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The α-subunit of eukaryotic initiation factor 2 (eIF2α) is a key translation regulator that plays an important role in cellular stress responses. In the present study, we investigated how eIF2α phosphorylation can be regulated by a tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) and how such regulation is used by PTEN-deficient hepatocytes to adapt and cope with oxidative stress. We found that eIF2α was hyperphosphorylated when Pten was deleted, and this process was AKT dependent. Consistent with this finding, we found that the Pten-null cells developed resistance to oxidative glutamate and H2O2-induced cellular toxicity. We showed that the messenger level of CReP (constitutive repressor of eIF2α phosphorylation), a constitutive phosphatase of eIF2α, was downregulated in Pten-null hepatocytes, providing a possible mechanism through which PTEN/AKT pathway regulates eIF2α phosphorylation. Ectopic expression of CReP restored the sensitivity of the Pten mutant hepatocytes to oxidative stress, confirming the functional significance of the downregulated CReP and upregulated phospho-eIF2α in the resistance of Pten mutant hepatocytes to cellular stress. In summary, our study suggested a novel role of PTEN in regulating stress response through modulating the CReP/eIF2α pathway. Mol Cancer Res; 9(12); 1708–17. ©2011 AACR.

Liver malignancies consist of hepatocellular carcinoma (HCC) with the highest occurrence, intrahepatic cholangiocarcinoma (iCCA), and serval rare subtypes, which is the third lethal cause among all cancer types worldwide. PTEN is a well-known tumor suppressor gene, liver-specific loss of PTEN leads to the development of liver tumors from tumor-initiating cells (TICs). A mouse model that specifically mutant PTEN in hepatocytes (PM mice, PTENloxP/loxP; Alb-Cre+) has been used to mimic the natural progression of liver malignancy and study the mechanism of liver tumorigenesis. AKT, also known as protein kinase B, is a downstream kinase that is negatively regulated by PTEN. PTEN loss will unequivocally result in AKT phosphorylation and activation of the AKT pathway. In this study, we explored the role of AKT2, the most abundant liver isoform of AKT in the PTEN loss-driven liver malignancy by generating a new double mutant mouse model (DM mice, PTENloxP/loxP; AKT2loxP/loxP; Alb-Cre+). Our data demonstrated that only PM mice developed tumors starting from a 6-month age. A moderate reactive duct/oval cell accumulation phenotype is observed in the PM livers with Von Meyenbury complex (VMC) formation. And both HCC and iCCA phenotypes are observed following steatosis development in PM mice. AKT2 loss arrested tumor development at the pre-malignant stage. The DM mice also developed VMC condition with minimum steatosis starting from 9-month age and some of them manifest an advanced stage called polycystic liver disease. No tumors are observed in these mice up to 16 months of age. Our preliminary data showed that the deletion of AKT2 attenuated the accumulation of TICs marked by Sox9 suggesting a potential role of SOX9 in the regulation of PTEN-driven tumorigenesis. In summary, our result shows that AKT2 is a determining factor in PTEN loss-induced liver malignancy. Citation Format: Qi Tang, LIna He, Chien-Yu Chen, Shefali Chopra, Bangyan L. Stiles. AKT2 as the determining factor for PTEN loss-induced liver malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2583.

Liver carcinoma is the 6th most prevalent cancer worldwide in 2020. Moreover, it is the 3rd leading cause of cancer related deaths. In addition to the genomic and transcriptomic heterogeneity of liver tumor cells which is recognized as a major driver in liver cancer progression, the liver immune system is also fundamental to liver carcinogenesis and presents a promising target for therapy. The liver immune response is orchestrated by cytokines and chemokines. Recent studies suggest that chemokines not only recruit immune cells but also regulate various liver functions. In partial hepatectomy, CXCL2 has been shown to promote hepatocyte proliferation. CXCL1, 2, 5 and 8 can induce endothelial cells chemotaxis to promote angiogenesis through binding to CXCR2. These diverse functions suggest that chemokines could play multifaceted roles in liver cancer development. However, chemokines that are commonly associated with liver cancer is still unknown.We analyzed HCC patient data from the GEO database, and we categorized the datasets based on HCC etiologies including HBV, HCV, alcoholic and NASH. We identified CXCL5 as the only chemokine consistently upregulated in HCC with different etiologies compared to healthy or cirrhotic livers. Immunohistochemistry (IHC) analysis reveals that CXCL5 was produced by immune cells but not tumor cells in human HCC tissues. To further study HCC associated CXCL5 expression, the liver-specific Pten deletion mouse model (PM mice) that recapitulates NAFLD-NASH-HCC progression was used. A gradual increase of hepatic CXCL5 expression is observed during HCC development, reaching nearly 100-fold upregulation of CXCL5 mRNA expression in 12-month-old PM mice livers carrying tumors. Examination of liver immune cell populations showed that macrophages were significantly enriched in Pten deleted livers bearing tumors than wild type livers without tumors. Flow cytometry and IHC analysis further identified Kupffer cells (KCs), the liver resident macrophages as the source of CXCL5 in tumor bearing livers using these mice. Since increased LPS is a prominent feature in most chronic liver diseases, we isolated and treated mouse KCs with LPS and found that LPS treatment robustly increased CXCL5 expression by nearly 20-fold. Interestingly, neither murine macrophage cell lines nor primary peritoneal macrophages displayed induced CXCL5 expression in response to LPS. These data suggest that induction of CXCL5 in KCs is likely a unique function of the KCs but not of other macrophages. To explore the function of CXCL5 in HCC development, we treated mouse hepatocytes and HCC cells with CXCL5 and showed that CXCL5 induces the proliferation of these cells. This effect is further blocked by the inhibition of CXCR2, the receptor of CXCL5, demonstrating the specificity for CXCL5 mediated effects. Together we show here for the first time that CXCL5 expression is a unique property of Kupffer cells and the induction of CXCL5 promotes HCC progression. Citation Format: Taojian Tu, Handan Hong, Lina He, Mario Alba, Curtis T. Okamoto, Bangyan L. Stiles. Kupffer cells secrete CXCL5 to promote liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1350.

Liver cancer is hallmarked with chronic inflammation resulting from underlying liver diseases such as liver steatosis. Steatotic liver injury recruits inflammatory cells and establishes the tumor immune environment that can promote the development of liver cancer. Previously, we showed that macrophages produce growth factors to promote liver cancer growth in a genetic model where steatosis and liver tumors were induced by the deletion of tumor suppressor Pten (phosphatase and tensin homologue deleted on chromosome 10). In this model, we showed that attenuating steatosis and depletion of macrophages leads to inhibition of liver cancer development. To investigate the recruitment and functions of immune cells in liver cancer, we analyzed the expression profiles of these tumors as well as tumors from other mouse models together with human liver samples. Our analysis of the inflammatory cytokines identified one chemokine that was commonly upregulated in all tumor model and attenuated in non-tumor models. CXCL5 is a member of the neutrophil-activating chemokines. In the Pten deleted liver tumors, CXCL5 is significantly upregulated whereas its expression is inhibited when steatosis is attenuated via caloric restriction or deletion of a metabolic kinase, Akt2. We explored the cell types that may produce CXCL5. To our surprise, the upregulation of CXCL5 is concurrent with increases in macrophage but not neutrophil populations. We further observed significant upregulation of CXCL5 mRNA expression in Kupffer cells, the liver resident macrophages isolated from the Pten deleted mice. We explored the hypothesis that macrophages secrets CXCL5 to establish the tumor environment and promote tumor growth. Our data showed that CXCL5 treatment increased mouse hepatocytes and human HepG2 cells proliferation. This effect is blocked by inhibition of CXCR2, the receptor for CXCL5. Additionally, we explored the mechanisms of CXCL5 upregulation in Kupffer cells and discovered that lipopolysaccharide (LPS) induces the expression of CXCL5 by nearly 20-fold in Kupffer cells. This is in contrast with the lack of induction in murine macrophage cell lines and primary peritoneal macrophages. This unique response of Kupffer cells suggests that the source of CXCL5 in the Pten deletion mouse model is likely Kupffer cells. This data is collaborated by bioinformatic analysis of datasets where steatotic liver injury is induced by diet feeding. Furthermore, analysis of patient proteomic data detected CXCL5 as the primary chemokine that is induced in tumors among other neutrophil-activating peptides. In summary, our data identified CXCL5 as a novel chemokine produced by Kupffer cells that plays key roles in steatosis driven liver cancer development. Chronic liver diseases such as steatohepatitis can establish tumorigenesis environment through accumulation of LPS stimulated CXCL5 release from Kupffer cells. Citation Format: Bangyan L. Stiles, Taojian Tu, Lina He, Mario Alba. Steatosis promote liver cancer development by inducing chemokine production from Kupffer cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P048.

Liver cancer is an extremely deadly disease ranked as the third most common cancer and the second leading cause of male cancer-related death worldwide. Among primary liver cancers, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are the two most frequent subtypes, accounting for 85-95% of the total liver cancer cases, and both types of liver cancer have been found to originate from the expansion and differentiation of tumor initiating cells (TICs). Thus, it is critically important to understand mechanisms that regulate liver TICs activation. By utilizing a Pten(loxp/loxp); Albumin-Cre+ mouse liver cancer model (L-PKO), our previous studies have confirmed the activation of TICs during liver tumorigenesis. We also found that SOX9 was expressed in both HCC and CC, suggesting that SOX9 might serve as a liver TICs marker. We thus used the Pten(loxp/loxp); SOX9-CreER+ mouse model (TIC-PKO) to study the role of PTEN in TICs during liver tumorigenesis. Liver tumor development was observed in TIC-PKO mice at the age of 12 months old, and immunohistochemical analysis revealed that the tumors were heterogeneous, indicating TICs were involved in the tumorigenesis process. In addition, treatment of DDC, which is a liver toxin and thus causes hepatocyte death, dramatically increased the incidence and led to the early onset of liver tumorigenesis in the mutant but not the control mice, suggesting that liver injury served as an essential microenvironmental niche in promoting liver tumor development. Together, our observations suggested that the PTEN signaling is critical for TICs regulation and Pten deletion synergizes with liver injury to drive both TICs activation and liver tumorigenesis. Citation Format: Ni Zeng, Anketse Kassa, Janel Kopp, Lina He, Maike Sander, Bangyan Stiles. Pten deletion in SOX9+ cells synergizes with hepatic injury to drive tumor development in mouse liver. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-145. doi:10.1158/1538-7445.AM2015-LB-145

Estrogen-related receptor α (ERRα), an orphan nuclear receptor, is a prominent regulator of mitochondrial biogenesis and function. We previously showed that liver-specific PTEN deletion and the subsequent activation of PI3K/AKT pathway lead to tumorigenesis. Here we show that Pten-null hepatocytes established from Pten-null mice exhibit dramatically increased ERRα expression, accompanied by elevated mitochondrial mass, cellular oxygen consumption and reactive oxygen species production. Silencing ERRα by siRNA in Pten-null hepatocytes not only suppresses mitochondrial biogenesis and respiration but also attenuates hepatocytes' proliferation and colony-forming potential. Cohort analyses of clinical datasets from liver tissues revealed a negative correlation between expressions of PTEN and ERRα in patients with liver cancer. Therefore, targeting ERRα might provide therapeutic implication for treating tumors bearing abnormal PTEN-ERRα signaling. ERRα exerts transcriptional activity towards its target genes via binding to consensus DNA sequences named estrogen-related receptor α response elements (ERRE). The DNA-binding activity of ERRα could be modulated by polyamides, a class of synthetic amino acid oligomers capable of binding to DNA minor groove with sequence specificity. By designing and employing polyamide that specifically targets ERRE in Pten-null hepatocytes, we found the mRNA levels of two ERRα target genes, cytochrome c (cyt c) and ATP synthase β (ATP5b), are reduced. Biochemical gel-shift assay showed the binding between ERRα and ERRE is significantly diminished by polyamides in vitro. Moreover, polyamide treatment in hepatocytes reduced mitochondrial respiration and colony-forming ability. In conclusion, our findings show that ERRα is significantly induced via PTEN-AKT signaling pathway and in turn activates mitochondrial bioenergetics, which likely provides growth and survival advantages. The crucial role in mitochondrial metabolism and the association with PTEN signaling make ERRα a useful target for cancer therapy. Synthetic polyamide that disrupts transcription factor-DNA interaction can be utilized to modulate ERRα's activity, highlighting its potential as a future therapeutic target. Citation Format: Yang Li, John Gallagher, Bogdan Olenyuk, Bangyan Stiles, Ankeste Kassa. Targeting estrogen-related receptor alpha using pyrrole-imidazole polyamide. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2430. doi:10.1158/1538-7445.AM2014-2430

Liver cancer is one of the most aggressive malignancies with a five-year survival rate less than 10%. There is thus an urgent unmet need to understand the process of liver tumorigenesis in order to develop early diagnosis and effective therapeutic treatments. Clinical studies have suggested that tumor initiating cell (TIC) activation is observed in over 70% of liver cancer samples and is closely linked with poor survival outcome after treatment. Consistent with these observations, our previous studies utilizing a Pten (loxp/loxp); Albumin-Cre+ mouse liver cancer model also confirmed the expansion of TICs during liver tumorigenesis. However, how TICs are activated and what mechanisms contribute to liver tumor development remains unclear. Here, we studied the role of tumor suppressor PTEN in TICs using the Pten (loxp/loxp); SOX9-CreER+ mouse model. Lineage tracing results indicated that in the liver SOX9+ cells possess the progenitor cell characteristics and give rise to bile duct cells and peri-portal hepatocytes later on. Pten deletion in SOX9+ cells at 4 weeks of age leads to expansion of both CK+ duct cells and HNF4+ hepatocytes in the peri-portal areas. Interestingly, we also observed tumor development at 12 months old mutant mice and close examinations revealed that the tumors are heterogeneous, indicating the contribution of TICs during this process. Together, our study suggested that the PTEN signaling is critical for TICs regulation and that TICs activation plays a key role in liver tumorigenesis. Citation Format: Ni Zeng, Janel Kopp, Lina He, Maike Sander, Bangyan Stiles. Pten deletion in SOX9+ cells leads to tumor initiating cell expansion and tumor development in mouse liver. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3900. doi:10.1158/1538-7445.AM2014-3900

Liver cancer is one of the most common solid tumors with poor prognosis and high mortality. Mutation or deletion of the tumor suppressor PTEN is strongly correlated with human liver cancer. Glucose-regulated protein 94 (GRP94) is a major endoplasmic reticulum (ER) chaperone protein, but its in vivo function is still emerging. To study the role of GRP94 in maintaining liver homeostasis and tumor development, we created two liver-specific knockout mouse models with the deletion of Grp94 alone, or in combination with Pten, using the albumin-cre system. We demonstrated that while deletion of GRP94 in the liver led to hyperproliferation of liver progenitor cells, deletion of both GRP94 and PTEN accelerated development of liver tumors, including both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) suggestive of progenitor cell origin. Furthermore, at the premalignant stage, we observed disturbance of cell adhesion proteins but no detectable overt liver injury. We further discovered that when GRP94 was deleted in PTEN-null livers, ERK was selectively activated, suggesting that this could be a potential molecular mechanism for enhanced liver tumorigenesis in this mouse model. Conclusion: We identify GRP94 as a novel regulator of cell adhesion, liver homeostasis, and tumorigenesis. Citation Format: Wan-Ting Chen, Chun-Chih Tseng, Kyle Pfaffenbach, Gary Kanel, Biquan Luo, Bangyan L. Stiles, Amy S. Lee. Liver-specific knockout of GRP94 in mice disrupts cell adhesion, activates liver progenitor cells, and accelerates liver tumorigenesis. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B09.

Glucose-regulated protein 78 (GRP78), a molecular chaperone widely elevated in human cancers, is critical for endoplasmic reticulum (ER) protein folding, stress signaling, and PI3K/AKT activation. Genetic knockout models of GRP78 revealed that GRP78 maintains homeostasis of metabolic organs, including liver, pancreas, and adipose tissues. Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are the most common liver cancers. There is a lack of effective therapeutics for HCC and CC, highlighting the need to further understand liver tumorigenic mechanisms. Phosphatase and tensin homolog (PTEN), a tumor suppressor that antagonizes the PI3K/AKT pathway, is inactivated in a wide range of tumors, including 40-50% of human liver cancers. To elucidate the role of GRP78 in liver cancer, we created a mouse model with biallelic liver-specific deletion of Pten and Grp78 mediated by Albumin-Cre-recombinase (cPf/f78f/f). Interestingly, in contrast to PTEN, deletion of GRP78 was progressive but incomplete. At 3 months cPf/f78f/f livers showed hepatomegaly, activation of lipogenic genes, exacerbated steatosis, and liver injury, implying that GRP78 protects the liver against PTEN-null mediated pathogenesis. Furthermore, in response to liver injury, we observed increased proliferation and expansion of bile duct and liver progenitor cells in cPf/f78f/f livers. Strikingly, bile duct cells in cPf/f78f/f livers maintained wild-type GRP78 level while adjacent areas showed GRP78 reduction. Analysis of signaling pathways revealed selective c-Jun N-terminal protein kinase activation, β-catenin downregulation, along with platelet-derived growth factor receptor α upregulation, which was unique to cPf/f78f/f livers at 6 months. Development of both HCC and CC were accelerated and evident in cPf/f78f/f livers at 8-9 months, coinciding with intense GRP78 expression in the cancer lesions, and GRP78 expression in adjacent normal areas reverted back to the WT level. In contrast, livers of Albumin-Cre; Grp78f/f (c78f/f) mice showed no malignancy even at 14 months. These studies reveal GRP78 is a novel regulator for PTEN-loss mediated liver injury and cancer progression. Citation Format: Wan-Ting Chen, Genyuan Zhu, Kyle Pfaffenbach, Gary Kanel, Bangyan Stiles, Amy S. Lee. GRP78 as a regulator of liver steatosis and cancer progression mediated by loss of the tumor suppressor PTEN. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C61.

Liver cancer is one of the most common malignant tumors. It is reported to be the third most lethal malignancy worldwide. Recent studies including our own identified CD133+ cell population as the tumor initiating cells for liver cancer. Tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten) is aberrantly expressed in liver cancers. Liver specific Pten (Pm) null mice develop liver cancer following an extensive phase of chronic lipid accumulation and demonstrate escalating levels of hepatic injury markers from 6-12M, prior to hepatic progenitor cell proliferation. Concomitantly, expression of mRNA levels for Wnt ligands and receptors also increased progressively. Wnt/β-Catenin signaling pathway has various roles in regulating embryonic development and tumorigenesis. In Pten null liver progenitor cell line and tissues; we observed high levels of βcatenin, a downstream target component of the Wnt signaling pathway compared to control cells. In this study, we investigate the role of the Wnt/β-Catenin pathway in the activation of hepatic progenitor cells in the Pten deletion liver cancer model using two methods: a novel Wnt/β-Catenin inhibitor ICG-001 and β-Catenin specific RNAi. ICG-001 is a small molecule, which specifically inhibits β-Catenin/CBP interaction. Coactivator CBP/β-Catenin/T cell factor (TCF) mediated transcription has been reported as critical for stem cell/progenitor cell proliferation. ICG-001 significantly attenuates the proliferation of hepatic cancer stem cells in vitro and in vivo. Our preliminary results demonstrate inhibition of Wnt/β-Catenin pathway using sh-β-Catenin in vitro can effectively inhibit the proliferation of CD133+ hepatic cancer stem cells. These observations indicate that Wnt/β- Catenin pathway likely mediates proliferation of hepatic cancer stem cell. Targeting this pathway using ICG-001 and/or β-Catenin directed shRNA holds promise for the treatment and eradication of liver cancer. Citation Format: Anketse D. Kassa, Vivian G. Medina, Ni Zeng, Lina He, Cu Nguyen, Goar Smbatyan, Megan Rieger, Michael Kahn, Bangyan L. Stiles. The therapeutic potential of ICG-001 and β-catenin specific RNAi in liver cancer development. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4014. doi:10.1158/1538-7445.AM2013-4014

Progenitor or tumor initiating cells (TICs) are “altered” stem cells with the capacity to form solid tumors. Tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten) is aberrantly expressed in liver cancers. Liver specific Pten (Pm) mice develop liver cancer following an extensive phase of chronic lipid accumulation and demonstrate escalating levels of hepatic injury markers from 6-12M, prior to hepatic progenitor cell proliferation. In addition, TUNEL analysis revealed that hepatocytes from Pm mice undergo extensive apoptosis relative to control mice. We hypothesize that hepatocyte cell death induced by hepatic injury presents an opportunity for TICs to proliferate and consequently form mixed lineage tumors. Attenuation of hepatic injury by Akt2 deletion reduces progenitor cell proliferation and delays tumor development. Our analyses also revealed that the Wnt/β-Catenin signaling pathway is the likely molecular mediator of TIC proliferation in our Pm model. Wnt ligands including 7a and 10a as well as Wnt signaling receptor Fzd 2 are induced. In this study, we investigate the role of the Wnt/β-Catenin pathway in the activation of hepatic progenitor cells in the Pten deletion liver cancer model using novel Wnt/β-catenin inhibitor ICG-001. ICG-001 is a small molecule which specifically inhibits β-Catenin/CBP interaction. Coactivator CBP/β-Catenin/T cell factor (TCF) mediated transcription has been reported as critical for stem cell/progenitor cell proliferation. Here we demonstrate that ICG-001 significantly attenuates proliferation of hepatic cancer stem cell line P0 in vitro and also inhibits hepatotoxin induced proliferation of hepatic TICs in vivo in liver Pten null mice. Our preliminary results indicate that targeting the cancer stem cell niche may prove to be a viable approach to inhibit liver tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3473. doi:1538-7445.AM2012-3473

We used a liver specific Pten (phosphatase and tensin homologue deleted on chromosome ten) deletion murine model to investigate the mechanism of hepatic cancer stem cell activation in vivo. We have shown that loss of PTEN induces the transformation of liver progenitor cells to tumor initiating cells (TICs). Activation of TIC in this model involves death of hepatocytes resulting from PTEN loss induced liver steatosis. Such hepatocytes death presents a selection pressure and altered the progenitor cell niche for mutant progenitor cells to proliferate and replace liver parenchyma. We found that hepatic injury is followed by expansion of hepatic progenitors and upregulation of hepatic progenitor cell markers. In addition, we also found factors governing the progenitor niche such as Wnt ligands including Wnt5a, 7a and 10a as well as Wnt signaling receptor Fzd are induced. Furthermore, β-catenin activation is observed in vivo as well as in progenitor cells isolated from these mice. Thus, we hypothesize that activation of Wnt serves as the niche signal to allow proliferation of mutant progenitors. To test this hypothesis, we treated the isolated progenitor cells with a small molecule compound ICG-001 to interrupt b-catenin/CBP binding. β-catenin/CBP signal allows the activation of progenitor niche by inducing selfrenewal signals while inhibiting differentiation signals. We showed that ICG-001 treatment blocked the self-renewal signals and proliferation of the isolated progenitors in vitro. The in vivo experiment is ongoing and will also be presented at the meeting. Our study demonstrated that β-catenin activation, either directly through PTEN loss or indirectly through PTEN loss induced lipotoxic death of hepatocytes, serves as an important mediator for the activation of Pten mutant progenitor cells. Targeting such niche with our inhibitor may prove to be a viable approach to inhibit liver tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-102. doi:10.1158/1538-7445.AM2011-LB-102

Cancer stem cells have been identified in solid and aqueous tumors and are defined as “altered” stem cells with the capacity to form tumors. We used the liver specific Pten (phosphatase and tensin homologue deleted on chromosome ten) deletion murine model to investigate the role of hepatic cancer stem cells in vivo. Proliferation of hepatic progenitor cells in other rodent models has been observed as a consequence of chronic liver injury. Pten-null mice demonstrate escalating levels of hepatic injury markers from 6-12M of age. In addition, TUNEL analysis revealed that hepatocytes from mutant mice are undergoing extensive apoptosis relative to control mice. We hypothesize that hepatocyte cell death induced by lipotoxicity promotes progenitor cell proliferation to replace liver parenchyma. We show here that activation of hepatic progenitor cells (oval cells) in this model occurs progressively. In mutant mice hepatic injury is followed by elevated expression of hepatic progenitor cell markers Cytokeratin 19 (CK 19), Epithelial cell adhesion molecule (EpCAM), α-fetoprotein (AFP) as well as activation of the Wnt/β-Catenin signaling pathway. Furthermore, we report that liver specific Pten-null mice develop both cholangiocellular carcinoma (CC) and hepatocellular carcinoma (HCC) by 12-15 months of age. Collectively these results suggest that liver tumors in this model arise from hepatic progenitor cells. Our studies also demonstrate that when hepatic injury is attenuated tumor onset is significantly delayed, highlighting the relevance of hepatic injury to progenitor cell induced carcinogenesis. In addition, we show that reactive oxygen species (ROS) is the likely molecular mediator for hepatocyte cell death. This study presents in vivo evidence for the contribution of hepatic injury to progenitor cell mediated liver tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4260.