Tumor–stroma interactions in hepatocellular carcinoma (HCC) are of key importance to tumor progression. In this study, we show that HCC invasive cells produce high levels of connective tissue growth factor (CTGF) and generate tumors with a high stromal component in a xenograft model. A transforming growth factor β (TGF-β) receptor inhibitor, LY2109761, inhibited the synthesis and release of CTGF, as well as reducing the stromal component of the tumors. In addition, the TGF-β–dependent down-regulation of CTGF diminished tumor growth, intravasation, and metastatic dissemination of HCC cells by inhibiting cancer-associated fibroblast proliferation. By contrast, noninvasive HCC cells were found to produce low levels of CTGF. Upon TGF-β1 stimulation, noninvasive HCC cells form tumors with a high stromal content and CTGF expression, which is inhibited by treatment with LY2109761. In addition, the acquired intravasation and metastatic spread of noninvasive HCC cells after TGF-β1 stimulation was blocked by LY2109761. LY2109761 interrupts the cross-talk between cancer cells and cancer-associated fibroblasts, leading to a significant reduction of HCC growth and dissemination. Interestingly, patients with high CTGF expression had poor prognosis, suggesting that treatment aimed at reducing TGF-β–dependent CTGF expression may offer clinical benefits. Conclusion: Taken together, our preclinical results indicate that LY2109761 targets the cross-talk between HCC and the stroma and provide a rationale for future clinical trials. (Hepatology 2009.)

Background and aims: Myofibroblast-like cells, originating from activation of hepatic stellate cells (HSC/MFs), play a key role in liver fibrosis, a potentially reversible process that may rely on induction of HSC/MFs apoptosis. While this possibility has been shown in cultured rat HSC, very limited data are currently available for human HSC/MFs. Methods: Cultured human HSC/MFs were exposed to several proapoptotic stimuli, including those known to induce apoptosis in rat HSC/MFs, and induction of cell death and related mechanisms were investigated using morphology, molecular biology, and biochemical techniques. Results: In this study we report that fully activated human HSC/MFs did not undergo spontaneous apoptosis and survived to prolonged serum deprivation, Fas activation, or exposure to nerve growth factor, tumour necrosis factor α (TNF-α), oxidative stress mediators, doxorubicin, and etoposide. Induction of caspase dependent, mitochondria driven apoptosis in HSC/MFs was observed only when protein synthesis or transcription were inhibited. Importantly, the process of HSC activation was accompanied by changes in expression of a set of genes involved in apoptosis control. In particular, activated human HSC/MFs in culture overexpressed Bcl-2. The role of Bcl-2 was crucial as Bcl-2 silenced cells became susceptible to TNF-α induced apoptosis. Finally, Bcl-2 was markedly expressed in HSC/MFs present in liver tissue obtained from patients with hepatitis C virus related cirrhosis. Conclusions: Human activated HSC/MFs are resistant to most proapoptotic stimuli due to Bcl-2 overexpression and this feature may play a key role in the progression of fibrosis in chronic liver diseases.

Tumor cell invasion is a process regulated by integrins, matrix-degrading enzymes, and interactions with host tissue stromal cells. The ADAM family of proteins plays an important role in modulating various cellular responses. Here, we show that an alternatively spliced variant of ADAM9 is secreted by hepatic stellate cells and promotes carcinoma invasion. ADAM9-S induced a highly invasive phenotype in several human tumor cell lines in Matrigel assays, and the protease activity of ADAM9-S was required for invasion. ADAM9-S binds directly to α₆β₄ and α₂β₁ integrins on the surface of colon carcinoma cells through the disintegrin domain. ADAM9-S was also able to cleave laminin and promote invasion. Analysis of human liver metastases revealed that ADAM9 is expressed by stromal liver myofibroblasts, particularly those that are localized within the tumor stroma at the invasive front. These results emphasize the importance of tumor-stromal interactions in invasion and suggest that ADAM9-S can be an important determinant in the ability of cancer cells to invade and colonize the liver.

Pathogenic mechanisms of B-cell lymphoproliferative disorders in chronic hepatitis C virus (HCV) infection are unclear. We studied t(14;18) translocation by polymerase chain reaction in peripheral blood mononuclear cells from 50 patients with HCV-related liver disease (group A), 7 with mixed cryoglobulinemia syndrome (group B), 55 with HCV-negative liver disease (group C), and 30 with HCV-negative chronic rheumatic disorders or chronic infection by nonhepatotropic agents (group D). T(14;18) was significantly more frequent in group A (13/50 patients = 26 %) and group B (5/7 = 71.4%) patients than in group C (1/55 = 3.6%) and group D (1/30 = 3.3%) ones. Immunoblot analysis showed bcl-2 over-expression in all t(14;18)-positive samples. In group A, 10/13 (77%) patients with t(14;18) and 13/37 (35%) without t(14;18) had serum cryoglobulins in the absence of mixed cryoglobulinemia symptoms (P < .05). These data indicate that t(14;18) and bcl-2 over-expression in lymphoid cells are frequent in chronic HCV infection and suggest that this event may contribute to the pathogenesis of HCV-related lymphoproliferative disorders.

Vascular invasion is one of the major negative prognostic factors in patients with hepatocellular carcinoma (HCC), leading to cancer recurrence. To invade, HCC cells must penetrate the vessel wall, consisting of endothelial cells and extracellular matrix components, including fibronectin and fibrinogen. Employing invasive and noninvasive HCC cells, we studied the mechanism underlying vascular invasion. We show that HCC cells invade blood vessels via α5β1, that is equally expressed in invasive and noninvasive cells. However, in the former, the intracytoplasmic tail of β1 integrin is constitutively phosphorylated at threonine 788‐789 and the extracellular part is conformationally activated. In noninvasive cells, β1 integrin is not activated. Transforming growth factor (TGF)‐β1 specifically phosphorylates β1 integrin (threonine 788‐789) via Smad‐2 and Smad‐3, causing a conformational change of the extracellular component with an inside‐out mechanism. This leads noninvasive HCC cells to behave like invasive cells. A selective TGF‐βRI inhibitor inhibits phosphorylation of the β1 integrin intracytoplasmic tail, and blocks invasion of HCC cells, both constitutively invasive and with acquired invasive properties. In human HCC tissues with microvascular invasion, phospho‐β1 integrin was detected as well as TGF‐β1, p‐Smad‐2, and E‐cadherin. Conclusion: TGF‐β1 promotes vascular invasion by activating β1 integrin. This suggests a rationale for targeting TGF‐βRI in future clinical trials. (HEPATOLOGY 2009.)

Hepatocellular carcinoma (HCC) occurs in fibrotic liver as a consequence of underlying cirrhosis. The goal of this study was to investigate how the interaction between HCC cells and stromal fibroblasts affects tumor progression. We isolated and characterized carcinoma-associated fibroblasts (CAFs) and paired peritumoral tissue fibroblasts (PTFs) from 10 different patients with HCC and performed coculture experiments. We demonstrated a paracrine mechanism whereby HCC cells secrete lysophostatidic acid (LPA), which promotes transdifferentiation of PTFs to a CAF-like myofibroblastic phenotype. This effect is mediated by up-regulation of specific genes related to a myo/contractile phenotype. After transdifferentiation, PTFs expressed α-smooth muscle actin (α-SMA) and enhanced proliferation, migration, and invasion of HCC cells occur. A pan-LPA inhibitor (α-bromomethylene phosphonate [BrP]-LPA), or autotaxin gene silencing, inhibited this PTF transdifferentiation and the consequent enhanced proliferation, migration, and invasion of HCC cells. In vivo, PTFs coinjected with HCC cells underwent transdifferentiation and promoted tumor progression. Treatment with BrP-LPA blocked transdifferentiation of PTFs, down-regulated myofibroblast-related genes, and slowed HCC growth and progression. Patients with larger and metastatic HCC and shorter survival displayed higher serum levels of LPA. Analysis of microdissected tissues indicated that stroma is the main target of the LPA paracrine loop in HCC. As a consequence, α-SMA–positive cells were more widespread in tumoral compared with paired peritumoral stroma. Conclusion: Our data indicate that LPA accelerates HCC progression by recruiting PTFs and promoting their transdifferentiation into myofibroblasts. Inhibition of LPA could prove effective in blocking transdifferentiation of myofibroblasts and tumor progression. (HEPATOLOGY 2011;)