Pomila Singh - US grants

hormone receptor; receptor binding; gastrointestinal hormones; peptide hormone; biomedical facility; inositol phosphates; cyclic AMP; calcium; cell membrane; growth factor; molecular weight; radiotracer; autoradiography;

The central hypothesis of our proposal is that gastrin, which is known to be trophic for normal gastrointestinal (GI) tissues, is also trophic for GI cancers & that gastrin receptors (GR) mediate the trophic effects of gastrin. The intracellular mechanism of gastrin mediated trophic effects is, however, unknown. Various hormones & hormone antagonists have been observed to modulate the growth stimulatory effects of gastrin on colon cancer cells in vivo, the mechanisms of which are largely unknown. We have recently established an in vitro system to study the trophic effects of gastrin directly on colon cancer cells. One of the major objectives of the present grant proposal is to study, 1) the intracellular mechanism of action of gastrin on cancer cells (role of cyclic nucleotides, inositol phosphates, protein kinases & growth factors will be examined), & 2) to examine the cellular & intracellular mechanism by which various steroid & peptide hormones regulate the growth of GI cancer cells. The hetero- regulation of the growth of mouse colon cancer (MC-26) cells by various hormones & hormone antagonists, in relation to concentration & binding kinetics of GR on cancer cells, has been observed by us in vivo, which could be due to either a direct or indirect interaction of the hormone/hormone antagonists with the cancer cells; indirect mechanisms involved will be investigated & the nature of direct interation defined. The physico-chemical characteristics of GR on normal tissues & GI cancers will be defined in order to determine whether the molecular characteristics of GR on cancer cells are similar or different from those on normal cells. A possible autocrine role of peptides & growth factors in the growth of MC- 26 cells will be additionally examined. In clinically relevant studies, GR levels in GI cancers from patients were found to correlate with the stage & differentiation of tumors, & may become useful in predicting the survival of patients. A second major objective of this grant proposal, therefore, is to isolate & purify GR from GI cancers & prepare mono-specific antibodies against GR which can be used for localization & identification of tumors positive for GR. We will continue to quantitate GR on freshly resected GI cancers & adjacent normal tissues from patients (using membrane binding assays & autoradiographic procedures), & correlate the receptor levels with clinical parameters including survival of the patients. The above studies will help us to further understand role of hormones in the growth of colon cancers & help define the cellular & intracellular mechanisms involved.

We have established direct and specific effects of LHRH on nuclear estradiol receptor (ERn) in the anterior pituitaries (AP) of female rats. ERn is known to increase dramatically during proestrus (PR), coincident with a steep rise in AP responsiveness to the action of LHRH for LH release, the mechanism of which may be related to the increasing ERn levels under the influence of rising LHRH and estradiol (E2) levels during PR. To test this hypothesis it is proposed to: 1) examine the role of in situ LHRH on the known increases in ERn during PR. Endogenous LHRH release will be blocked by phenobarbital at various time points during PR, E2 maintained at control levels, and the effect of 1/2-4 h LHRH block examined on ERn and LH levels; 2) determine interrelationship, if any, between ERn and corresponding LH levels within AP in response to LHRH. Changes in ERn levels as a dose and temporal response to LHRH and related peptide hormones will be monitored in vivo and in vitro (tissue and dispersed cell perifusion model) in relation to corresponding LH levels; 3) evaluation of the effects of LHRH in other target glands of E2 in comparison to its effects on AP, to determine if tissue-specific or non-specific mechanisms are involved. It is hoped that the above investigations will highlight the physiological significance of the effects of LHRH on ERn in AP. The molecular events, which precede the increase in ERn activity within AP, on exposure to LHRH, remains an intriguing question. The fourth objective, therefore, is to explore the various molecular mechanisms that could be mediating the effects of LHRH on ERn, including i) possible retention/stabilization of ERn by LHRH, ii) decreased/increased proteolytic activity, iii) activation/deactivation of ERn by way of possible phosphorylation/dephosphorylation, iv) evaluation of the presence of LHRH receptors on the nuclear membranes and possible involvement of secondary messengers (Ca++, cAMP, cGMP), and v) delineation of the sub-population of ERn being specifically affected by LHRH. The proposed studies should help in furthering our understanding of the mechanism of LHRH action in AP and provide further insight into the relationship between the actions of LHRH and E2 within the broader goal of synergizing the events of estrous cycle by way of various feedback loops, which may importantly include dynamic changes in the levels of ERn.

Colon cancer continues to be a leading cause of death in men and women. Other than surgical resection no other systemic or adjuvant therapy is available. Cumulative evidence in literature suggests that gastrins play a role in the proliferation of colon cancers. Colon cancer cells express the gastrin gene and a significant percent bind gastrins suggesting an autocrine role of gastrins. In Aim 1 we will test the hypothesis that gastrin gene products function as autocrine growth factors for colon cancer cells by using antisense strategies. Specific antibodies will be used to assess the functional role of autocrine vs endocrine gastrins. In Aim 2 we will test the hypothesis that src-like kinases mediate mitogenic/autocrine effects of gastrins. We will use microinjection technology to experimentally test the obligatory role of src-like kinases. Intestinal cells, overexpressing mutated src proteins will also be used. Recent studies suggest that novel receptors (GP-R), that bind both amidated and gly extended gastrins, mediate proliferative effects of gastrins on intestinal cells. Four other receptor-subtypes have been identified that mediate biological effects of gastrin on several target cells. In Aim 3 we will examine the role of the various receptor-subtypes, including GP-R, in mediating mitogenic effects of gastrins. Since the novel GP-R have yet to be purified and cloned, we will purify GP-R for purposes of peptide sequencing and cDNA cloning in Aim 4. If studies under Aim 1 confirm a functional role of autocrine/endocrine gastrins in the growth and tumorigenicity of colon cancer cells then one can begin to develop treatment strategies that include reduction of gastrin gene expression. If studies under Aim 2 confirm a mediatory role of src PTKs then one can further develop strategies that block the signaling pathway. If we are successful in purifying the novel GP-R (and confirm its role in mediating the mitogenic effects of gastrins) then one can develop systemic strategies to block the receptor mediated effects in future funding periods.

Role of IGFs in the transformation of several cancer cells is by now well established. Both IGFs (IGF-I and IGF-II) are potent mitogenic agents for several normal and neoplastic cells. IGF binding proteins (IGF-BPs), on the other hand are believed to modulate the mitogenic effects of IGFs. Within the past two years we and others have established that primary human colon cancers and established colon cancer cell lines express and secrete the mitogen, IGF-II, and the modulating proteins, IGF-BPs. It is, however, not known if IGFs and IGF-BPs play a major role in the growth and differentiation of colon cancer cells. It is speculated that the mitogenic potential of a colon cancer cell line will depend upon the relative expression of the mitogen and the modulator. The major hypothesis of this grant proposal therefore is that the relative expression and secretion of IGF-II and IGF-BPs dictates the growth differentiation potential of colon cancer cells. Our major strategy is to investigate the individual role of IGF-II and IGF-BPs in the growth and differentiation of colon cancers. Towards this goal we will use stable and inducible transfectants that either express the sense or the antisense form of the mitogen/modulatory proteins to confirm the relative role of IGFs and IGF-BPs in initiating or supporting the proliferative/differentiated state of the cells. Alternatively, we will measure the effect of IGF peptides, specific antibodies and antisense oligonucleotides on proliferation of a representative colon cancer cell line (Colo-205) (that does not differentiate in culture), and differentiation of a representative colon cancer cell line (CaCo2 cells) (that spontaneously differentiates in culture). In a second set of studies, the proliferation of colon cancer cells will be measured in vivo in specific mouse models, in the presence or absence of IGFs, IGF-BPs and IGF-receptor antagonists. Alternatively, growth and differentiation of cancer cells, transfected with vectors expressing the sense or antisense IGFs and BPs, will be measured in nude mice. These studies will help us to define the role of circulating vs autocrine (endogenous) IGFs and IGF- BPs in the growth and differentiation of colon cancers in vivo. At the end of these studies we expect to have defined the mitogenic/differentiation potential of IGFs in the presence of modulating IGF-BPs. The results of these studies will help us to move a step closer towards our overall goal of understanding the role of the IGF system in the proliferation and differentiation of neoplastic colonic mucosal cells, and provide the basic information for developing more appropriate, clinically relevant, protocols.

DESCRIPTION (provided by applicant): Colorectal cancer represents one of the leading causes of cancer-related mortality in the U.S. Hyperproliferation of colonic epithelium is now recognized as a risk factor for colorectal cancer. Factors that increase proliferation of colonic epithelium are believed to play a role in colon carcinogenesis. We now know that fully processed amidated gastrins (G17) and unprocessed non-amidated gastrins (gly-gastrin and progastrin, PG) are mitogenic for normal and cancerous intestinal cells. Our recent studies with mutant mice that over-express either PG or G17 or lack the functional gastrin gene (GAS-KO), suggest the novel possibility that PG is a co-carcinogen, while amidated gastrins inhibit rather than stimulate colon carcinogenesis. Differential effects of amidated (G17) vs non-amidated (PG) gastrins on colon carcinogenesis is a novel finding that will be further investigated in this application. The major hypothesis of this proposal is that PG increases the risk while G17 decreases the risk of colon carcinogenesis in response to chemical carcinogens. In Aim 1 we will confirm if PG is equally co-carcinogenic at physiological concentrations using more appropriate mutant mouse models. Possible dose-dependent effects of PG on colon carcinogenesis will be examined by treating GAS-KO mice with increasing concentrations of PG. In Aim 2 we will investigate the novel possibility that G17 reduces the risk of colon carcinogenesis, by either using mutant mouse models that over-express G17, treating GAS-KO mice and their wild type (WT) littermates with increasing concentrations of G17, or reducing endogenous levels of gastrins in WT mice. In Aim 3 we will investigate a role, if any, of gastrin receptor subtypes in mediating differential effects of PG vs G17. We recently reported a novel finding that PG may function as an anti-apoptotic factor for colon cancer cells and intestinal epithelial cells. In Aim 4, we will examine relative effects of PG vs G17 on apoptotic potential of colonic mucosal cells, that may help to explain differential effects between the two peptides. Results of the studies in the four Aims will allow us to confirm our novel hypotheses, and provide important mechanistic clues that will form the basis of studies in future funding periods. These results are expected to impact diagnosis, prognosis and clinical management of patients with colon cancer.

DESCRIPTION (provided by applicant): Colorectal cancer represents one of the leading causes of cancer-related mortality in the U.S. Hyperproliferation of colonic epithelium is now recognized as a risk factor for colorectal cancer. Factors that increase proliferation of colonic epithelium are believed to play a role in colon carcinogenesis. We now know that fully processed amidated gastrins (G17) and unprocessed non-amidated gastrins (gly-gastrin and progastrin, PG) are mitogenic for normal and cancerous intestinal cells. Our recent studies with mutant mice that over-express either PG or G17 or lack the functional gastrin gene (GAS-KO), suggest the novel possibility that PG is a co-carcinogen, while amidated gastrins inhibit rather than stimulate colon carcinogenesis. Differential effects of amidated (G17) vs non-amidated (PG) gastrins on colon carcinogenesis is a novel finding that will be further investigated in this application. The major hypothesis of this proposal is that PG increases the risk while G17 decreases the risk of colon carcinogenesis in response to chemical carcinogens. In Aim 1 we will confirm if PG is equally co-carcinogenic at physiological concentrations using more appropriate mutant mouse models. Possible dose-dependent effects of PG on colon carcinogenesis will be examined by treating GAS-KO mice with increasing concentrations of PG. In Aim 2 we will investigate the novel possibility that G17 reduces the risk of colon carcinogenesis, by either using mutant mouse models that over-express G17, treating GAS-KO mice and their wild type (WT) littermates with increasing concentrations of G17, or reducing endogenous levels of gastrins in WT mice. In Aim 3 we will investigate a role, if any, of gastrin receptor subtypes in mediating differential effects of PG vs G17. We recently reported a novel finding that PG may function as an anti-apoptotic factor for colon cancer cells and intestinal epithelial cells. In Aim 4, we will examine relative effects of PG vs G17 on apoptotic potential of colonic mucosal cells, that may help to explain differential effects between the two peptides. Results of the studies in the four Aims will allow us to confirm our novel hypotheses, and provide important mechanistic clues that will form the basis of studies in future funding periods. These results are expected to impact diagnosis, prognosis and clinical management of patients with colon cancer.

[unreadable] DESCRIPTION (provided by applicant): We now know that progastrins (PG) and insulin-like growth factors (IGFs) exert potent proliferative and anti- apoptotic effects on colon cancer (CRC) cells and can potentially function as co-carcinogens during all phases of colorectal carcinogenesis. Dietary agents, such as curcumin, inhibit the growth of CRC cells and inhibit colon carcinogenesis at both the pre-malignant and post-malignant stages of the disease. It is, however, not known if curcumin can inhibit the growth factor effects of autocrine and/or endocrine PG and IGF-II during colon carcinogenesis. Our preliminary studies suggest that the inhibitory effects of curcumin are attenuated in the presence of growth factors, such as IGF-II and PG; surprisingly the degree of attenuation was significantly higher in the presence of IGF-II than in the presence of PG. Therefore, the major hypothesis of our grant proposal is that the inhibitory efficacy of curcumin will be dictated by either the circulating growth factor profile of animal models or by the autocrine growth factors in CRC cells. To address this hypothesis, in Aim 1, we will develop isogenic cell lines that either express PG or IGF-II, and examine the pro-apoptotic and anti-proliferative potency of curcumin on these cells. Inhibitory effects of curcumin on intact colonic crypt cells, prepared from either transgenic mice over-expressing PG or IGFs or prepared from wild type mice, will also be examined. In Aim 2, we will examine dose-dependent effects of dietary curcumin against all phases of colon carcinogenesis in transgenic mice over-expressing PG or IGF-II, either in the circulation or locally within the intestinal mucosa. The intracellular pathways that mediate anti-apoptotic vs proliferative effects of PG and IGFs, on CRC and intestinal epithelial (IEC) cells, are being currently examined in our laboratory. The mechanisms by which curcumin inhibits the growth factor effects of IGFs and PG are unknown at the present time. In Aim 3 we will examine the effect of curcumin on the phosphorylation/dephosphorylation of several kinases/phosphatases that are activated in response to PG or IGF-II in isogenic CRC and IEC cells. The above experiments will allow us to learn for the first time the relative effectiveness of curcumin on colon carcinogenesis in the presence of growth factors relevant to the etiology of the disease. The results of these studies are expected to help in developing mechanism-based strategies for preventative/treatment protocols for CRC using curcumin like agents. [unreadable] [unreadable]

In the past funding period we confirmed our major hypothesis that progastrin peptides (PG) exert proliferative/anti-apoptotic/co-carcinogenic effects on intestinal/colonic epithelial cell lines, in vitro and in vivo, and increase the risk of colon carcinogenesis in response to azoxymethane (AOM). Additionally, we made the novel discovery that Annexin II (ANXII) functions as a high affinity receptor for PG and is required for mediating growth factor effects of PG on target cells. An important role of [unreadable]-catenin, c-Src, PI3K/Akt, MAPK/ERK and NFkB in mediating growth factor effects of PG on normal and neoplastic intestinal epithelial cells was also identified. Based on these findings, the major hypothesis of our current grant proposal is that ANXII facilitates the activation of one or more of the above indicated kinases/transcription factors in response to PG. Experiments in Aims 1 and 2 will examine this hypothesis. In Aim 1, PG responsive cell lines, altered for ANX-II expression will be used, as an in vitro model of investigation. For the in vivo studies, we will either use transgenic mice over-expressing PG (Fabp-PG), before or after modulation of ANXII expression. We will either use ANX-II knock out mice, or use specific siRNA for down regulating ANXII expression. In preliminary studies co-localization of PG with ANXII in situ, in cells over-expressing autocrine PG or responsive to exogenous PG was observed. Therefore in Aim 2, pathways mediating intracellular translocation of ANXII/PG, in response to binding of PG to extracellular ANXII (and its functional significance) will be examined;fluorescence based detection techniques and biochemical analysis will be used. In Aim 3 the clinical relevance of our findings will be examined in a pilot correlative study with samples obtained from patients, consented at the time of colonoscopy or at the time of surgical resection of colonic tumors, at different stages of colon carcinogenesis. Our initial studies strongly implicate PG peptides in the progression of the colon cancer disease. Therefore, understanding the role of ANX-II and the various signaling molecules in mediating the actions of PG are clinically important goals. Data obtained from the above studies will facilitate the development of more effective strategies for targeting the actions of PG for preventative and treatment purposes.

DESCRIPTION (provided by applicant): Cumulative evidence strongly suggests that precursor peptides such as progastrins (PG) exert mitogenic/co-carcinogenic effects on target cells and increase the risk of tumorigenesis. Many tumors express PG, and down-regulation of gastrin gene/PG expression attenuates growth of PG-dependent cancer cells. We recently discovered that extra-cellular membrane-associated annexin A2 (ANXA2) represents a novel receptor for PG. At the same time, ANXA2 expression has been reported to be critical for maintaining the growth of epithelial cancer cells. More recently, we discovered that over-expression of PG in HEK-293 cells significantly increased stem cell populations, positive for DCAMKL+1/Lgr5/CD44, and surprisingly resulted in tumorigenic transformation of the cells. In an important related study we reported potent inhibition of PG stimulated growth on cancer cells via the inhibition of critical signaling pathways. Our preliminary studies suggest that curcumin significantly reduces expression of ANXA2 and stem cell markers; intriguingly opposite effects of curcumin were measured on DCAMKL+1 expression in transformed versus non-transformed epithelial cells, Based on these novel findings we will test the hypothesis that 'chemo/dietary preventive agents differentially modulate growth factor-mediated expression of ANXA2 and stem cell markers in non-tumorigenic versus tumorigenic epithelial cells'. Towards this goal, we will focus on examining the effects of curcumin. In Aim 1, relative inhibitory efficacy of curcumin on growth of non-transformed/transformed epithelial cells, enriched for ANXA2/stem cell markers will be examined using in vitro and in vivo models of investigation. Regulatory effects of curcumin on expression of ANXA2/stem cell markers in non-tumorigenic/tumorigenic cells will be examined in relation to growth inhibitory effects of curcumin. Target-specific siRNA/shRNA against stem cell markers will be used to augment inhibitory effects of curcumin. Results of these studies will likely have therapeutic implications. In Aim 2 mechanisms by which curcumin reduces stimulatory effects of PG on the expression of ANXA2/stem cell markers will be examined in transformed/non-transformed cell lines at protein and RNA levels; inhibitory effects at the transcriptional level will be examined in promoter-reporter assays. These experiments will expand our understanding of the interplay between growth factors, curcumin and stem cell markers. In Aim 3, dose-dependent effects of dietary curcumin against initiation, promotion, and progression phases of colon carcinogenesis in transgenic mice over-expressing PG, in relation to efects on ANXA2/stem cell markers will be examined. Pre-neoplastic and neoplastic growths and surrounding normal mucosa wil be analyzed for relative expression of ANXA2/stem cell markers in relation to activation of 2- catenin/NF:Bp65 and other transcriptional factors. Results of these studies are expected to help us develop mechanism-based strategies for prevention and/or treatment of cancers in patients, positive for circulating PG (growth factors) and/or ANXA2 expressing tumors.