinase 3 regulates adherens junctions and epithelial mesenchymal transition through β-catenin

Compromise in adherens junctions (AJs) is associated with several chronic inflammatory diseases. We reported previously that Janus kinase 3, a non-receptor tyrosine kinase, plays a crucial role in AJ formation through its interaction with β-catenin. In this report, we characterize the structural determinants responsible for Jak3 interactions with β-catenin and determine the functional implications of previously unknown tyrosine residues on β-catenin phosphorylated by Jak3. We demonstrate that Jak3 autophosphorylation was the rate-limiting step during Jak3 trans-phosphorylation of β-catenin, where Jak3 directly phosphorylated three tyrosine residues, viz. Tyr30, Tyr64, and Tyr86 in the N-terminal domain (NTD) of β-catenin. However, prior phosphorylation of β-catenin at Tyr654 was essential for further phosphorylation of β-catenin by Jak3. Interaction studies indicated that phosphorylated Jak3 bound to phosphorylated β-catenin with a dissociation constant of 0.28 μm, and although both the kinase and FERM (Band 4.1, ezrin, radixin, and moesin) domains of Jak3 interacted with β-catenin, the NTD domain of β-catenin facilitated its interactions with Jak3. Physiologically, Jak3-mediated phosphorylation of β-catenin suppressed EGF-mediated epithelial–mesenchymal transition and facilitated epithelial barrier functions by AJ localization of phosphorylated β-catenin through its interactions with α-catenin. Moreover, loss of Jak3-mediated phosphorylation sites in β-catenin abrogated its AJ localization and compromised epithelial barrier functions. Thus, we not only characterize Jak3 interaction with β-catenin but also demonstrate the mechanism of molecular interplay between AJ dynamics and EMT by Jak3-mediated NTD phosphorylation of β-catenin.

Structure and Function of Jak3- SH2 Domain

Janus kinase 3 (Jak3) is a non-receptor tyrosine kinase expressed in hematopoietic and non-hematopoietic cells. Though expression of Jak3 is necessary for adaptive immune functions, non-hematopoietic cell expression of Jak3 is also essential for the regulation of several physiological functions of gastrointestinal mucosa. Both in immune cells and intestinal epithelial cells (IEC), Jak3 regulates cell migration and mucosal restitution respectively by cytoskeletal remodeling through interactions with actin binding proteins (ABPs). Jak3 also regulates chronic inflammation of gastrointestinal tract through intestinal differentiation, mucosal homeostasis, regulation of proliferation and apoptosis, and maintenance of mucosa barrier functions. Molecularly, these functions are regulated through posttranslational and transcriptional regulation of Jak3, ABPs, and adapter proteins p52ShcA. For all these functions of Jak3, tyrosine phosphorylation of the SH2 domain plays a central role not only during common gamma chain induced auto-phosphorylation of Jak3 but also during phosphatases mediated deactivation of Jak3. In this chapter, we discuss the intramolecular and trans-molecular switches that regulate Jak3 interactions with other proteins and several aspects of its functions. Since SH2 domain plays a central role not only in Jak3 functions but the functions of all non-receptor tyrosine kinases, last section of the chapter discusses SH2 domain as drug target for the amelioration of different chronic inflammatory and other diseases.

Jak3 regulates IL-2 induced mucosal homeostasis through regulation of ShcA expression: P-092.

(group A), DSS induced colitis (group B), low does rebamipide-treated of 200mg/kg (group c), middle does rebamipide-treated of 400mg/kg (group D), high does rebamipide of 800mg/kg (group E). The mice in group A were given the distilled water. The mice in group B were given 5% DSS by oral administration from day 1 to day 7.The mice in group C,D,E were treated with different does of rebamipide from day 1 to day 7,and drank 5% DSS from day 2 to day 7. Diarrhea and bloody stool as well as colonic histology were observed. The level of SOD, MPO, GSH-PX, MDA were determined in colonic mucosa. Colonic mucosa permeability was determined by Evans blue. RESULTS: In mice treated with rebamipide, clinical (body weightloss, bloody diarrhea, reduced physical activity, and shortened colonic length) and histopathological (pathological lesion score) findings of DSS colitis were significantly less than in mice with DSS colitis not treated with rebamipide. The level of SOD and GSH-PX were high in rebamipide-treated groups, as the level of MPO and MDA were low in these groups. The Colonic mucosa permeability was improved in the rebamipide-treated groups. CONCLUSIONS: Rebamipide can improve colonic mucosa permeability. This effect is attributable to its inhibition of peroxidation in colonic mucosa.