Gen-Sheng Feng, PhD - US grants

Syp is a mammalian SH2-containing phosphotyrosine phosphatase (PTP) that was recently discovered during my post-doctoral studies, and is homologous to the Drosophila corkscrew gene product. Interestingly, this phosphatase appears to be a common target of receptor and cytoplasmic protein tyrosine kinases (PTKs), since Syp is able to physically bind to and is tyrosine phosphorylated by a number of ligand-activated growth factor receptors. Syp is constitutively phosphorylated on tyrosine in v-Src or Bcr-Abl transformed cells and forms a stable complex with Bcr-Abl and Grb2 in Bcr- Abl positive cells, suggesting a putative role of the PTP in neoplastic diseases. The PTP might also participate in insulin signaling since it binds to phosphorylated insulin receptor substrate 1 (IRS1) in adipocytes treated with insulin. While these biochemical evidences suggest that Syp is an important signaling component in normal and cancer cells, little is known about its biological significance. The objective of this project is therefore to uncover the Syp functions by a gene targeting approach. A Syp null mutation has been created in mouse embryonic stem (ES) cells and germ-line transmission of the mutant allele has been achieved from three independent ES cell clones. Preliminary results indicated that the homozygous Syp-/mutants die around day 8.5 of gestation. Our specific aims are: a). to analyze the phenotype of the Syp-/- mutant embryos, by focusing on the mesodermal development and patterning; b). to establish the Syp-deficient fibroblast cell lines from the knockout mice and to determine their signaling defects in response to mitogenic stimuli; c). to confirm the defective phenotype of the Syp- cell by rescue with the wild type Syp cDNA; d). to dissect the structure and functions of the PTP by transfecting the Syp- cells with Syp cDNAs mutated in its regulatory or catalytic domains and evaluating the ability of these mutants to rescue the Syp- phenotype; e). to examine the interaction of Syp with other signaling molecules in vivo by crossing the Syp knockout with various mutant mice. This study should provide fundamental insight into the molecular mechanism by which Syp acts in the control of signal transduction that regulates cell growth and differentiation. It will help us to understand the origin of cancer cells and to design new types of anti-cancer drugs.

PROJECT SUMMARY Blood cell development is orchestrated by the dynamic interplay between environmental cues and intrinsic genetic contents. However, the intracellular signaling mechanisms underlying regulation of hematopoiesis by extracellular signals are not well understood. The focus of this project is on deciphering Shp2 functions in control of hematopoietic cell development. Murine Shp2 is a cytoplasmic tyrosine phosphatase that was originally cloned by the applicant, and a main interest of this lab is to understand the fundamental cell signaling mechanisms involving tyrosine phosphatases. Work accomplished in the previous funding cycles has demonstrated a pivotal role of Shp2 in genesis of all blood cell lineages. This is the first example that a cytoplasmic tyrosine phosphatase promotes hematopoietic stem cell (HSC) commitment and differentiation. A general positive action of Shp2 in hematopoiesis defined by this lab has contributed significantly to most recent determination of a causal role of dominant active PTPN11/Shp2 mutants in child leukemia, leading to identification of PTPN11 as the first proto-oncogene that encodes a tyrosine phosphatase. The goal of this competing renewal application is to decipher biological functions of Shp2 in control of normal blood cell development in adults, using a conditional somatic gene deletion strategy. Our specific aims are: 1) to elucidate mechanisms by which Shp2 regulates adult HSC self- renewal, differentiation and homing, and also the HSC-niche interaction; 2) to determine Shp2 action and mechanism in T lymphocyte development and functions; 3) to determine Shp2 action and mechanism in B lymphocyte development and functions. We have generated new animal models and interesting preliminary data to support ALL three Aims. Completion of these studies will provide new insights into general mechanisms underlying coordinated regulation of HSC activities and specification/development of cell lineages in the hematopoietic compartment. Elucidation of Shp2 functions in normal hematopoiesis will also lead to a new paradigm that significantly advances our understanding of and fighting against leukemogenesis.

DESCRIPTION (adapted from applicant's abstract): A central question to be addressed in cell regulation is the biochemical mechanism by which many different kinds of signaling proteins and enzymes work in concert to mediate cellular responses to a specific extracellular stimulus. We are just beginning to appreciate that scaffold or adapter proteins may play important roles in signal relay from the plasma membrane to intracellular targets by aggregating a variety of proteins into specific signaling pathways or networks. We and others have recently identified a novel human scaffold protein, Gab2, that is closely related to Gab I (Grb2-associated binder 1) and Drosophila Dos (daughter of sevenless). Both Gabi and Gab2 contain a PH domain and mutliple potential tyrosine phosphorylation sites for SH2 proteins as well as proline-rich motifs for SH3 binding. Interestingly, we have found that Gabi and Gab2 exhibit reciprocal functions in coupling cytoplasmic-nuclear signaling, and that Gab2 acts to suppress the activity of the transcription factor Elk-i induced by oncogenic RasV 12 or epidermal growth factor, without down-regulating extracellular signal-regulated kinase (Erk) activity. We hypothesize that Gab2 acts to aggregate a unique set of enzymes and their specific substrates for signal relay, which represents a novel and unexplored pathway in intracellular signaling. The goal of this proposal is to dissect this pathway for the negative effect of Gab2 in signal transduction. This will be accomplished by: 1). identification of the structural domain in Gab2 involved in the negative regulatory role; 2). isolation and functional analysis of proteins that interact with Gab2 through the "negative effect domain"; 3). elucidation of the physiological consequence of the Gab2 interaction with its partners; and 4). determination of the biological function of Gab2 in vivo by generating a Gab2-deficient mouse model. This work will enable us to understand better the mechanism whereby the specificity of intracellular signaling is achieved through organization of multimolecular complexes (signalsomes) by scaffold proteins, such as Gab2, and will also aid in designing efficient pharmaceutical intervention of certain cellular disorders associated with heart failure, diabetes and malignant diseases.

Reversible tyrosine phosphorylation, a major biochemical event in cell regulation, is controlled by the opposing activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). However, relatively little is known about the mechanism for regulation and functions of PTPs, as compared to the extensive attention received for PTKs. The goal of this project is to determine the role of Shp-2, a PTP with two src-homology 2 (SH2) domains, in intracellular signal transduction. Work from this laboratory and others implicates the involvement of Shp-2 in different signaling pathways as a positive or a negative regulator in the control of cell growth, differentiation, migration and death. In particular, Shp-2 acts to promote growth factor stimulation of extracellular signal regulated kinase (ERK) activity. However, it remains a mystery how a PTP can act positively downstream of a receptor PTK to enhance the induction of ERK activity. Our most recent data present a fresh view that Shp-2 works in concert with Gab1 scaffold protein in a multimeric protein complex, in promoting the activation of the Ras-Raf-MEK-ERK cascade by epidermal growth factor. In this competitive renewal of the previously funded R29 grant, we propose to determine the biochemical basis for Shp-2 function in Ras-ERK activation by identifying its specific substrate(s). We will further investigate the physiological role of Shp-2 in cytoplasmic signaling, particularly in the modulation of information flow along the Ras pathway, and we will also determine the phosphatase-dependent and independent activities of the Shp-2 protein. Finally, we will define the biological function of Shp-2 in different cell types. Results from this study will allow us understand better how PTPs are regulated and how PTP activities are executed during intracellular signal relay in general.

Ultraviolet (UV) light is a complete carcinogen that can induce skin cancer. Among many effects, UV irradiation rapidly induces activation of the c-Jun NH2-terminal protein kinase (JNK) in mammalian cells and causes cell apoptosis. However, the molecular mechanism for JNK activation by UV exposure is poorly understood. We have found that the multisubstrate adapter Gab1 plays an essential role in the signaling cascade that is upregulated by UV irradiation. Gab1-deficient fibroblast cells are defective in the induction of JNK activity by UV light, and this defect is rescued by re-introduction of Gab1. We have also found that Gab1 is constitutively associated with JNK, and that UV irradiation of cells induces tyrosine phosphorylation of Gab 1. Although preliminary data suggest a possible role for the hepatocyte growth factor (HGF) receptor c-Met, the upstream kinase(s) that phosphorylates Gab 1 in response to UV remains to be unequivocally identified. We hypothesize that Gab1, a scaffold protein without catalytic activity, orchestrates a unique combination of enzymes and signaling proteins in the cellular responses to UV exposure, which represents a novel and unexplored signaling pathway. We will take a multidisciplinary approach to elucidate the molecular mechanism for Gab 1 activity in cellular responses to UV exposure. Our specific aims are: 1) to define the signaling events upstream of Gab1 in UV-irradiated cells; 2) to dissect the biochemical machinery linking Gabl with JNK; 3) to examine the role of Gab1 in the cellular responses to UV light; and 4) to determine the role of Gab1 in the development of UV-induced skin cancer in mice. The long term goal of this work is to identify targets for pharmaceutical intervention to block pathological processes elicited by UV light.

DESCRIPTION (provided by applicant): Obesity has become a widespread health problem reaching an epidemic level in our society, with the affected individuals having a high risk for diabetes, heart disease, hypertension and cancer. The adipocyte- derived hormone leptin regulates energy balance by controlling food intake and metabolism. The leptin receptor long form (LepRb, or ObRb), which mediates important leptin responses, is highly expressed in hypothalamic nuclei known to regulate body weight and appetite. It has become clear that most obese individuals develop leptin resistance with dramatically increased plasma leptin levels. The lack of mechanistic understanding of leptin signaling has prevented us from designing effective therapeutic intervention of leptin resistance. It was proposed that inactivation of the tyrosine phosphatase Shp2 might overcome obesity and enhance leptin sensitivity, based on in vitro data showing a negative effect of Shp2 on the Jak2/Stat3 pathway. We have demonstrated recently, however, that mice deficient for Shp2 expression in forebrain neurons developed early-onset obesity and leptin resistance, but were not hyperphagic. Thus, we believe the novel obese mouse model created in this laboratory offers an unprecedented opportunity to delineate the neuronal control mechanism of energy homeostasis. We hypothesize that Shp2 acts to promote hypothalamic leptin signal flow and that enhancing the Shp2 phosphatase activity may improve leptin sensitivity. For this project, we will elucidate the physiological role of Shp2 in signaling events downstream of LepRb. We will also determine the concerted functions of Shp2 and StatS, two important players downstream of LepRb, in relay of hypothalamic leptin signals. Finally, we will determine whether selective expression of a dominant active Shp2 mutant in the hypothalamus potentiates leptin signals and alleviates diet-induced obesity in mice. A new paradigm will likely emerge from the proposed studies, which will illuminate the molecular signaling mechanism for leptin action in control of energy homeostasis.

DESCRIPTION (provided by applicant): The goal of this project is to decipher molecular signaling mechanisms for control of insulin biosynthesis and secretion, and the immediate focus is on dissecting the function of Shp2 tyrosine phosphatase in orchestrating signaling cascades in ( cells. Although pancreatic ( cell failure is a critical component in all forms of diabetes, the molecular basis underlying ( cell dysfunction is poorly understood. This is mainly because that little is known for the cytoplasmic components mediating glucose and insulin signals in (-cells. Shp2 is a cytoplasmic tyrosine phosphatase with two SH2 domains that is implicated in regulation and coordination of signaling pathways. In particular, Shp2 has been shown to promote insulin-stimulated Erk activation in vitro, although the physiological significance of Shp2 function in insulin signaling is unclear. In recent studies, we have successfully created a conditional Shp2 knockout allele, Shp2flox, in mice, which allows us to investigate specific Shp2 functions in a specific cell type or tissue in vivo. We have generated mutant mice with Shp2 deleted in mature (-cells or in Pdx1+ pancreatic precursor cells, and will characterize these novel mouse models to test the working hypothesis that Shp2 acts to coordinate and control the strength of several signaling pathways in orchestrating insulin biosynthesis and secretion in (-cells. In complement with the gene targeting approach in vivo, we will also use siRNA- mediated gene knockdown technique to decipher the molecular signaling mechanisms in ( cells. Our specific aims are: 1) to determine the physiological role of Shp2 in (-cell function and glucose homeostasis;2) to dissect the molecular mechanism for Shp2 action in (-cells;and 3) to investigate the Shp2 function in pancreatic development and (-cell regeneration. Successful completion of the proposed experiments will fill in a gap in our knowledge for coordinated regulation of cytoplasmic signaling events in (-cells, and may even lead to a new paradigm on regulation of (-cell functions in glucose homeostasis and also in pathogenesis of type 2 diabetes. PUBLIC HEALTH RELEVANCE The goal of this project is to understand the intracellular signaling mechanisms for control of insulin biosynthesis in ( cells and also the molecular basis for ( cell failure in Type 2 diabetes, the most common metabolic disease in the world. Type 2 diabetes is characterized by defective pancreatic ( cell insulin release in response to glucose and impaired insulin action on its target tissues. (-cell failure is likely caused by inadequate expansion of the (-cell mass and/or failure of the (-cells to respond to glucose. Now, a crucial issue is to understand the molecular signaling scheme for (-cell sensing of glucose and secretion of insulin. We originally cloned murine Shp2 (Syp) as a protein tyrosine phosphatase that contains two Src homology 2 (SH2) domains (Feng et al., Science 1993). Shp2 is a cytoplasmic enzyme and has been implicated in regulation of signaling events triggered by growth factors, cytokines and hormones. In particular, Shp2 binds the insulin receptor substrate (IRS) and Grb2-associated binder (GAB) proteins. Several groups have demonstrated a positive effect of Shp2 in mediating insulin-stimulated Erk activation in vitro. While these studies suggest a putative role of Shp2 in insulin action, the physiological evidence in vivo is yet to be obtained. To determine Shp2 function in (-cells, we have generated mutant mouse lines with Shp2 selectively deleted in the pancreas or (-cells. These mutant mice displayed impaired glucose tolerance and defective insulin secretion and production. Our central working hypothesis is that Shp2 acts as a coordinator to fine-tune and integrate multiple signals for insulin biosynthesis and secretion in (-cells. Our preliminary experimental data suggest that Shp2 indeed has a critical role in promoting insulin biosynthesis and secretion in (-cells for control of glucose homeostasis. Successful completion of the proposed experiments in these three Aims will not only fill in a gap in our knowledge in molecular signaling events in (- cells, but may even refresh our current view on (-cell functions in glucose-stimulated insulin secretion and also on the etiology of type 2 diabetes.

DESCRIPTION (provided by applicant): The goal of this project is to delineate the molecular and cellular mechanisms that drive hepatocarcinogenesis, and the immediate focus is on deciphering dual roles of molecules in liver tumorigenesis. Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide, although the underlying mechanisms are poorly understood. In most recent experiments, we have found an unanticipated HCC- suppressing effect of Shp2, a tyrosine phosphatase previously known to be pro-oncogenic. Ablation of Shp2 promotes hepatic damage, inflammation, and spontaneous development of hepatocellular tumors in aged mice. Shp2 loss dramatically sensitizes the mice to chemical carcinogen-induced liver tumorigenesis. We have also found pro- and anti-oncogenic actions of Stat3 in HCC development. Consistent with our observations on Shp2 and Stat3, several other groups have identified HCC-inhibitory effects in genes previously identified as pro-tumorigenic. Although the underlying mechanisms remain to be elucidated, one common phenotype is the augmented HCC development following removal of a pro-survival molecule from hepatocytes. Of note, these mouse tumor models closely recapitulate many aspects of the pathogenic process in human HCCs, involving chronic hepatic injury-inflammation-compensatory proliferation-hepatocarcinogenesis. Therefore, we believe that common mechanisms are shared between the mouse models and human patients in HCC initiation and development. On this project, we will use the established animal models to dissect the molecular and cellular events in the liver at initial, early and late stages of hepatocarcinomas. Specifically, we propose the following 3 Aims: 1) to determine the nature of cell origin in HCC initiation and cell-cell communications driving tumor progression; 2) to determine the tumorigenic properties and aberrant signaling pathways of isolated hepatoma cells; and 3) to decipher the dual functions of Stat3 in HCC development. Success of this project will illustrate a general mechanism underlying HCC initiation and progression, and will also facilitate design of novel diagnostic and therapeutic strategies for hepatocarcinoma.

? DESCRIPTION (provided by applicant): It is well known that one gene mutation is not sufficient to trigger tumorigenesis, and therefore one urgent issue is to elucidate how various pro-oncogenic events work cooperatively in driving tumor initiation. Primary liver cancers, in particular hepatocellular carcinoma (HCC), are the 2nd leading cause of cancer-related deaths. Lack of understanding of the molecular pathogenesis for HCC has prevented us from designing mechanism-based therapeutic strategies. Mutations and silencing of Pten tumor suppressor have been detected in many liver cancer patients, but it is unclear how Pten deficiency interacts with other cell signaling disorders in promoting HCC development. Epidemiological analyses clearly indicate a strong association of HCC with chronic hepatitis B or C virus (HBV or HCV) infection in 80% of the diagnosed cases worldwide. However, the majority of hepatitis patients do NOT develop HCCs, indicating requirement of host cell defects in inducing hepato-oncogenesis. In most recent experiments, we found that additional deletion of Shp2 (a tyrosine phosphatase) in hepatocytes dramatically enhanced and accelerated HCC development induced by Pten loss. The Pten and Shp2 double knockout (DKO) mice developed HCCs at 100% penetrance in 7 months. Using this new compound mutant mouse line with defined kinetics of liver tumorigenesis and clear genetic defects, we will determine how Pten deficiency cooperates with additional tumor-promoting events in HCC development. We will also generate new mouse lines by crossing hepatocyte-specific Pten or Shp2 KO mouse with HBV transgenic mouse, to determine the dynamic interplay of viral infection with host cell defects in driving hepatopathogenesis. We will perform RNA-seq and bioinformatics analyses, to understand signaling pathways driving HCC initiation and also tumor cell-intrinsic as well as hepatic environmental signals required for tumorigenesis. We believe that in-depth molecular and cellular analyses of animal tumor models, using multidisciplinary tools, will be a most powerful approach to decipher the mechanisms underlying HCC initiation and progression.

? DESCRIPTION (provided by applicant): The goal of this project is to decipher cell type-specific crosstalk of signaling pathways in hematopoiesis and hematological diseases. This application is compelled by our new and surprising data indicating that two well-known signal regulators, Pten and Shp2, can work cooperatively or antagonistically in different blood cell lineages. Thus, the outcome of signal interplay in different cell types cannot be simply deduced from previously known functions for each molecule. Shp2 is a non-receptor tyrosine phosphatase possessing two SH2 domains that promotes Erk signaling, and dominantly activating mutations in PTPN11/Shp2 have leukemogenic effect. Ablating Shp2 suppressed hematopoietic stem cell (HSC) and progenitor cell proliferation and differentiation in mice, defining a positive role of Shp2 in hematopoiesis. In contrast, Pten (phosphatase and tensin homolog) is a tumor suppressor that negatively regulates the PI3K/Akt pathway and is frequently mutated in human leukemia. Targeted deletion of Pten resulted in dramatic expansion of short-term HSCs (ST-HSCs), excessive myeloid cell proliferation and development of leukemia. To test whether Pten and Shp2 have directly opposing functions in leukemogenesis, we generated conditional Pten and Shp2 double knockout (DKO) mice. Preliminary data suggest that additional deletion of Shp2 indeed suppressed excessive myeloid cell proliferation and leukemia induced by Pten-deficiency as expected, but surprisingly the Pten and Shp2 DKO mice developed lethal anemia. These results argue that although Shp2 and Pten have antagonistic roles in myeloid cells, these two regulators act in concert to promote red blood cell (RBC) development and maturation. This application seeks to elucidate the opposing as well as cooperating functions of Shp2 and Pten in blood cells. We will elucidate the molecular mechanisms underlying the cell type-specific crosstalk of Shp2- and Pten-modulated signals. The success of this project will not only advance our understanding of the interactive molecular pathways driving hematopoiesis in mammals, but also suggest better therapeutic strategies for leukemia and anemia.

Liver cancer, mainly hepatocellular carcinoma (HCC), has become a most deadly malignant disease worldwide. So far, pharmaceutic inhibition of major oncogenic pathways has achieved little therapeutic benefit to liver cancer patients. We believe this is due to under- appreciation of the complexity in mechanisms of hepato-oncogenesis. In recent experiments, we and others have identified paradoxically anti-oncogenic effects of classical oncogenic molecules, such as c-Met, EGFR, ?-catenin, Ikkb, Jnk, and Shp2, in the liver. Ablating these molecules in hepatocytes enhanced HCC induced by chemical carcinogen DEN. To test a theory that loss of the oncogenic molecules generates an oncogenic microenvironment that promotes DEN-induced HCC, we have established another mouse HCC model, by transfection of oncogenic ?-catenin (CAT), c-Met (MET) and PIK3CA (PIK), oncoproteins frequently detected in human HCCs. As expected, MET/CAT-driven HCC was aggravated in ?-catenin-deficient liver, due to tumor-promoting factors induced by ?-catenin removal. In contrast, Shp2 deletion dramatically suppressed HCC driven by MET/CAT or MET/PIK, despite a similar pro-tumorigenic environment in Shp2-deficient liver. Based on these novel unanticipated data, we propose a new hypothesis that although removal of Shp2 or ?-catenin generates cell-extrinsic tumorigenic factors in the hepatic environment, the endogenous Shp2 is indispensable for oncogenic signaling in hepatocytes. To test this hypothesis, we propose three specific aims on this project. Aim 1 is to determine the cell-intrinsic role of Shp2 in hepato-oncogenic signaling. Aim 2 is to determine the cell-autonomous effect of ?-catenin in liver tumorigenesis. Aim 3 is to search and identify cell-extrinsic factors induced by loss of the oncoproteins in hepatocytes. The results are expected to be instrumental for design of novel therapeutic strategies for liver cancer by inhibiting both cell-intrinsic oncogenic signals and the secondary environmental factors.

The goal of this project is to decipher how various liver injuries and disorders can accelerate and exacerbate development of hepatocellular carcinoma (HCC), one leading cause of cancer-related mortality worldwide. The immediate focus is on elucidating the tumorigenic liver damages generated ironically by loss of pro-oncogenic molecules in hepatocytes. In recent experiments, we found that deletion of Shp2/Ptpn11, previously known to be pro-oncogenic, aggravated HCC development induced by diethylnitrosamine (DEN) or by Pten deficiency and NASH. Consistently, several other groups reported that targeted removal of oncoproteins, such as c-Met, Ikkb, and b-catenin, from hepatocytes indeed aggravated HCC induced by DEN or other oncogenic drivers. However, the underlying mechanisms for the anti-oncogenic effect of these oncoproteins are unclear. Our hypothesis is that loss of the pro-oncogenic molecules generates a variety of tumor-promoting factors in the liver microenvironment, resulting in exacerbated tumorigenesis. Of note, these mouse tumor models closely recapitulate many aspects of the pathogenic process in liver cancer patients. Therefore, we believe that common mechanisms or oncogenic liver disorders are shared between the mouse models and human patients in tumor initiation and progression. On this project, we will pursue a comprehensive analysis of the molecular and cellular events that drive hepato-carcinogenesis using several mouse models. We propose the following three Specific Aims: 1) to search and identify tumorigenic factors in livers deficient for c-Met, Ikkb, Shp2 or b-catenin; 2) to determine the mutation profiles and HCC initiation in these mutants; and 3) to characterize DEN-induced and spontaneous tumorigenesis in liver deficient for both Shp2 and Ikkb. Success of this project will decipher common and distinctive mechanisms that drive liver tumorigenesis, and will facilitate design of novel and effective therapeutic strategies for liver cancer.

The goal of this project is to elucidate a new mechanism for compensatory hepatocyte proliferation under stress. Liver regeneration in mammals has been extensively interrogated, although it is unclear how hepatocytes with proliferative signaling defect strive to proliferate in response to hepatic damages. To address this question, we investigated cellular dynamics in regenerating livers with hepatocyte-specific deletion of Shp2, a signal transmitter of receptor tyrosine kinases. Following partial hepatectomy (PHx), a few Shp2-deficient hepatocytes grouped together, and proliferated in colony-like structures. These proliferating hepatocytes in colonies were characterized by high levels of CD133 expression but lack of other progenitor cell markers such as EpCAM, Sox9 or AFP. The CD133+ hepatocytes apparently communicated via tight cell-cell contact and CD133+ vesicles. The hepatocyte clusters emerged transiently in Shp2-deficient livers following PHx and disappeared quickly after completion of liver regeneration. CD133 has been known as a biomarker for stem/progenitor cells and also as a physical marker for cancer stem cells (CSCs), although its function and mechanism are poorly understood. Based on the preliminary results, we hypothesize that CD133-mediated intercellular communication is an inherent function with which cells strive to proliferate under proliferative signaling deficit, given that cells strive to survive via the process of autophagy under nutritional deficit. To test this hypothesis, we propose three Specific Aims. Aim 1 is to characterize the distinctive CD133+ hepatocyte proliferation pattern in livers deficient for different proliferative signaling molecules. Aim 2 is to determine the functional requirement of CD133 and CD133+ vesicles for compensatory hepatocyte proliferation. Aim 3 is to investigate this compensatory cell proliferation mechanism in drug resistance of cancer cells. Success of this project will elucidate a long-sought mechanism of CD133 function in normal and cancer cell proliferation under stress, independent of stemness, which we discovered unexpectedly in preliminary experiments.