The role of pregnancy associated plasma protein-A in triple negative breast cancer: a promising target for achieving clinical benefits.

Pregnancy associated plasma protein-A (PAPP-A) plays an integral role in breast cancer (BC), especially triple negative breast cancer (TNBC). This subtype accounts for the most aggressive BC, possesses high tumor heterogeneity, is least responsive to standard treatments and has the poorest clinical outcomes. There is a critical need to address the lack of effective targeted therapeutic options available. PAPP-A is a protein that is highly elevated during pregnancy. Frequently, higher PAPP-A expression is detected in tumors than in healthy tissues. The increase in expression coincides with increased rates of aggressive cancers. In BC, PAPP-A has been demonstrated to play a role in tumor initiation, progression, metastasis including epithelial-mesenchymal transition (EMT), as well as acting as a biomarker for predicting patient outcomes. In this review, we present the role of PAPP-A, with specific focus on TNBC. The structure and function of PAPP-A, belonging to the pappalysin subfamily, and its proteolytic activity are assessed. We highlight the link of BC and PAPP-A with respect to the IGFBP/IGF axis, EMT, the window of susceptibility and the impact of pregnancy. Importantly, the relevance of PAPP-A as a TNBC clinical marker is reviewed and its influence on immune-related pathways are explored. The relationship and mechanisms involving PAPP-A reveal the potential for more treatment options that can lead to successful immunotherapeutic targets and the ability to assist with better predicting clinical outcomes in TNBC.

Crosstalk between Immune Checkpoint Modulators, Metabolic Reprogramming and Cellular Plasticity in Triple-Negative Breast Cancer.

Breast cancer is one of the major causes of mortality in women worldwide. Accounting for 15-20% of all breast cancer diagnoses, the triple-negative breast cancer (TNBC) subtype presents with an aggressive clinical course, heightened metastatic potential and the poorest short-term prognosis. TNBC does not respond to hormonal therapy, only partially responds to radio- and chemotherapy, and has limited targeted therapy options, thus underlining the critical need for better therapeutic treatments. Although immunotherapy based on immune checkpoint inhibition is emerging as a promising treatment option for TNBC patients, activation of cellular plasticity programs such as metabolic reprogramming (MR) and epithelial-to-mesenchymal transition (EMT) causes immunotherapy to fail. In this report, we review the role of MR and EMT in immune checkpoint dysregulation in TNBCs and specifically shed light on development of novel combination treatment modalities for this challenging disease. We highlight the clinical relevance of crosstalk between MR, EMT, and immune checkpoints in TNBCs.

CD73 and PD-L1 as Potential Therapeutic Targets in Gallbladder Cancer.

Gallbladder cancer (GBC) is one of the most common and aggressive biliary tract cancers with a dismal prognosis. Ongoing clinical trials are evaluating a few selected immune checkpoint inhibitors (ICIs) as monotherapy for the treatment of GBC patients. However, only a subset of patients benefits from these treatments. To improve ICI therapy response, molecular mechanisms that confer resistance to immune checkpoint (IC) blockade needs to be explored. Epithelial-to-mesenchymal transition (EMT) program and cancer stem cells (CSCs) have been implicated as key processes that confer ICI treatment resistance. However, in GBC the EMT-CSC-IC axis has not yet been clearly elucidated. This study aims to examine the aberrant expression of ICs associated with CSC and EMT. We successfully enriched CSCs by utilizing a 3-dimensional culture system and established a reversible EMT model with human GBC NOZ cell line. Notably, ICs CD73 and PD-L1 were closely associated with both CSC and EMT phenotypes. Knockdown of CD73 or PD-L1 reduced the proliferative and motile abilities of both adherent monolayers and anchorage-free spheroids. In conclusion, blocking CD73 and PD-L1 offer a promising therapeutic strategy for targeting highly aggressive populations with CSC and EMT phenotype to improve GBC patient prognosis.

Dual Targeting of Sorafenib-Resistant HCC-Derived Cancer Stem Cells.

Sorafenib, an oral multi-tyrosine kinase inhibitor, has been the first-line therapy for the treatment of patients with advanced HCC, providing a survival benefit of only three months in approximately 30% of patients. Cancer stem cells (CSCs) are a rare tumour subpopulation with self-renewal and differentiation capabilities, and have been implicated in tumour growth, recurrence and drug resistance. The process of epithelial-to-mesenchymal transition (EMT) contributes to the generation and maintenance of the CSC population, resulting in immune evasion and therapy resistance in several cancers, including HCC. The aim of this study is to target the chemoresistant CSC population in HCC by assessing the effectiveness of a combination treatment approach with Sorafenib, an EMT inhibitor and an immune checkpoint inhibitor (ICI). A stem-cell-conditioned serum-free medium was utilised to enrich the CSC population from the human HCC cell lines Hep3B, PLC/PRF/5 and HepG2. The anchorage independent spheres were characterised for CSC features. The human HCC-derived spheres were assessed for EMT status and expression of immune checkpoint molecules. The effect of combination treatment with SB431542, an EMT inhibitor, and siRNA-mediated knockdown of programmed cell death protein ligand-1 (PD-L1) or CD73 along with Sorafenib on human HCC-derived CSCs was examined with cell viability and apoptosis assays. The three-dimensional spheres enriched from human HCC cell lines demonstrated CSC-like features. The human HCC-derived CSCs also exhibited the EMT phenotype along with the upregulation of immune checkpoint molecules. The combined treatment with SB431542 and siRNA-mediated PD-L1 or CD73 knockdown effectively enhanced the cytotoxicity of Sorafenib against the CSC population compared to Sorafenib alone, as evidenced by the reduced size and proliferation of spheres. Furthermore, the combination treatment of Sorafenib with SB431542 and PD-L1 or CD73 siRNA resulted in an increased proportion of an apoptotic population, as evidenced by flow cytometry analysis. In conclusion, the combined targeting of EMT and immune checkpoint molecules with Sorafenib can effectively target the CSC tumour subpopulation.

Prognostic Role of Immune Checkpoint Regulators in Cholangiocarcinoma: A Pilot Study.

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy associated with steadily increasing incidence and poor prognosis. Ongoing clinical trials are assessing the effectiveness and safety of a few immune checkpoint inhibitors (ICIs) in CCA patients. However, these ICI treatments as monotherapies may be effective for a proportion of patients with CCA. The prevalence and distribution of other immune checkpoints (ICs) in CCA remain unclear. In this pilot study, we screened databases of CCA patients for the expression of 19 ICs and assessed the prognostic significance of these ICs in CCA patients. Notably, expression of immune modulator IDO1 and PD-L1 were linked with poor overall survival, while FASLG and NT5E were related to both worse overall survival and progression-free survival. We also identified immune modulators IDO1, FASLG, CD80, HAVCR2, NT5E, CTLA-4, LGALS9, VTCN1 and TNFRSF14 that synergized with PD-L1 and correlated with worse patient outcomes. In vitro studies revealed that the expression of ICs was closely linked with aggressive CCA subpopulations, such as cancer stem cells and cells undergoing TGF-ß and TNF-α-mediated epithelial-to-mesenchymal transition. These findings suggest that the aforementioned IC molecules may serve as potential prognostic biomarkers and drug targets in CCA patients, leading to lasting and durable treatment outcomes.

Immune checkpoint molecules are regulated by transforming growth factor (TGF)-ß1-induced epithelial-to-mesenchymal transition in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with a high mortality rate. Epithelial-to-mesenchymal transition (EMT) confers cancer cells with immune evasive ability by modulating the expression of immune checkpoints in many cancers. Thus, the aim of our study is to examine the interplay between EMT and immune checkpoint molecules in HCC. A reversible EMT model was utilised with transforming growth factor (TGF)-ß1 as an EMT inducer for HCC cell lines Hep3B and PLC/PRF/5. HCC cells were treated with TGF-ß1 for 72 h and the EMT status and immune checkpoint expression were examined. In addition, the migratory ability of HCC cells were examined using wound healing and transwell migration assays in the reversible EMT model. siRNA-mediated knockdown of immune checkpoint molecule, B7-H3, was further utilised to validate the association between TGF-ß1-mediated EMT and immune checkpoint expression in HCC. In addition, a web-based platform, SurvExpress, was utilised to evaluate the association between expression of TGF-ß1 in combination with immune checkpoint molecules and overall survival in HCC patients. We observed induction of EMT upon treatment of HCC cells with TGF-ß1 revealed by reduced expression of epithelial markers along with increased expression of mesenchymal markers. Withdrawal of TGF-ß1 reversed the process of EMT with elevated expression of epithelial markers and reduced expression of mesenchymal markers. TGF-ß1 treatment elevated the migratory potential of HCC cells which was reversed following reversal assay. Notably, during TGF-ß1-induced EMT, there was upregulation of immune checkpoint molecules PD-L1 and B7-H3. However, the reversal of EMT decreased the expression of PD-L1 and B7-H3. In addition, TGF-ß1 driven EMT was reversed following knockdown of B7-H3 in both HCC cells further validating the interplay between TGF-ß1-mediated EMT and immune checkpoint expression in HCC. Furthermore, the coordinate expression of TGF-ß1 with PD-L1 (p=0.01487) and B7-H3 (p=0.009687) was correlated with poor overall survival in 422 HCC patients. Our study has demonstrated a close association between TGF-ß1-mediated EMT and regulation of immune checkpoints in HCC.

Combined Inhibition of TGF-ß1-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)-ß 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-ß1 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-ß1-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.

Aberrant pregnancy-associated plasma protein-A expression in breast cancers prognosticates clinical outcomes.

Elevated levels of pregnancy-associated plasma protein-A (PAPP-A) have been implicated in the pathogenesis of various malignancies, including breast cancers. Breast cancer is one of the most frequent carcinomas and is the second most common cancer type detected in women of child-bearing age. Throughout pregnancy PAPP-A is produced and secreted by the placental syncytiotrophoblast cells; co-incidentally pregnancy-associated breast cancers often have an aggressive clinical course. The components of the PAPP-A/IGF axis was assessed in a panel of breast cancer cell lines. Using neutralising antibodies the impact of PAPP-A/IGF axis on cell motility was evaluated. PAPP-A was expressed in four of the twelve breast cancer cell lines tested. Blocking PAPP-A and IGFBP4 with neutralising antibodies significantly decreased motiliy of MDA-MB-231 cells. Upregulation of PAPP-A expression in breast tumours resulted in a trend towards worse overall survival. Notably, PAPP-A expression also positively correlated with epithelial-to-mesenchymal transition markers. In conclusion, these results indicate that PAPP-A plays an important role in breast cancer progression and it may be a promising therapeutic target in breast cancer.

TNF­α­mediated epithelial­to­mesenchymal transition regulates expression of immune checkpoint molecules in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fastest growing cause of cancer­related deaths globally. Epithelial­to­mesenchymal transition (EMT) is a cellular process that confers HCC tumor cells with the ability to evade the immune system. Immune escape in most tumors, including HCC, is controlled by immune checkpoint molecules. The aim of the present study was to investigate the association between EMT and immune checkpoint in HCC, and identify novel therapeutic targets for HCC. An in vitro model of reversible EMT was utilized based on cytokine tumor necrosis factor (TNF)­α treatment of HCC cell lines Hep3B and PLC/PRF/5. Hep3B and PLC/PRF/5 cells were treated with TNF­α, and the EMT status and the expression of immune checkpoint molecules was assessed by reverse transcription­quantitative PCR, western blotting and immunofluorescence. To confirm an association between EMT and immune modulators, cells were exposed to culture medium with TNF­α for 3 days to induce EMT, following which a reversal assay was performed. The expression of immune modulators and mesenchymal­to­epithelial transition (MET) status was investigated upon reversal of EMT. Furthermore, SurvExpress, a web­based platform was utilized to analyze survival and recurrence in a dataset of patients with HCC. TNF­α treatment for 3 days induced EMT in Hep3B and PLC/PRF/5 cells, as demonstrated by the downregulation of epithelial markers along with upregulation in mesenchymal markers. An EMT reversal assay was able to induce MET by increasing epithelial markers and decreasing mesenchymal markers. TNF­α­induced EMT led to the upregulation of immune modulators, including programmed death receptor ligand (PD­L)1, PD­L2, CD73 and B7­H3. In contrast, reversal of EMT suppressed the expression of PD­L1, PD­L2, CD73 and B7­H3. In addition, high expression of TNF­α and PD­L1 in 422 patients with HCC was associated with poor overall survival. The coordinate expression of TNF­α with PD­L2 in this patient cohort was associated with increased HCC recurrence. In conclusion, the present study demonstrated a close association between immune modulator expression and EMT induction/reversal driven by TNF­α.

Spotlight on Bortezomib: potential in the treatment of hepatocellular carcinoma.

INTRODUCTION: This study reviews the evidence for the use of Bortezomib (BZB), a first-in-class proteasome inhibitor in advanced Hepatocellular carcinoma (HCC). This review aims to delineate the role of BZB within the management of non-surgical and metastatic HCC, either as an alternative or as an adjunct to the current treatment paradigm. AREAS COVERED: In addition to BZB pharmacology and mechanism of action, safety and tolerance profiles of the drug obtained from clinical trials are explored. The utility of BZB as a therapeutic agent either alone or in combination with other therapies against HCC, including its application in both preclinical and clinical settings has been reviewed. In particular, we highlight the importance of preclinical evaluation of BZB as a combinatorial agent in synergism with other therapies for the use in the management of HCC. EXPERT OPINION: There has been much interest surrounding the use of BZB, a first-in-class proteasome inhibitor for HCC therapy. The discernment of outcomes of BZB clinical trials for HCC need to take into consideration the disease-specific factors that can affect survival outcomes including patient selection and aetiological differences. Further preclinical testing of BZB in combination with other therapeutic modalities can be important for eliciting enhanced anti-HCC effects.

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.

Evaluation of the Glypican 3 promoter for transcriptional targeting of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major health problem as evidenced by its increasing incidence and high morbidity and mortality rates. Most patients with HCC have underlying liver disease and dysfunction which limits the current therapeutic options. Treatments that spare the liver and destroy the HCC are needed. Targeting transcriptional differences between HCC and liver cells may provide this therapeutic window. In this study, we examine the potential of the Glypican 3 (GPC3) promoter as a targeting strategy. GPC3 is an oncofetal protein belonging to the proteoglycan family which is normally only expressed during fetal development. However, in HCC, the expression of this protein is reactivated. Here, we show that GPC3 is expressed primarily in HCC and not in normal liver lines. We show that the GPC3 promoter can be used to drive expression of significantly more luciferase and eYFP in HCC cell lines compared to normal liver cells. Further, we show that vectors containing cytosine deaminase (CD) under GPC3 promotor control induced significantly more killing of HCC cell lines after treatment with 5-FC compared to normal liver cell lines. These data suggest that transcriptionally targeted delivery of transgene in HCC cells can be achieved using the GPC3 promoter and this targeting strategy produces limited toxicity to normal liver cells.

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.

Murine hepatocellular carcinoma derived stem cells reveal epithelial-to-mesenchymal plasticity.

AIM: To establish a model to enrich and characterize stem-like cells from murine normal liver and hepatocellular carcinoma (HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesenchymal transition (EMT). METHODS: In this study, we utilized a stem cell conditioned serum-free medium to enrich stem-like cells from mouse HCC and normal liver cell lines, Hepa 1-6 and AML12, respectively. We isolated the 3-dimensional spheres and assessed their stemness characteristics by evaluating the RNA levels of stemness genes and a cell surface stem cell marker by quantitative reverse transcriptase-PCR (qRT-PCR). Next, we examined the relationship between stem cells and EMT using qRT-PCR. RESULTS: Three-dimensional spheres were enriched by culturing murine HCC and normal hepatocyte cell lines in stem cell conditioned serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor and heparin sulfate. The 3-dimensional spheres had enhanced stemness markers such as Klf4 and Bmi1 and hepatic cancer stem cell (CSC) marker Cd44 compared to parental cells grown as adherent cultures. We report that epithelial markers E-cadherin and ZO-1 were downregulated, while mesenchymal markers Vimentin and Fibronectin were upregulated in 3-dimensional spheres. The 3-dimensional spheres also exhibited changes in expression of Snai, Zeb and Twist family of EMT transcription factors. CONCLUSION: Our novel method successfully enriched stem-like cells which possessed an EMT phenotype. The isolation and characterization of murine hepatic CSCs could establish a precise target for the development of more effective therapies for HCC.

Epithelial-to-Mesenchymal Transition: A Mediator of Sorafenib Resistance in Advanced Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide and its incidence is steadily rising. Currently, sorafenib remains the only approved standard treatment for patients with advanced HCC, as it has proven to increase survival in these patients. However, clinical and preclinical observations indicate that sorafenib treatment may have limited efficacy due to tumor progression from the rapid development of acquired resistance. Elucidation of the underlying mechanisms of evasive resistance to sorafenib is a major challenge in HCC research. In recent years, the role of epithelial-to-mesenchymal transition (EMT) in the advancement of HCC and development of drug resistance has gained increasing attention. EMT is a developmental multistep molecular and cellular reprogramming process that is hijacked by cancer cells to enable aggressiveness. In this review, we provide an overview of the currently available preclinical studies on the EMT mechanisms underlying resistance to sorafenib treatment. Recent studies report enrichment of cancer stem cells (CSCs) after sorafenib treatment. Interestingly, EMT process has been implicated in the generation of CSCs associated with therapy resistance. We discuss how combination of sorafenib with EMT inhibitors could enhance the clinical response to sorafenib, resulting in longer duration of responses, than observed with sorafenib monotherapy. In particular, we discuss how these new insights may facilitate rational development of combination therapies in the future to impact survival of patients with advanced HCC.

Two-photon dual imaging platform for in vivo monitoring cellular oxidative stress in liver injury.

Oxidative stress reflects an imbalance between reactive oxygen species (ROS) and antioxidants, which has been reported as an early unifying event in the development and progression of various diseases and as a direct and mechanistic indicator of treatment response. However, highly reactive and short-lived nature of ROS and antioxidant limited conventional detection agents, which are influenced by many interfering factors. Here, we present a two-photon sensing platform for in vivo dual imaging of oxidative stress at the single cell-level resolution. This sensing platform consists of three probes, which combine the turn-on fluorescent transition-metal complex with different specific responsive groups for glutathione (GSH), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl). By combining fluorescence intensity imaging and fluorescence lifetime imaging, these probes totally remove any possibility of crosstalk from in vivo environmental or instrumental factors, and enable accurate localization and measurement of the changes in ROS and GSH within the liver. This precedes changes in conventional biochemical and histological assessments in two distinct experimental murine models of liver injury. The ability to monitor real-time cellular oxidative stress with dual-modality imaging has significant implications for high-accurate, spatially configured and quantitative assessment of metabolic status and drug response.

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.

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.

Identifying and targeting determinants of melanoma cellular invasion.

Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells. This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion. We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells. These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro. Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells. Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients.

Dual Targeting of Bromodomain and Extraterminal Domain Proteins, and WNT or MAPK Signaling, Inhibits c-MYC Expression and Proliferation of Colorectal Cancer Cells.

Inhibitors of the bromodomain and extraterminal domain (BET) protein family attenuate the proliferation of several tumor cell lines. These effects are mediated, at least in part, through repression of c-MYC. In colorectal cancer, overexpression of c-MYC due to hyperactive WNT/ß-catenin/TCF signaling is a key driver of tumor progression; however, effective strategies to target this oncogene remain elusive. Here, we investigated the effect of BET inhibitors (BETi) on colorectal cancer cell proliferation and c-MYC expression. Treatment of 20 colorectal cancer cell lines with the BETi JQ1 identified a subset of highly sensitive lines. JQ1 sensitivity was higher in cell lines with microsatellite instability but was not associated with the CpG island methylator phenotype, c-MYC expression or amplification status, BET protein expression, or mutation status of TP53, KRAS/BRAF, or PIK3CA/PTEN Conversely, JQ1 sensitivity correlated significantly with the magnitude of c-MYC mRNA and protein repression. JQ1-mediated c-MYC repression was not due to generalized attenuation of ß-catenin/TCF-mediated transcription, as JQ1 had minimal effects on other ß-catenin/TCF target genes or ß-catenin/TCF reporter activity. BETi preferentially target super-enhancer-regulated genes, and a super-enhancer in c-MYC was recently identified in HCT116 cells to which BRD4 and effector transcription factors of the WNT/ß-catenin/TCF and MEK/ERK pathways are recruited. Combined targeting of c-MYC with JQ1 and inhibitors of these pathways additively repressed c-MYC and proliferation of HCT116 cells. These findings demonstrate that BETi downregulate c-MYC expression and inhibit colorectal cancer cell proliferation and identify strategies for enhancing the effects of BETi on c-MYC repression by combinatorial targeting the c-MYC super-enhancer. Mol Cancer Ther; 15(6); 1217-26. ©2016 AACR.