Lopa Mishra

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.)

Progenitor/stem cells give rise to liver cancer due to aberrant TGF-β and IL-6 signaling

Cancer stem cells (CSCs) are critical for the initiation, propagation, and treatment resistance of multiple cancers. Yet functional interactions between specific signaling pathways in solid organ “cancer stem cells,” such as those of the liver, remain elusive. We report that in regenerating human liver, two to four cells per 30,000–50,000 cells express stem cell proteins Stat3, Oct4, and Nanog, along with the prodifferentiation proteins TGF-β-receptor type II (TBRII) and embryonic liver fodrin (ELF). Examination of human hepatocellular cancer (HCC) reveals cells that label with stem cell markers that have unexpectedly lost TBRII and ELF. elf+/− mice spontaneously develop HCC; expression analysis of these tumors highlighted the marked activation of the genes involved in the IL-6 signaling pathway, including IL-6 and Stat3, suggesting that HCC could arise from an IL-6-driven transformed stem cell with inactivated TGF-β signaling. Similarly, suppression of IL-6 signaling, through the generation of mouse knockouts involving a positive regulator of IL-6, Inter-alpha-trypsin inhibitor-heavy chain-4 (ITIH4), resulted in reduction in HCC in elf+/− mice. This study reveals an unexpected functional link between IL-6, a major stem cell signaling pathway, and the TGF-β signaling pathway in the modulation of mammalian HCC, a lethal cancer of the foregut. These experiments suggest an important therapeutic role for targeting IL-6 in HCCs lacking a functional TGF-β pathway.

The STAT3 inhibitor NSC 74859 is effective in hepatocellular cancers with disrupted TGF-β signaling

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide, with few effective therapeutic options for advanced disease. At least 40% of HCCs are clonal, potentially arising from STAT3+, NANOG+ and OCT3/4+ liver progenitor/stem cell transformation, along with inactivation of transforming growth factor-beta (TGF-β) signaling. Here we report significantly greater signal transducer and activator of transcription 3 (STAT3) and tyrosine phosphorylated STAT3 in human HCC tissues (P<0.0030 and P<0.0455, respectively) than in human normal liver. Further, in HCC cells with loss of response to TGF-β, NSC 74859, a STAT3-specific inhibitor, markedly suppresses growth. In contrast, CD133+ status did not affect the response to STAT3 inhibition: both CD133+ Huh-7 cells and CD133– Huh-7 cells are equally sensitive to NSC 74859 treatment and STAT3 inhibition, with an IC50 of 100 μM. Thus, the TGF-β/beta2 spectrin (β2SP) pathway may reflect a more functional ‘stem/progenitor’ state than CD133. Furthermore, NSC 74859 treatment of Huh-7 xenografts in nude mice significantly retarded tumor growth, with an effective dose of only 5 mg/kg. Moreover, NSC 74859 inhibited tyrosine phosphorylation of STAT3 in HCC cells in vivo. We conclude that inhibiting interleukin 6 (IL6)/STAT3 in HCCs with inactivation of the TGF-β/β2SP pathway is an effective approach in management of HCCs. Thus, IL6/STAT3, a major signaling pathway in HCC stem cell renewal and proliferation, can provide a novel approach to the treatment of specific HCCs.

Tgf-Beta signaling in development.

The transforming growth factor–β (TGF-β) superfamily comprises nearly 30 growth and differentiation factors that include TGF-βs, activins, inhibins, and bone morphogenetic proteins (BMPs). Multiple members of the TGF-β superfamily serve key roles in stem cell fate commitment. The various members of the family can exhibit disparate roles in regulating the biology of embryonic stem (ES) cells and tumor suppression. For example, TGF-β inhibits proliferation of multipotent hematopoietic progenitors, promotes lineage commitment of neural precursors, and suppresses epithelial tumors. BMPs block neural differentiation of mouse and human ES cells, contribute to self-renewal of mouse ES cells, and also suppress tumorigenesis. ES cells and tumors may be exposed to multiple TGF-β members, and it is likely that the combination of growth factors and cross-talk among the intracellular signaling pathways is what precisely defines stem cell fate commitment. This Connections Map Pathway in the Database of Cell Signaling integrates signaling not only from TGF-β and BMP but also from the ligands nodal and activin, and describes the role of the signaling pathways activated by these ligands in mammalian development. Much of the evidence for the connections shown comes from studies on mouse and human ES cells or mouse knockouts. This pathway is important for understanding not only stem cell biology, but also the molecular effectors of TGF-β and BMP signaling that may contribute to cancer suppression or progression and thus are potential targets for therapeutic intervention.

The role of TGF-β and Wnt signaling in gastrointestinal stem cells and cancer

The past three decades have seen an unremitting quest to identify and understand gastrointestinal stem cells, their plasticity in differentiating across cell types, as well as their role in normal, regenerative, and cancer cells. A fascinating hallmark of stem cells is their ability to undergo assymetric cell division, which entails replication of the DNA followed by division of the nucleus and partitioning of the cytoplasm to yield two different daughter cells: a stem cell as well as a committed progenitor cell, the latter proliferating into differentiated progeny. We are only just beginning to understand how normally quiescent, tissue-specific stem cells interpret a vast array of signals to develop into the gastrointestinal system. These signaling pathways include the transforming growth factor-β (TGF-β) superfamily, Wnt, FGFs, Hedgehog, Hox proteins that originate from surrounding mesodermal/stromal tissue as well as endodermal/epithelial tissue. TGF-β and wnt proteins are key morphogens that ultimately influence cell division and cell fate, so that gut endodermal stem cells enter the cell cycle, and undergo cell division that ultimately leads to differentiated cells such as functional hepatocytes, gastric parietal cells, or gut epithelial cells. Disruptions and errors in this process usually lead to tissue-specific gastrointestinal cancers such as hepatocellular cancers, gastric adenocarcinomas, and colonic adenocarcinomas. An increasingly complex and coherent view of stem/progenitor cell signaling networks, which coordinate cell growth, proliferation, stress management, and survival, is helping to define the fragile areas where malignancies are likely to develop and shows promise for the development of better cancer therapies.

NOD2-expressing bone marrow-derived cells appear to regulate epithelial innate immunity of the transplanted human small intestine

Background: Intestinal allograft rejection resembles Crohn’s disease clinically and pathologically. An understanding of its mechanism could impact this life-saving procedure, as well as provide insight into the pathophysiology of inflammatory bowel disease. The NOD2 protein has been implicated as a key player in intestinal immune health, as a consequence of the discovery of three polymorphisms linked with Crohn’s disease. An investigation was carried out to determine whether epithelial immune function and graft survival were influenced by NOD2 mutations in an intestinal transplant population. Methods: The NOD2 genotypes of 34 transplants performed consecutively over the past 3 years were determined. The NOD2 genotypes were related to clinical outcomes and the expression of certain intestinal antimicrobial peptides (AMPs) believed to protect the epithelium. Results: An unexpectedly high percentage of recipients, 35%, possessed NOD2 polymorphisms, while 8.6% of donors had comparable mutations. The likelihood of allograft failure was about 100-fold higher in recipients with mutant NOD2 alleles compared with recipients with wild-type NOD2 loci. Rejection in NOD2 mutant recipients was characterised by decreased expression of certain Paneth cell and enterocyte AMPs, prior to the onset of epithelial injury and inflammation. Conclusions: Crohn’s disease-associated polymorphisms in the NOD2 gene in the recipient represent a critical immunological risk factor for intestinal allograft rejection. Compromised epithelial defences precede visible epithelial injury and inflammatory infiltration. The association of impaired epithelial immunity with the recipient’s genotype suggests that certain NOD2-expressing cells of haematopoietic origin play a role in the process, perhaps by regulating expression of certain epithelial AMPs within the allograft.

Smad Proteins and Hepatocyte Growth Factor Control Parallel Regulatory Pathways That Converge on β1-Integrin To Promote Normal Liver Development

ABSTRACT Smads serve as intracellular mediators of transforming growth factor β (TGF-β) signaling. After phosphorylation by activated type I TGF-β receptors, Smad proteins translocate to the nucleus, where they serve as transcription factors and increase or decrease expression of TGF-β target genes. Mice lacking one copy each ofSmad2 and Smad3 suffered midgestation lethality due to liver hypoplasia and anemia, suggesting essential dosage requirements of TGF-β signal components. This is likely due to abnormal adhesive properties of the mutant hepatocytes, which may result from a decrease in the level of the β1-integrin and abnormal processing and localization of E-cadherin. Culture of mutant livers in vitro revealed the existence of a parallel developmental pathway mediated by hepatocyte growth factor (HGF), which could rescue the mutant phenotype independent of Smad activation. These pathways merge at the β1-integrin, the level of which was increased by HGF in the cultured mutant livers. HGF treatment reversed the defects in cell proliferation and hepatic architecture in theSmad2 +/− ; Smad3 +/− livers.

Mutation Profiling in Cholangiocarcinoma: Prognostic and Therapeutic Implications

Background Cholangiocarcinoma (CCA) is clinically heterogeneous; intra and extrahepatic CCA have diverse clinical presentations. Next generation sequencing (NGS) technology may identify the genetic differences between these entities and identify molecular subgroups for targeted therapeutics. Methods We describe successful NGS-based testing of 75 CCA patients along with the prognostic and therapeutic implications of findings. Mutation profiling was performed using either a) NGS panel of hotspot regions in 46 cancer-related genes using a 318-chip on Ion PGM Sequencer or b) Illumina HiSeq 2000 sequencing platform for 3,769 exons of 236 cancer-related genes plus 47 introns from 19 genes to an average depth of 1000X. Clinical data was abstracted and correlated with clinical outcome. Patients with targetable mutations were referred to appropriate clinical trials. Results There were significant differences between intrahepatic (n = 55) and extrahepatic CCA (n = 20) in regard to the nature and frequency of the genetic aberrations (GAs). IDH1 and DNA repair gene alterations occurred more frequently in intrahepatic CCA, while ERBB2 GAs occurred in the extrahepatic group. Commonly occurring GAs in intrahepatic CCA were TP53 (35%), KRAS (24%), ARID1A (20%), IDH1 (18%), MCL1 (16%) and PBRM1 (11%). Most frequent GAs in extrahepatic CCA (n = 20) were TP53 (45%), KRAS (40%), ERBB2 (25%), SMAD4 (25%), FBXW7 (15%) and CDKN2A (15%). In intrahepatic CCA, KRAS, TP53 or MAPK/mTOR GAs were significantly associated with a worse prognosis while FGFR GAs correlated with a relatively indolent disease course. IDH1 GAs did not have any prognostic significance. GAs in the chromatin modulating genes, BAP1 and PBRM1 were associated with bone metastases and worse survival in extrahepatic CCA. Radiologic responses and clinical benefit was noted with EGFR, FGFR, C-met, B-RAF and MEK inhibitors. Conclusion There are significant genetic differences between intra and extrahepatic CCA. NGS can potentially identify disease subsets with distinct prognostic and therapeutic implications.

Transforming growth factor-beta and wound healing.

Acute and chronic wounds are a source of significant morbidity for patients, and they demand a growing portion of health-care time and finances to be devoted to their care. Transforming growth factor-beta (TGF-beta) has surfaced from abundant research as a key signal in orchestrating wound repair. In beginning this review, we discuss the inflammatory, proliferative, and maturational phases of wound healing. We then focus on TGF-beta by first discussing the pathway from its production to the target cell where Smad proteins execute an intracellular signaling cascade. To review TGF-betas role in wound healing, we discuss the actions of it individually on keratinocytes, fibroblasts, endothelial cells, and monocytes, which are the major cell types involved in wound repair. From illustrating these cellular actions of TGF-beta, we summarize its multipotent role in the process of wound repair. As a clinical correlation, we also review research dedicated to the involvement of TGF-beta in venous stasis ulcers.

Disruption of transforming growth factor-β signaling through β-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation

Transforming growth factor-β (TGF-β) signaling members, TGF-β receptor type II (TBRII), Smad2, Smad4 and Smad adaptor, embryonic liver fodrin (ELF), are prominent tumor suppressors in gastrointestinal cancers. Here, we show that 40% of elf+/− mice spontaneously develop hepatocellular cancer (HCC) with markedly increased cyclin D1, cyclin-dependent kinase 4 (Cdk4), c-Myc and MDM2 expression. Reduced ELF but not TBRII, or Smad4 was observed in 8 of 9 human HCCs (P<0.017). ELF and TBRII are also markedly decreased in human HCC cell lines SNU-398 and SNU-475. Restoration of ELF and TBRII in SNU-398 cells markedly decreases cyclin D1 as well as hyperphosphorylated-retinoblastoma (hyperphosphorylated-pRb). Thus, we show that TGF-β signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.