Mechanisms of epithelial-to-mesenchymal transition in experimental and idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by accumulation of activated myofibroblasts and excessive extracellular matrix deposition, in part mediated through enhanced TGF-beta signaling. TGF-beta1 is a potent inducer of epithelial-to-mesenchymal transition (EMT), the reversible phenotypic switching of epithelial to fibroblast-like cells. Recently, EMT has been demonstrated in alveolar epithelial cells (AECs) and has been proposed as a causative factor in lung fibrosis, but its precise mediators and mechanisms in IPF remains to be resolved. During developmental and disease settings, the phenotypic conversion of the epithelium is under tight transcriptional control, however, the transcription factors eliciting EMT in IPF have yet to be identified. Putative roles for SNAI transcription factors as regulators of EMT during development and a wide variety of diseases including cancer and organ fibrosis have been documented. This study is based on the hypothesis that in AECs, TGF-beta1-induced SNAI transcription factors facilitate the acquisition of new morphology and motility, based on their ability to influence EMT marker gene expression. Thus, the objective of this study was to analyze the molecular mediators of TGF-beta1-induced EMT in vitro, in human A549 and primary mouse AT2 cells, and to assess their contribution to the development of fibrosis in experimental and idiopathic pulmonary fibrosis in vivo. Immunofluorescent costaining of Tjp1 and a-SMA (an epithelial and mesenchymal marker, respectively) demonstrated TGF-beta1-induced EMT in AECs. Furthermore, in vitro, TGF-beta1 treatment increased the expression and nuclear accumulation of the zinc finger transcription factors SNAI1 (Snail) and SNAI2 (Slug), as assessed by RT-PCR and immunofluorescence. Ectopic expression of SNAI1 and SNAI2 proteins was sufficient to induce EMT in A549 cells, even in the absence of TGF-beta1 stimulation. In contrast, the siRNA-mediated depletion of SNAI1 and SNAI2 attenuated TGF-b1-induced AEC migration and EMT in A549 cells. The detection of EMT in vitro, with an increase in SNAI transcription factors was substantiated in vivo in the bleomycin model of pulmonary fibrosis early in disease. In vivo, SNAI expression was elevated in primary AECs isolated from fibrotic lungs, seven days after bleomycin challenge. An indication of occurrence of EMT with an increase in SNAI transcription factors was also corroborated in IPF patient lungs compared to control lungs. Furthermore, the occurrence of EMT, as well as the involvement of transcriptional control of SNAI factors was clarified in a unilateral ureteral obstruction (UUO) mouse model of renal fibrosis. This study shows that (1) TGF-beta1-induced EMT in alveolar epithelial cells is accompanied by elevated expression of SNAI transcription factors, (2) EMT in AECs is essentially controlled by SNAI transcription factors, as ectopic expression of SNAI1 and SNAI2 triggers EMT, whereas depletion of these factors abrogates TGF-beta1-induced EMT, (3) increased expression of these zinc finger transcription factors are detected in an experimental model of lung fibrosis, with indication of the occurrence of EMT, (4) SNAI1 and SNAI2 upregulation have important implications for the development of IPF, (5) the detection of SNAI transcription factors early in EMT in a UUO model of renal fibrosis and the inhibition of EMT by leukocyte blocker treatment further emphasizes the significance of SNAI transcription factors in EMT as a causal factor in disease. Thus, reversal and/or inhibition of EMT may present a valid therapeutic option in lung fibrosis.Die idiopathische pulmonale Fibrose (IPF) ist eine fatale interstitielle Lungenerkrankung, die durch Ansammlung von aktivierten Myofibroblasten und verstärkter extrazellulärer Matrixbildung gekennzeichnet ist. An diesem Vorgang ist der TGF-beta Signalweg beteiligt. Zudem induziert TGF-beta1 die epitheliale-mesenchymale Transition (EMT), die reversible phänotypische Umwandlung von epithelialen zu fibroblasten-ähnlichen Zellen. Kürzlich konnte EMT in Alveolarepithelzellen in der Lungenfibrose gezeigt werden. Während des Entwicklungsprozesses und bei vielen Krankheiten, wie Krebs und Fibrose unterliegt die phänotypische Umwandlung des Epithels strengen Transkriptionsvorgängen. Die einzelnen Transkriptionsfaktoren, die EMT in IPF hervorrufen, sind noch zu identifizieren. Die vermeintliche Rolle von SNAI Transkriptionsfaktoren als EMT Regulatoren in anderen Erkrankungen wurde schon dokumentiert. Die vorliegende Studie basiert auf der Hypothese, dass TGF-beta-induzierte SNAI Transkriptionsfaktoren in alveolaren Epithelzellen EMT-Markerexpression beeinflussen können und somit die Morphologie und Motilität der Zellen verändern können. Das Ziel dieser Arbeit war es, die molekularen Mechanismen von TGF-beta1-induzierter EMT in vitro, in humanen A549 Zellen und primären murinen Alveolarepithelzellen zu untersuchen. Dabei galt es ihre Beteiligung bei der Entwicklung in experimenteller und idiopathischer pulmonaler Fibrose in vivo zu beurteilen. Die TGF-beta1-induzierte EMT in Alveolarepithelzellen wurde anhand epithelialer Proteinen (Tjp1) und mesenchymaler Proteinen (a-SMA) durch Immunfluoreszenz nachgewiesen. Des weiteren wurde in vitro, nach Behandlung mit TGF-beta1, eine erhöhte Expression und nukleäre Ansammlung der Zinkfingerproteine SNAI1 und SNAI2 mittels quantitative RT-PCR und Immunfluoreszenz ermittelt. Die ektopische Expression von SNAI1 und 2 war bereits ausreichend, um EMT in A549 Zellen zu induzieren; auch ohne TGF-beta1 Stimulation. Im Gegensatz dazu, führte eine siRNA vermittelte Verringerung von SNAI1 und 2 zur Abschwächung einer TGF-beta1 induzierten Migration von Alveolarepithelzellen und EMT in A549 Zellen. Der Nachweis von EMT in vitro mit Anstieg an SNAI Transkriptionsfaktoren konnte in vivo im Bleomycinmodell der pulmonalen Fibrose belegt werden. Sieben Tage nach Bleomycin Exposition zeigten primäre Alveolarepithelzellen aus fibrotischen Lungen eine Erhöhung der SNAI Expression. Auch in IPF Lungen bestätigte sich dieses Ergebnis im Vergleich zu gesunden Lungen. Außerdem konnte EMT, sowie die Beteiligung von transkriptionskontrollierten SNAI Faktoren im Mausmodell der renalen Fibrose, einer unilateralen urethralen Obstruktion (UUO), nachgewiesen werden. Zusammenfassend konnte gezeigt werden, dass die TGF-beta1-induzierte EMT in Alveolarepithelzellen mit einer erhöhten Expression von SNAI Transkriptionsfaktoren einher geht. Die Überexpression von SNAI1 und 2 zeigte, dass diese Transkriptionsfaktoren den EMT-Prozeß im wesentlichen triggern, während eine Verringerung dieser Faktoren die TGF-beta1-induzierte EMT aufhebt. Die Erhöhung dieser Zinkfingerproteine, mit dem Hinweis auf EMT, konnte zudem in einem experimentellen Modell der Lungenfibrose nachgewiesen werden. Diese Beobachtung bestätigte sich ebenfalls in humanen IPF-Proben. Der Nachweis von SNAI Transkriptionsfaktoren in EMT im renalen Fibrosemodell, der UUO, und die Hemmung von EMT durch Behandlung mit Leukozytenblockern, heben die Signifikanz dieser Faktoren als Ursache im Krankheitsmechanismus hervor. Eine Aufhebung und/oder Hemmung von EMT könnte somit eine Therapiemöglichkeit in der Behandlung der Lungenfibrose darstellen

Combined inhibition of TGF-beta 1-induced EMT and PD-L1 silencing re-sensitizes hepatocellular carcinoma to sorafenib treatment

Hepatocellular carcinoma (HCC) is the most common type of primary hepatic malignancy. HCC is one of the leading causes of cancer deaths worldwide. The oral multi-tyrosine kinase inhibitor Sorafenib is the standard first-line therapy in patients with advanced unresectable HCC. Despite the significant survival benefit in HCC patients post treatment with Sorafenib, many patients had progressive disease as a result of acquiring drug resistance. Circumventing resistance to Sorafenib by exploring and targeting possible molecular mechanisms and pathways is an area of active investigation worldwide. Epithelial-to-mesenchymal transition (EMT) is a cellular process allowing epithelial cells to assume mesenchymal traits. HCC tumour cells undergo EMT to become immune evasive and develop resistance to Sorafenib treatment. Immune checkpoint molecules control immune escape in many tumours, including HCC. The aim of this study is to investigate whether combined inhibition of EMT and immune checkpoints can re-sensitise HCC to Sorafenib treatment. Post treatment with Sorafenib, HCC cells PLC/PRF/5 and Hep3B were monitored for induction of EMT and immune checkpoint molecules using quantitative reverse transcriptase (qRT)- PCR, western blot, immunofluorescence, and motility assays. The effect of combination treatment with SB431542, a specific inhibitor of the transforming growth factor (TGF)-β receptor kinase, and siRNA mediated knockdown of programmed cell death protein ligand-1 (PD-L1) on Sorafenib resistance was examined using a cell viability assay. We found that three days of Sorafenib treatment activated EMT with overexpression of TGF-β1 in both HCC cell lines. Following Sorafenib exposure, increase in the expression of PD-L1 and other immune checkpoints was observed. SB431542 blocked the TGF-β1-mediated EMT in HCC cells and also repressed PD-L1 expression. Likewise, knockdown of PD-L1 inhibited EMT. Moreover, the sensitivity of HCC cells to Sorafenib was enhanced by combining a blockade of EMT with SB431542 and knockdown of PD-L1 expression. Sorafenib-induced motility was attenuated with the combined treatment of SB431542 and PD-L1 knockdown. Our findings indicate that treatment with Sorafenib induces EMT and expression of immune checkpoint molecules, which contributes to Sorafenib resistance in HCC cells. Thus, the combination treatment strategy of inhibiting EMT and immune checkpoint molecules can re-sensitise HCC cells to Sorafenib

Promoter hypomethylation of NY-ESO-1, association with clinicopathological features and PD-L1 expression in non-small cell lung cancer.

Cancer-Testis antigens (CTA) are immunogenic molecules with normal tissue expression restricted to testes but with aberrant expression in up to 30% of non-small cell lung cancers (NSCLCs). Regulation of CTA expression is mediated in part through promoter DNA methylation. Recently, immunotherapy has altered treatment paradigms in NSCLC. Given its immunogenicity and ability to be re-expressed through demethylation, NY-ESO-1 promoter methylation, protein expression and its association with programmed death receptor ligand-1 (PD-L1) expression and clinicopathological features were investigated. Lung cancer cell line demethylation resulting from 5-Aza-2'-deoxycytidine treatment was associated with both NY-ESO-1 and PD-L1 re-expression in vitro but not increased chemosensitivity. NY-ESO-1 hypomethylation was observed in 15/94 (16%) of patient samples and associated with positive protein expression (P < 0.0001). In contrast, PD-L1 expression was observed in 50/91 (55%) but strong expression in only 12/91 (13%) cases. There was no association between NY-ESO-1 and PD-L1 expression, despite resultant re-expression of both by 5-Aza-2'-deoxycytidine. Importantly, NY-ESO-1 hypomethylation was found to be an independent marker of poor prognosis in patients not treated with chemotherapy (HR 3.59, P = 0.003) in multivariate analysis. In patients treated with chemotherapy there were no differences in survival associated with NY-ESO-1 hypomethylation. Collectively, these results provided supporting evidence for the potential use of NY-ESO-1 hypomethylation as a prognostic biomarker in stage 3 NSCLCs. In addition, these data highlight the potential to incorporate demethylating agents to enhance immune activation, in tumours currently devoid of immune infiltrates and expression of immune checkpoint genes

Monitoring Immune Checkpoint Regulators as Predictive Biomarkers in Hepatocellular Carcinoma

The global burden of hepatocellular carcinoma (HCC), one of the frequent causes of cancer-related deaths worldwide, is rapidly increasing partly due to the limited treatment options available for this disease and recurrence due to therapy resistance. Immune checkpoint inhibitors that are proved to be beneficial in the treatment of advanced melanoma and other cancer types are currently in clinical trials in HCC. These ongoing trials are testing the efficacy and safety of a few select checkpoints in HCC. Similar to observations in other cancers, these immune checkpoint blockade treatments as monotherapy may benefit only a fraction of HCC patients. Studies that assess the prevalence and distribution of other immune checkpoints/modulatory molecules in HCC have been limited. Moreover, robust predictors to identify which HCC patients will respond to immunotherapy are currently lacking. The objective of this study is to perform a comprehensive evaluation on different immune modulators as predictive biomarkers to monitor HCC patients at high risk for poor prognosis. We screened publically available HCC patient databases for the expression of previously well described immune checkpoint regulators and evaluated the usefulness of these immune modulators to predict high risk, patient overall survival and recurrence. We also identified the immune modulators that synergized with known immune evasion molecules programmed death receptor ligand-1 (PD-L1), programmed cell death protein-1 (PD-1), and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and correlated with worse patient outcomes. We evaluated the association between the expression of epithelial-to-mesenchymal transition (EMT) markers and PD-L1 in HCC patient tumors. We also examined the relationship of tumor mutational burden with HCC patient survival. Notably, expression of immune modulators B7-H4, PD-L2, TIM-3, and VISTA were independently associated with worse prognosis, while B7-H4, CD73, and VISTA predicted low recurrence-free survival. Moreover, the prognosis of patients expressing high PD-L1 with high B7-H4, TIM-3, VISTA, CD73, and PD-L2 expression was significantly worse. Interestingly, PD-L1 expression in HCC patients in the high-risk group was closely associated with EMT marker expression and prognosticates poor survival. In HCC patients, high tumor mutational burden (TMB) predicted worse patient outcomes than those with low TMB

Epithelial-to-mesenchymal plasticity of cancer stem cells: therapeutic targets in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) remains one of the most common and lethal malignancies worldwide despite the development of various therapeutic strategies. A better understanding of the mechanisms responsible for HCC initiation and progression is essential for the development of more effective therapies. The cancer stem cell (CSC) model has provided new insights into the development and progression of HCC. CSCs are specialized tumor cells that are capable of self-renewal and have long-term repopulation potential. As they are important mediators of tumor proliferation, invasion, metastasis, therapy resistance, and cancer relapse, the selective targeting of this crucial population of cells has the potential to improve HCC patient outcomes and survival. In recent years, the role of epithelial-to-mesenchymal transition (EMT) in the advancement of HCC has gained increasing attention. This multi-step reprograming process resulting in a phenotype switch from an epithelial to a mesenchymal cellular state has been closely associated with the acquisition of stem cell-like attributes in tumors. Moreover, CSC mediates tumor metastasis by maintaining plasticity to transition between epithelial or mesenchymal states. Therefore, understanding the molecular mechanisms of the reprograming switches that determine the progression through EMT and generation of CSC is essential for developing clinically relevant drug targets. This review provides an overview of the proposed roles of CSC in HCC and discusses recent results supporting the emerging role of EMT in facilitating hepatic CSC plasticity. In particular, we discuss how these important new insights may facilitate rational development of combining CSC- and EMT-targeted therapies in the future

Epithelial-to-mesenchymal plasticity of cancer stem cells: Therapeutic targets in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) remains one of the most common and lethal malignancies worldwide despite the development of various therapeutic strategies. A better understanding of the mechanisms responsible for HCC initiation and progression is essential for the development of more effective therapies. The cancer stem cell (CSC) model has provided new insights into the development and progression of HCC. CSCs are specialized tumor cells that are capable of self-renewal and have long-term repopulation potential. As they are important mediators of tumor proliferation, invasion, metastasis, therapy resistance, and cancer relapse, the selective targeting of this crucial population of cells has the potential to improve HCC patient outcomes and survival. In recent years, the role of epithelial-to-mesenchymal transition (EMT) in the advancement of HCC has gained increasing attention. This multi-step reprograming process resulting in a phenotype switch from an epithelial to a mesenchymal cellular state has been closely associated with the acquisition of stem cell-like attributes in tumors. Moreover, CSC mediates tumor metastasis by maintaining plasticity to transition between epithelial or mesenchymal states. Therefore, understanding the molecular mechanisms of the reprograming switches that determine the progression through EMT and generation of CSC is essential for developing clinically relevant drug targets. This review provides an overview of the proposed roles of CSC in HCC and discusses recent results supporting the emerging role of EMT in facilitating hepatic CSC plasticity. In particular, we discuss how these important new insights may facilitate rational development of combining CSC- and EMT-targeted therapies in the future

Seek and destroy: targeted adeno-associated viruses for gene delivery to hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer with high incidence globally. Increasing mortality and morbidity rates combined with limited treatment options available for advanced HCC press for novel and effective treatment modalities. Gene therapy represents one of the most promising therapeutic options. With the recent approval of herpes simplex virus for advanced melanoma, the field of gene therapy has received a major boost. Adeno-associated virus (AAV) is among the most widely used and effective viral vectors today with safety and efficacy demonstrated in a number of human clinical trials. This review identifies the obstacles for effective AAV based gene delivery to HCC which primarily include host immune responses and off-target effects. These drawbacks could be more pronounced for HCC because of the underlying liver dysfunction in most of the patients. We discuss approaches that could be adopted to tackle these shortcomings and manufacture HCC-targeted vectors. The combination of transductional targeting by modifying the vector capsid and transcriptional targeting using HCC-specific promoters has the potential to produce vectors which can specifically seek HCC and deliver therapeutic gene without significant side effects. Finally, the identification of novel HCC-specific ligands and promoters should facilitate and expedite this process

Association between hereditary hemochromatosis and hepatocellular carcinoma: a comprehensive review

Hepatocellular carcinoma (HCC) is a significant global health problem with high morbidity and mortality. Its incidence is increasing exponentially worldwide with a close overlap between annual incidence and death rates. Even though significant advances have been made in HCC treatment, fewer than 20% of patients with HCC are suitable for potentially curative treatment. Hereditary hemochromatosis (HH) is an important genetic risk factor for HCC. HH is an autosomal recessive disorder of iron metabolism, characterised by elevated iron deposition in most organs including the liver, leading to progressive organ dysfunction. HCC is a complication of HH, nearly always occurring in patients with cirrhosis and contributes to increased mortality rates. Identifying the susceptibility of development of HCC in HH patients has gained much traction. This review summarises the current knowledge with regard to the association of HH and HCC in order to encourage further research. In this review, we focus particularly on HFE gene-related HH. Herein, we highlight and discuss emerging clinical research which addresses the prevalence of HCC in HH patients and the coincidence of HH with other risk factors for HCC development. We also focus on the therapeutic tools in the management of HCC associated with HH