Francesco Dituri

Down‐regulation of connective tissue growth factor by inhibition of transforming growth factor β blocks the tumor–stroma cross‐talk and tumor progression in hepatocellular carcinoma

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.)

Tumor‐secreted lysophostatidic acid accelerates hepatocellular carcinoma progression by promoting differentiation of peritumoral fibroblasts in myofibroblasts

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;)

Role of epithelial to mesenchymal transition in hepatocellular carcinoma

The epithelial to mesenchymal transition (EMT) is a multistep biological process whereby epithelial cells change in plasticity by transient de-differentiation into a mesenchymal phenotype. EMT and its reversal, mesenchymal to epithelial transition (MET), essentially occur during embryogenetic morphogenesis and have been increasingly described in fibrosis and cancer during the last decade. In carcinoma progression, EMT plays a crucial role in early steps of metastasis when cells lose cell-cell contacts due to ablation of E-cadherin and acquire increased motility to spread into surrounding or distant tissues. Epithelial plasticity has become a hot issue in hepatocellular carcinoma (HCC), as strong inducers of EMT such as transforming growth factor-β are able to orchestrate both fibrogenesis and carcinogenesis, showing rising cytokine levels in cirrhosis and late stage HCC. In this review, we consider the significance of EMT-MET in malignant hepatocytes as well as changes in the plasticity of hepatic stellate cells for cellular heterogeneity of HCC, and further aim at explaining the current limiting insights into EMT by snapshot analyses of HCC tissues. Recent advances in the identification of clinically relevant mechanisms that impinge on important EMT-transcription factors, as well as on miRNAs causing EMT signatures and HCC progression are highlighted. In addition, we draw particular attention to framing EMT in the context of potential clinical relevance for HCC patients. We conclude that some aspects of EMT are still elusive and further studies are required to better link the clinical management of HCC with biomarkers and targeted therapies related to EMT.

Moving towards personalised therapy in patients with hepatocellular carcinoma: the role of the microenvironment

The goal of personalised therapy based on hepatocellular carcinoma (HCC) molecular characteristics is still beyond our grasp. Systemic treatments show poor efficacy, mainly because of the great heterogeneity of the tumour. Indeed, differences in aetiology, disease stage and biochemical composition of the fibrotic liver make cirrhosis itself a highly dyshomogeneous disease. Cancer cells grow in a cirrhotic microenvironment, interacting with stromal cells and engaging matrix components that differ from patient to patient, hampering the development of drugs to treat all patients. Growing evidence suggests a role for the cross-talk between HCC and the host stroma in driving disease progression and hence prognosis and survival. Many efforts have been devoted to identifying genes responsible for good or bad prognosis, but no study has yet proven helpful in guiding therapeutic choices and management over time, also taking into account the development of drug resistance. The questions of what to target and in which patient are still unsolved. In the personalised therapy scenario, the patient rather than the disease becomes the target of the therapy. However, this still requires an evidence-based medical approach. Herein, we will discuss how individual differences in terms of quality and quantity of the tissue microenvironment components affect progression of HCC. Then, we will highlight potential druggable pathways, also considering ongoing clinical trials. The development of biomarkers will be discussed in the light of new experimental research conducted with the aim of moving towards personalised therapy in patients with HCC.

Differential Inhibition of the TGF-β Signaling Pathway in HCC Cells Using the Small Molecule Inhibitor LY2157299 and the D10 Monoclonal Antibody against TGF-β Receptor Type II

We investigated blocking the TGF-β signaling pathway in HCC using two small molecule inhibitors (LY2157299, LY2109761) and a neutralizing humanized antibody (D10) against TGF-βRII. LY2157299 and LY2109761 inhibited HCC cell migration on Laminin-5, Fibronectin, Vitronectin, Fibrinogen and Collagen-I and de novo phosphorylation of pSMAD2. LY2157299 inhibited HCC migration and cell growth independently of the expression levels of TGF-βRII. In contrast to LY2157299, D10 showed a reduction in pSMAD2 only after a short exposure. This study supports the use of LY2157299 in clinical trials, and presents new insights into TGF-β receptor cycling in cancer cells.

Hepatic stellate cells stimulate HCC cell migration via laminin-5 production

Activated HSCs (hepatic stellate cells) are the main source of extracellular matrix proteins present in cirrhotic liver on which HCC (hepatocellular carcinoma) commonly develops. HCC cells behave differently according to differences in the surrounding microenvironment. In the present study, we have investigated a mechanism whereby HSCs modulate the migratory activity of HCC cells. We used primary cultures of human HSCs to investigate their effect on Hep3B, Alexander, HLE and HLF HCC cells. The expression of Ln-5 (laminin-5) was documented at transcript and protein levels both in vitro and in vivo. HCC cells strongly adhere, migrate and spread in the presence of HSC-conditioned medium and of co-culture. HSCs produce and secrete Ln-5 in the CM (conditioned medium). The electrophoretic pattern of secreted Ln-5 is consistent with that of a migratory substrate, showing the presence of the γ2x fragment. Blocking antibodies against Ln-5 inhibit HCC migration in the presence of HSC-CM. HCC cells migrate very poorly in the presence of Ln-5 immunodepleted HSC-CM. HCC migration in the presence of HSCs is dependent on the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathway, but not the PI3K (phosphoinositide 3-kinase)/Akt pathway. HSC-CM, as well as Ln-5, activates the MEK/ERK but not the PI3K/Akt pathway. In human HCC tissues, Ln-5 is mainly distributed along α-SMA (smooth muscle actin)-positive cells, whereas in peritumoural tissues, Ln-5 is absent. HSCs stimulate HCC migration via the production and secretion of Ln-5.

PI3K Functions in Cancer Progression, Anticancer Immunity and Immune Evasion by Tumors

The immunological surveillance of tumors relies on a specific recognition of cancer cells and their associate antigens by leucocytes of innate and adaptive immune responses. However, a dysregulated cytokine release can lead to, or be associated with, a failure in cell-cell recognition, thus, allowing cancer cells to evade the killing system. The phosphatidylinositol 3-kinase (PI3K) pathway regulates multiple cellular processes which underlie immune responses against pathogens or malignant cells. Conversely, there is accumulating evidence that the PI3K pathway is involved in the development of several malignant traits of cancer cells as well as their escape from immunity. Herein, we review the counteracting roles of PI3K not only in antitumor immune response but also in the mechanisms that cancer cells use to avoid leukocyte attack. In addition, we discuss, from antitumor immunological point of view, the potential benefits and disadvantages arising from use of anticancer pharmacological agents targeting the PI3K pathway.

PI3K class IB controls the cell cycle checkpoint promoting cell proliferation in hepatocellular carcinoma

Alterations of the cell cycle checkpoint frequently occur during hepatocarcinogenesis. Dysregulation of the phosphatidylinositol‐3‐kinases (PI3K) signaling pathway is believed to exert a potential oncogenic effect in hepatocellular carcinoma (HCC), ultimately promoting tumor cell proliferation. However, the impact of PI3K on cell cycle regulation remains unclear. We used a combined loss‐ and gain‐of‐function approach to address the involvement of p110γ in HCC cell proliferation, apoptosis and the cell cycle. We also investigated the correlation between p110γ and Ki‐67 in 24 HCC patients. Finally, we analyzed the expression levels of p110γ and cell cycle regulators in HCC tissues. We found that PI3K class IB, but not class IA, is required for HCC cell proliferation. In particular, we found that knock‐down of p110γ inhibits cell proliferation because of an arrest of the cell cycle in the G0‐G1 phase. This effect is associated with an altered expression of proteins regulating the cell cycle progression, including p21, and with an increased apoptosis. By contrast, we found that ectopic expression of p110γ promotes HCC cell proliferation. Tissues analysis performed in HCC patients showed a positive correlation between the expression of p110γ and Ki‐67, a marker of proliferation, and, even more importantly, that p21 expression is up‐regulated in HCC patients with a lower p110γ expression. Our results emphasize the role of p110γ as a promoter of HCC proliferation and unveil an important cell cycle regulation function of this molecule.

Hepatic stellate cells induce hepatocellular carcinoma cell resistance to sorafenib through the laminin-332/α3 integrin axis recovery of focal adhesion kinase ubiquitination.

In patients with hepatocellular carcinoma (HCC) receiving sorafenib, drug resistance is common. HCC develops in a microenvironment enriched with extracellular matrix proteins including laminin (Ln)‐332, produced by hepatic stellate cells (HSCs). Ln‐332 is the ligand of α3β1 and α6β4 integrins, differently expressed on the HCC cell surface, that deliver intracellular pathways. The aim of this study was to investigate the effect of Ln‐332 on sorafenibs effectiveness. HCC cells were challenged with sorafenib in the presence of Ln‐332 and of HSC conditioned medium (CM). Sorafenib impaired HCC cell proliferation and induced apoptosis. HSC‐CM or Ln‐332 inhibited sorafenibs effectiveness in HCC cells expressing both α3β1 and α6β4. Inhibiting α3 but not α6 integrin subunit using blocking antibodies or small interfering RNA abrogated the protection induced by Ln‐332 and HSC‐CM. Hep3B cells expressing α6β4 but lacking the α3 integrin were insensitive to Ln‐332 and HSC‐CM protective effects. Hep3B α3‐positive, but not wild‐type and scramble transfected, cells acquired protection by sorafenib when plated on Ln‐332‐CM or HSCs. Sorafenib dephosphorylated focal adhesion kinase (FAK) and extracellular signal‐regulated kinases 1/2, whereas Ln‐332 and HSC‐CM partially restored the pathways. Silencing FAK, but not extracellular signal‐regulated kinases 1/2, abrogated the protection induced by Ln‐332 and HSC‐CM, suggesting a specific role for FAK. Sorafenib down‐regulated total FAK, inducing its proteasomal degradation, while Ln‐332 and HSC‐CM promoted the escape of FAK from ubiquitination, probably inducing a preferential membrane localization. Conclusion: This study unveils a novel mechanism of sorafenib resistance depending on the α3β1/Ln‐332 axis and requiring FAK ubiquitination, providing new insights into personalizing therapy for patients with HCC. (Hepatology 2016;64:2103‐2117).

Microenvironment inflammatory infiltrate drives growth speed and outcome of hepatocellular carcinoma: a prospective clinical study

In HCC, tumor microenvironment, heavily influenced by the underlying chronic liver disease, etiology and stage of the tissue damage, affects tumor progression and determines the high heterogeneity of the tumor. Aim of this study was to identify the circulating and tissue components of the microenvironment immune-mediated response affecting the aggressiveness and the ensuing clinical outcome. We analyzed the baseline paired HCC and the surrounding tissue biopsies from a prospective cohort of 132 patients at the first diagnosis of HCC for immunolocalization of PD-1/PD-L1, FoxP3, E-cadherin, CLEC2 and for a panel of 82 microRNA associated with regulation of angiogenesis, cell proliferation, cell signaling, immune control and autophagy. Original microarray data were also explored. Serum samples were analyzed for a panel of 19 cytokines. Data were associated with biochemical data, histopathology and survival. Patients with a more aggressive disease and shorter survival, who we named fast-growing accordingly to the tumor doubling time, at presentation had significantly higher AFP levels, TGF-β1 and Cyphra 21-1 levels. Transcriptomic analysis evidenced a significant downregulation of CLEC2 and upregulation of several metalloproteinases. A marked local upregulation of both PD-1 and PD-L1, a concomitant FoxP3-positive lymphocytic infiltrate, a loss of E-cadherin, gain of epithelial–mesenchymal transition (EMT) phenotype and extreme poor differentiation at histology were also present. Upregulated microRNA in fast-growing HCCs are associated with TGF-β signaling, angiogenesis and inflammation. Our data show that fast HCCs are characterized not only by redundant neo-angiogenesis but also by unique features of distinctively immunosuppressed microenvironment, prominent EMT, and clear-cut activation of TGFβ1 signaling in a general background of long-standing and permanent inflammatory state.