Arun Thenappan

Liver stem cells and hepatocellular carcinoma.

Although the existence of cancer stem cells (CSCs) was first proposed over 40 years ago, only in the past decade have these cells been identified in hematological malignancies, and more recently in solid tumors that include liver, breast, prostate, brain, and colon. Constant proliferation of stem cells is a vital component in liver tissues. In these renewing tissues, mutations will most likely result in expansion of the altered stem cells, perpetuating and increasing the chances of additional mutations and tumor progression. However, many details about hepatocellular cancer stem cells that are important for early detection remain poorly understood, including the precise cell(s) of origin, molecular genetics, and the mechanisms responsible for the highly aggressive clinical picture of hepatocellular carcinoma (HCC). Exploration of the difference between CSCs from normal stem cells is crucial not only for the understanding of tumor biology but also for the development of specific therapies that effectively target these cells in patients. These ideas have drawn attention to control of stem cell proliferation by the transforming growth factor beta (TGF‐β), Notch, Wnt, and Hedgehog pathways. Recent evidence also suggests a key role for the TGF‐β signaling pathway in both hepatocellular cancer suppression and endoderm formation, suggesting a dual role for this pathway in tumor suppression as well as progression of differentiation from a stem or progenitor stage. This review provides a rationale for detecting and analyzing tumor stem cells as one of the most effective ways to treat cancers such as HCC. (HEPATOLOGY 2009;49:318–329.)

Role of Transforming Growth Factor β Signaling and Expansion of Progenitor Cells in Regenerating Liver

Adult hepatic progenitor cells are activated during regeneration when hepatocytes and bile duct epithelium are damaged or unable to proliferate. On the basis of its role as a tumor suppressor and in the potential malignant transformation of stem cells in hepatocellular carcinoma, we investigated the role of key transforming growth factor beta (TGF‐β) signaling components, including the Smad3 adaptor protein β2‐Spectrin (β2SP), in liver regeneration. We demonstrate a streaming hepatocyte‐specific dedifferentiation process in regenerating adult human liver less than 6 weeks following living donor transplantation. We then demonstrate a spatial and temporal expansion of TGF‐β signaling components, especially β2SP, from the periportal to the pericentral zone as regeneration nears termination via immunohistochemical analysis. This expansion is associated with an expanded remaining pool of octamer 3/4 (Oct3/4)‐positive progenitor cells localized to the portal tract in adult human liver from more than 6 weeks posttransplant. Furthermore, disruption of TGF‐β signaling as in the β2SP (β2SP+/−) knockout mouse demonstrated a striking 2 to 4‐fold (P < 0.05) expanded population of Oct3/4‐positive cells with activated Wnt signaling occupying an alpha‐fetoprotein (AFP)+/cytokeratin‐19 (CK‐19)‐positive progenitor cell niche following two‐thirds partial hepatectomy. Conclusion: TGF‐β signaling, particularly β2SP, plays a critical role in hepatocyte proliferation and transitional phenotype and its loss is associated with activation of hepatic progenitor cells secondary to delayed mitogenesis and activated Wnt signaling. (HEPATOLOGY 2010.)

Liver allograft outcomes after laparoscopic-assisted and minimal access live donor hepatectomy for transplantation

BACKGROUND The critical shortage of deceased organ donors has led to live-donor hepatectomy as an alternative donor option for transplantation. Although laparoscopic hepatectomy has been well described for management of liver tumors and can be performed safely, few studies have examined early recipient allograft outcomes after laparoscopic live-donor hepatectomy. We describe our initial experience with laparoscopic-assisted and minimal-access donor hepatectomy and its potential as a safe alternative with graft function comparable with open resection in live-donor liver transplantation. METHODS We performed a retrospective analysis of our past 30 successive live-donor transplants between 2005 and 2009. Fifteen allografts were procured by standard open live-donor (OLD) hepatectomy, and 15 by laparoscopic-assisted (LALD) or minimal-access (MA) live-donor hepatectomy. Left lateral segment grafts were subcategorized and analyzed further. RESULTS Mean donor age, sex, and liver anatomy were comparable between donor groups. Early graft function as measured by peak total bilirubin level, aspartate aminotransferase level, alanine aminotransferase level, and international normalized ratio on postoperative days 2, 7, 30, and 90 were similar between groups, although the international normalized ratio was slightly more increased on postoperative day 7 in LALD grafts (1.75 ± .45 vs 1.28 ± .16; P = .02). Perioperative allograft biliary (2 of 15 vs 0 of 15; P = .48) and vascular (3 of 15 vs 1 of 15; P = .6) complication rates also were comparable between OLD and LALD/MA grafts. One-year graft and patient survival for LALD/MA was 100% compared with 93% for OLD. CONCLUSIONS Our experience shows that LALD or MA live-donor hepatectomy is a safe procedure and produces early graft function comparable with standard OLD hepatectomy. Multicenter, larger-volume experience will determine the widespread application of this technique.

Loss of transforming growth factor β adaptor protein β-2 spectrin leads to delayed liver regeneration in mice

Liver regeneration, following partial hepatectomy (PHx), occurs through precisely controlled and synchronized cell proliferation, in which quiescent hepatocytes undergo one to two rounds of replication, with restoration of liver mass and function. We previously demonstrated that loss of the Smad3/4 adaptor protein β‐2 spectrin (β2SP) is associated with faster entry into S phase, and hepatocellular cancer formation. These observations led us to further pursue the role of β2SP in cell cycle progression in vivo. Liver regeneration studies with PHx in β2SP+/− mice reveal a surprising and significant decrease in liver/body weight ratio at 48 hours after PHx in β2SP+/− mice in comparison to wildtype mice. At 48 hours after PHx we also observe decreased levels of cyclin E (2.4‐fold, P < 0.05), Cdk1 (7.2‐fold, P < 0.05), cyclin A, pRb (Ser249/Thr252), proliferative cell nuclear antigen (PCNA), cyclin D1 with elevated levels of pCdk1 (Thr14) (3.6‐fold, P < 0.05). Strikingly, at 24 hours elevated levels of p53 (4‐fold, P < 0.05), phospho‐p53 (ser15 and ser20), and p21 (200‐fold, P < 0.05) persisting to 48 hours after PHx further correlated with raised expression of the DNA damage markers pChk2 (Thr68) and γH2AX (S139). However, compromised cell cycle progression with loss of β2SP is not rescued by inhibiting p53 function, and that G2/M phase arrest observed is independent and upstream of p53. Conclusion: β2SP deficiency results in dysfunctional hepatocyte cell cycle progression and delayed liver regeneration at 48 hours after PHx, which is p53‐independent. β2SP loss may increase susceptibility to DNA damage, impair cell cycle progression, and ultimately lead to hepatocellular cancer. (HEPATOLOGY 2011;)

Stem Cells and Chronic Liver Failure: Potential New Therapeutics

Fulminant hepatic failure is a disease with high mortality. Prior to orthotopic liver transplantation (OLT), the mortality rate was greater than 80%. With improved intensive care, however, several series report a survival rate of 60% (1). At present, standard therapy is liver transplantation and approximately 6% of OLTs performed in the United States are for fulminant hepatic failure.

220 Targeting CEA Rescues Proliferation Inhibiting Effects of TGF-β Signaling Pathway in Colorectal Cancers

Carcinoembryonic antigen (CEA) is a tumor marker for the clinical management of colorectal cancer (CRC). The elevated blood levels of CEA are associated with metastasis and poor prognosis in CRC. There is mounting evidence that CEA enhances the metastatic potential of cancer cells. CEA increases the ability of weakly metastatic CRC to colonize the liver and to develop spontaneous hematogeneous liver and lung metastases. CEA expression has also been related with resistance to cytotoxic chemotherapy and to anoikis, a form of apoptosis caused by detachment from cell matrix. Yet the mechanism of CEA mediated metastasis is only partially understood. The TGF-β (transforming growth factor beta) signaling pathway contributes to tumorigenesis by controlling several biological processes, including cell proliferation, differentiation, migration and apoptosis. It has been reported that TGF-β regulates CEA transcription and secretion, however, little is known about the effects of CEA on TGFβ signaling. Aims: Based on the above facts, we focused on the influence of CEA on the TGF-β signaling in both normal cells and colorectal cancer cells. Results: Our preliminary data showed that CEA directly interacted with TGF-β receptors. Overexpression of CEA blocked TGF-β induced SMAD3 phosphorylation, SMAD3 translocation to nuclear and the downregulation of c-myc transcription. Targeting CEA with anti-CEA antibody rescued TGFβ response in CRC cell lines with elevated CEA expression, thereby restoring the inhibitory effects of TGF-β on the proliferation of these cancer cells. Finally, in animal experiment, we found that CEA enhanced survival of colorectal cancer cell in both local colonization and liver metastasis. Conclusion: Since CEA is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors, specific antibodies targeting CEA have been developed as a novel therapeutic approach for treatment of tumors expressing CEA on their surface. Based on our study, it may be helpful to combine CEA antibody and TGF-β to inhibit cancer cell proliferation and metastasis in some cases.

Abstract 3913: TGF-β signaling mediated suppression of stat3 transcription in hepatocellular cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC At least 40% of hepatocellular cancers (HCC) are clonal, suggesting that HCC may develop from malignant transformation of liver progenitor/stem cells. Several signaling pathways, including IL-6/STAT3 and TGF-β are known to be involved in stem cell renewal, differentiation and survival, and are commonly deregulated in HCC. We have previously demonstrated that human and mouse HCC tissues with aberrant TGF-β signaling show increased expression of STAT3. Moreover, down-regulation of the IL-6/STAT3 pathway in a TGF-β disrupted mouse model (β2SP+/−) by crossing with itih4—/— (Inter-alpha-trypsin inhibitor-heavy chain-4) mice resulted in a significant decrease in the incidence of HCC. Aims: This led us to hypothesize that the TGF-β signaling pathway is a strong candidate pathway for the transition from progenitor to differentiated cells and its disruption may activate the stem cell renewal IL-6/STAT3 pathway. We proceeded to investigate the role of key TGF-β signaling components, including the key Smad3/4 adaptor protein β2SP and Smad3, in the regulation of STAT3 expression. Methods and Results: First, using early passage mouse embryonic fibroblasts (MEFs) from wild type and β2SP−/− embryos, we demonstrate a four-fold increase in STAT3 mRNA by RT-PCR (p<0.001). Moreover, we demonstrate that overexpression of β2SP by transfection or inhibition of β2SP expression by siRNA in several HCC cell lines results in STAT3 suppression or induction, respectively, suggesting that β2SP and the TGF-β signaling pathway regulate STAT3 transcription. Then, using ChiP assay, we demonstrate that β2SP and Smad3 are bound to the STAT3 promoter only following TGF-β stimulation. To then further define the molecular mechanism of TGF-β-mediated STAT3 transcriptional regulation, we then used mutational analysis and demonstrate two transcription factor binding sites within the STAT3 promoter, the cAMP-responsive element (CRE) and STAT3 binding (SBE) sites, are essential for TGF-β-mediated regulation of the STAT3 transcription. Subsequent, electrophoresis mobility shift assays (EMSA) demonstrate that the CRE-binding protein ATF-2, Smad3, and β2SP proteins are major components of the TGF-β-mediated STAT3 transcriptional suppressor complex. Conclusion: These experiments demonstrate a clear link between the TGF-β and IL-6/STAT3 signaling pathways. TGF-β suppression of STAT3 transcription is mediated by a complex including [[Unsupported Character - ]]β2SP, Smad3 and ATF-2 at the CRE site of the STAT3 promoter. Inactivation of TGF-β signaling via disruption of β2SP decreases STAT3 suppression and suggests a potential mechanism for malignant transformation in TGF-β deficient progenitor/stem cells. STAT3 may also present an important target for new therapeutics in transformed cancer progenitor/stem cells. 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 3913.

Abstract 1491: CEA interacts with TGF-β receptor and inhibits TGF-β signaling in colorectal cancers

Carcinoembryonic antigen (CEA) is a tumor marker for the clinical management of colorectal cancer (CRC). The elevated blood levels of CEA are associated with metastasis and poor prognosis in CRC. There is mounting evidence that CEA enhances the metastatic potential of cancer cells. CEA increases the ability of weakly metastatic CRC to colonize the liver and to develop spontaneous hematogeneous liver and lung metastases. CEA expression has also been related with resistance to cytotoxic chemotherapy and to anoikis, a form of apoptosis caused by detachment from cell matrix. Yet the mechanism of CEA mediated metastasis is only partially understood. The TGF-β (transforming growth factor beta) signaling pathway contributes to tumorigenesis by controlling several biological processes, including cell proliferation, differentiation, migration and apoptosis. It has been reported that TGF-β regulates CEA transcription and secretion, however, little is known about the effects of CEA on TGF-β signaling. Aims: Based on the above facts, we focused on the influence of CEA on the TGF-β signaling in both normal cells and colorectal cancer cells. Results: Our preliminary data showed that CEA directly interacted with TGF-β receptors. Overexpression of CEA blocked TGF-β induced SMAD3 phosphorylation, SMAD3 translocation to nuclear and the downregulation of c-myc transcription. Targeting CEA with anti-CEA antibody rescued TGF-β response in CRC cell lines with elevated CEA expression, thereby restoring the inhibitory effects of TGF-β on the proliferation of these cancer cells. Finally, in animal experiment, we found that CEA enhanced survival of colorectal cancer cell in both local colonization and liver metastasis. Conclusion: Since CEA is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors, specific antibodies targeting CEA have been developed as a novel therapeutic approach for treatment of tumors expressing CEA on their surface. Based on our study, it may be helpful to combine CEA antibody and TGF-β to inhibit cancer cell proliferation and metastasis in some cases. 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 1491.