Molecular insights for overcoming Hepatocellular Carcinoma

2010/2011SUMMARY Introduction. Hepatocellular Carcinoma (HCC) ranks fifth in frequency of cancers in the world. Orthotopic Liver Transplantation (OLT) or liver resection represents the best treatments for HCC. However, most patients cannot be subjected to potential curative OLT or resection because of extensive tumor involvement of the liver, metastasis, invasion of the portal vein or advanced underlying hepatocellular disease at the time of diagnosis. Systemic chemotherapy or chemoembolization represent a good alternative for the treatment, however drug therapy of cancer in general is hampered by multidrug resistance (MDR) that is a phenomenon caused by the up-regulation of the ABC-transporters (ABC) leading to chemotherapy failure. To overcome these problems new therapeutic approaches, such gene therapy, are needed. Selective down-regulation of an essential and specific cancer gene such as telomerase (hTERT) could represent an emerging strategy that could prevent cancer progression and diminish numerous side effects derived from drug usage. The present study include two tasks whose aims are: Task 1: a) Assess if the extent of tumoral differentiation results in a different ABCB1, ABCC1 and ABCG2 expression. b) Assess whether the treatment with a chemotherapeutic drug(s) may affect the expression of the three ABC transporters under study. Task 2: To overcome the obstacle of MDR-induced chemoresistance using new therapeutic approaches such as gene therapy, silencing a cancer essential and specific gene. Results and discussion. Task 1: We assessed the ABCB1, ABCC1 and ABCG2 expression in three hepatic cell lines: IHH (non tumoral control), HuH7 (differentiated tumoral cells) and JHH6 (undifferentiated tumoral cells). Only ABCG2 expression correlates with the degree of tumoral differentiation. Through confocal microscopy analysis we observed that the Doxorubicin (Dox) is able to reach the cell’s nucleus within 10 min. After 24h and 48h Dox is completely concentrated into the nucleus where some nuclear damage occurs. The presence of damaged nuclei could explain the decreased mRNA in most of the ABCs under study. The treatment with Dox doses lower than the LC50 for 24h and 48h has different consequences for all the ABC considered in the three cell lines, with an mRNA expression pattern not in line with the protein one in most of the cases, suggesting that the possible mechanism that determines the ABCs protein upregulation in the tumoral cell lines (HuH7 and JHH6) is not the de-novo transcription but probably something related to the protein turnover. After the treatment ABCC1 protein expression increases in the tumoral cell lines but not in the non tumoral one (IHH). Regarding ABCB1 and ABCG2, these transporters seem to play a role only in HuH7 and JHH6 cells respectively. We were not able to correlate the tumorigenic potential of the two tumoral cell lines with the ABC expression since the different behaviour of ABCs and the different contribution to MDR. Thus in order to better clarify the contribution of each single ABC to MDR our future steps will consider the use specific inhibitors. Task 2: From our in vivo data, among four cancer related genes we selected hTERT as the best candidate for silencing experiments due to its exclusive expression in tumoral samples. A functional non-inflammatory siRNA targeting hTERT was designed: SirTel 1. Silencing experiments were conducted in JHH6 cell line. The hTERT silencing effect was dose dependent, at least at the three considered doses (25-50-100nM). For all the subsequent determinations the experimental concentration was 25nM. After 72h of silencing we observed a significant reduction in both hTERT mRNA expression and enzymatic activity (p<0.001). The effects observed in the cells after silencing are: - Morphological changes, from a fibroblast-like to an hepatocyte-like shape; - Increased albumin expression. The expression of this hepatic hallmark increases after silencing in JHH6 cells that, due to their poor degree of differentiation, at basal conditions do not express quantifiable levels of albumin. The peak of the higher albumin expression corresponds to the maximum hTERT silencing effect. - Decreased cell viability (p<0.01). Interestingly, the siRNA induced a reduction in cell viability higher than Dox. - Cell cycle arrest in G1 phase (p<0.01). All data were validated using a hTERT negative cell line (primary culture of human fibroblast). After 72h silencing, we observed that hTERT expression reaches its minimum, and the expression is recovered after 264h although it does not reach the initial expression levels. Re-exposing the cells to additional 25nM of siRNA induces a reduction of mRNA levels by 76% compared to the amount already present after the first treatment. Taken together all this results suggest the pivotal role of hTERT silencing in a HCC derived cell line. Therefore, hTERT represent a promising candidate for gene-therapy strategies in HCC.RIASSUNTO Introduzione. Il carcinoma epatico occupa il quinto posto per incidenza tra i diversi tipi di tumore a livello mondiale. Il trapianto di fegato o la resezione epatica rappresentano le terapie d’elezione per questo tipo di tumore, tuttavia molti dei pazienti non possono essere sottoposti a questo tipo di interventi visti l’estensione del tumore nell’organo, la presenza di metastasi, l’invasione portale e la presenza di altre disfunzioni epatiche al momento della diagnosi. La chemioterapia sistemica o la chemioembolizzazione rappresentano una valida alternativa per il trattamento della patologia, tuttavia l’utilizzo di farmaci chemioterapici può portare all’insorgenza di chemioresistenza (multidrug resi stance, MDR) determinando l’inefficacia del trattamento. Parte attiva del fenomeno della chemioresistenza è rappresentato dai trasportatori ACB che sono coinvolti nell’espulsione del farmaco dalla cellula, di fatto impedendone l’azione. Lo sviluppo di nuove terapie geniche potrebbe sostituire l’utilizzo di farmaci per il trattamento del tumore. L’inibizione selettiva di componenti essenziali e specifici per le cellule tumorali come lo è la Telomerasi può rappresentare una strategia emergente che potrebbe prevenire la progressione tumorale ed evitare l’insorgenza dei numerosi effetti collaterali che derivano dalla correnti terapie farmacologiche. Il presente studio comprende due task i cui obbiettivi sono i seguenti: Task 1: a) valutare se il diverso grado di differenziamento tumorale corrisponde ad una diversa espressione dei trasportatori ABCB1, ABCC1 e ABCG2. b) valutare se il trattamento farmacologico influisce sull’espressione dei trasportatori ABC analizzati in questo studio. Task 2: sviluppo di nuovi approcci terapeutici basati sulla terapia genica. Risultati e Discussion. Task 1: è stata valutata l’espressione di mRNA dei trasportatori ABCB1, ABCC1 e ABCG2 in tre linee cellulari epatiche: IHH (cellule epatiche immortalizzate, il controllo non tumorale), HuH7 (cellule tumorali differenziate) e JHH6 (cellule tumorali scarsamente differenziate). ABCB1 è il principale ABC espresso nelle Huh7 e solamente l’espressione di ABCG2 correla col grado di differenziamento tumorale delle cellule. Tramite l’utilizzo della microscopia confocale è stata osservata la capacità della Doxorubicina (Dox), uno tra i più utilizzati farmaci antineoplastici, di raggiungere il nucleo delle cellule entro 10 min dalla somministrazione. Trascorse 24h la Dox è totalmente localizzata all’interno del nucleo causando la morte delle cellule più sensibili. A 48h sono evidenti i danni nucleari e cellulari nelle cellule sopravissute. Il trattamento con dosi di Dox inferiori alla LC50 per 24h e 48h ha diverse conseguenze sull’espressione dei tre trasportatori nelle diverse linee cellulari studiate. In particolare in molti casi l’espressione di mRNA non correla con l’espressione proteica suggerendo che, in questi casi, la regolazione sia dovuta a fenomeni di diminuito turnover proteico piuttosto che di nuova sintesi. A seguito del trattamento con il farmaco, l’espressione di ABCC1 aumenta nelle sole linee cellulari tumorali (Huh7 e JHH6). ABCG2 sembra avere un ruolo rilevante nelle sole cellule JHH6 mentre ABCB1 è espresso unicamente nelle HuH7 dove vi è un aumento dell’espressione proteica a seguito di trattamenti con la Dox. In questo studio non è stato possibile correlare il potenziale tumorigenico delle linee cellulari con l’espressione degli ABC visto il loro peculiare profilo di espressione ed il loro diverso contributo nella chemioresistenza. Per questi motivi in futuro ci si propone di trattare le cellule con inibitori specifici per ciascun ABC al fine di meglio comprendere i loro diversi ruoli nell’insorgenza della resistenza alla Dox. Task 2: Da studi condotti in vivo, il gene della subunità catalitica della Telomerasi (hTERT) è stato selezionato per gli esperimenti di silenziamento genico vista la sua esclusiva espressione nei tessuti tumorali. Tramite analisi bioinformatiche è stato disegnato un siRNA anti-hTERT (SirTel 1) capace di evitare l’induzione di infiammazione nelle cellule che lo internalizzano. Gli esperimenti di silenziamento sono stati effettuati nella linea cellulare JHH6. Dopo 72h di silenziamento con 25nM di SirTel 1 si è osservata una riduzione significativa nell’espressione di hTERT e nella sua attività enzimatica (p<0,001). Gli effetti del silenziamento di hTERT riscontrati sono i seguenti: - Cambiamenti morfologici delle cellule silenziate le quali passano dall’avere un forma fibroblasto-simile ad una forma epatocita-simile. - Aumento dell’espressione di albumina, un marker epatocitario, il cui picco di espressione corrisponde al massimo effetto di silenziamento. - Diminuzione della vitalità cellulare (p<0,01). SirTel 1 risulta più efficace nel ridurre la vitalità cellulare della Dox, il farmaco antineoplastico più utilizzato nelle terapie tumorali. - Arresto del ciclo cellulare nella fase G1 (p<0,01). Tutti i risultati sono stati validati utilizzando cellule hTERT negative come i fibroblasti primari. Il silenziamento raggiunge i suoi massimi livelli trascorse le 72h dalla trasfezione e l’espressione della Telomerasi sembra ritornare ai livelli iniziali attorno alle 264h dalla iniziale somministrazione. La riesposizione con una seconda dose di siRNA riduce ancor più l’espressione di hTERT portandola a circa il 20% dell’espressione iniziale (p<0,001). Questi risultati dimostrano il ruolo fondamentale della Telomerasi per la sopravvivenza e la proliferazione delle cellule tumorali epatiche. Per questi motivi hTERT costituisce un candidato promettente per le future terapie geniche applicate all’HCC.XXIV Ciclo198

MiR-3201 as a Prognostic Blood Biomarker for Curative Treatments in Hepatocellular Carcinoma

Background: Hepatic resection, radiofrequency ablation (RF), and liver transplantation (LT) represent the only available curative treatments for early stage hepatocellular carcinoma (HCC). Various studies showed that the 5-year overall survival (OS) rate reaches ∼70% after resection and ∼60% after RF. Objective: To improve the success rate of curative therapies and consequently the OS, an improvement in patients' selection and management should be pursued. In this regard, microRNAs (miRNAs) can be helpful prognostic biomarkers. Materials and Methods: In this retrospective study, a miRNA array profiling was performed on 34 HCC blood samples which is collected before therapy (T0), 1 month (T1), and 6 months (T2) after curative treatments (resection and RF) to identify noninvasive biomarker candidates for therapy response and OS. MiRNAs were validated in 80 blood HCC samples using quantitative real-time PCR (qRT-PCR). Patients were divided into complete responder (CR) and partial responder and progressive disease (PRPD). Results: Among the selected miRNAs, miR-3201 is significantly associated with treatment response in the validation phase, showing a 23% reduction (P = .026) in CR compared to PRPD. MiR-3201 was able to distinguish CR from PRPD (area under the curve [AUC] = 0.69, 71% sensitivity, 70% specificity, P = .0036). Furthermore, lower levels of miR-3201 were associated with longer OS (hazard ratio [HR] = 2.61, P = .0006). Conclusions: Blood miR-3201 could be used as a prognostic biomarker for curative therapy response and OS in HCC

Serum AP-endonuclease 1 (sAPE1) as novel biomarker for hepatocellular carcinoma

Late diagnosis for Hepatocellular Carcinoma (HCC) remains one of the leading causes for the high mortality rate. The apurinic/apyrimidinic endonuclease 1 (APE1), an essential member of the base excision DNA repair (BER) pathway, contributes to cell response to oxidative stress and has other non-repair activities. In this study, we evaluate the role of serum APE1 (sAPE1) as a new diagnostic biomarker and we investigate the biological role for extracellular APE1 in HCC. sAPE1 level was quantified in 99 HCC patients, 50 non-HCC cirrhotic and 100 healthy controls. The expression level was significantly high in HCC (75.8 [67.3\u201387.9] pg/mL) compared to cirrhosis (29.8 [18.3\u201336.5] pg/mL] and controls (10.8 [7.5\u201313.2] pg/mL) (p < 0.001). The sAPE1 level corresponded with its protein expression in HCC tissue. sAPE1 had high diagnostic accuracy to differentiate HCC from cirrhotic (AUC = 0.87, sensitivity 88%, specificity 71%, cut-off of 36.3 pg/mL) and healthy subjects (AUC 0.98, sensibility 98% and specificity 83%, cut-off of 19.0 pg/mL). Recombinant APE1, exogenously added to JHH6 cells, significantly promotes IL-6 and IL-8 expression, suggesting a role of sAPE1 as a paracrine pro-inflammatory molecule, which may modulate the inflammatory status in cancer microenvironment. We described herein, for the first time to our knowledge, that sAPE1 might be considered as a promising diagnostic biomarker for HCC

Differences in circulating microRNA signature in Prader–Willi syndrome and non-syndromic obesity

Prader–Willi syndrome (PWS) represents the most common genetic-derived obesity disorder caused by the loss of expression of genes located on the paternal chromosome 15q11.2-q13. The PWS phenotype shows peculiar physical, endocrine and metabolic characteristics compared to those observed in non-syndromic essential obesity. Since miRNAs have now a well-established role in many molecular pathways, including regulatory networks related to obesity, this pilot study was aimed to characterize the expression of circulating miRNAs in PWS compared to essential obesity. The circulating miRNome of 10 PWS and 10 obese subjects, adequately matched for age, BMI and sex, was profiled throughout Genechip miRNA 4.0 microarray analysis. We identified 362 out of 2578 mature miRNAs to be expressed in serum of the studied population. The circulating miRNA signature significantly characterising the two populations include 34 differently expressed RNAs. Among them, miR-24-3p, miR-122 and miR-23a-3p highly differ between the two groups with a FC >10 in obese compared to PWS. In the obese subjects, miR-7107-5p, miR-6880-3p, miR-6793-3p and miR-4258 were associated to the presence of steatosis. A different signature of miRNAs significantly distinguished PWS with steatosis from PWS without steatosis, involving miR-619-5p, miR-4507, miR-4656, miR-7847-3p and miR-6782-5p. The miRNA target GO enrichment analysis showed the different pathway involved in these two different forms of obesity. Although the rarity of PWS actually represents a limitation to the availability of large series, the present study provides novel hints on the molecular pathogenesis of syndromic and non-syndromic obesity

MicroRNAs as Regulators of Neo-Angiogenesis in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a highly vascularized neoplasm. In the tumor niche, abundant angiogenesis is fundamental in providing nutrients for tumor growth and represents the first escape route for metastatic cells. Active angiogenesis, together with metastasis, are responsible for the reduction of recurrence-free survival of HCC. MicroRNAs (miRNAs) are small non-coding RNAs that have recently drawn attention in molecular targeted therapy or as diagnostic and prognostic biomarkers. MiRNA expression in HCC has been widely studied in the last decade. Some miRNAs have been found to be up- or down-regulated, besides association with apoptosis, metastasis progression and drug resistance have been found. This review article aims to summarize the angiogenetic process in tumor diseases and to update on what has been found in the vast world of HCC-related-miRNAs and, eventually, to report the latest finding on several miRNAs involved in HCC angiogenesis. We searched the state of the arts for the 12 miRNAs found to be involved with angiogenesis in HCC (miR-29b, miR-126-3p, miR-144-3p, miR-146a, miR-195, miR-199a-3p, miR-210-3p, miR-338- 3p, mir-491, mir-497, mir-638, mir-1301) and reported their main molecular targets and their overall effect in the sprouting of new vessels

Silencing efficacy prediction: a retrospective study on target mRNA features

Post-transcriptional gene silencing is a widely used method to suppress gene expression. Unfortunately only a portion of siRNAs do successfully reduce gene expression. Target mRNA secondary structures and siRNA-mRNA thermodynamic features are believed to contribute to the silencing activity. However, there is still an open discussion as to what determines siRNA efficacy. In this retrospective study, we analysed the target accessibility comparing very high (VH) compared with low (L) efficacy siRNA sequences obtained from the siRecords Database. We determined the contribution of mRNA target local secondary structures on silencing efficacy. Both the univariable and the multivariable logistic regression evidenced no relationship between siRNA efficacy and mRNA target secondary structures. Moreover, none of the thermodynamic and sequence-base parameters taken into consideration (H-b index, ΔG°overall, ΔG°duplex, ΔG°break-target and GC%) was associated with siRNA efficacy. We found that features believed to be predictive of silencing efficacy are not confirmed to be so when externally evaluated in a large heterogeneous sample. Although it was proposed that silencing efficacy could be influenced by local target accessibility we show that this could be not generalizable because of the diversity of experimental setting that may not be representative of biological systems especially in view of the many local protein factors, usually not taken into consideration, which could hamper the silencing process

Liquid biopsy: New opportunities for precision medicine in hepatocellular carcinoma care

Liquid biopsy, specifically the analysis of circulating tumor DNA (ctDNA), offers a non-invasive approach for hepatocellular carcinoma (HCC) diagnosis and management. However, its implementation in the clinical setting is difficult due to challenges such as low ctDNA yield and difficulty in understanding the mutation signals from background noise. This review highlights the crucial role of artificial intelligence (AI) in addressing these limitations and in improving discoveries in the field of liquid biopsy for HCC care. Combining AI with liquid biopsy data can offer a promising future for the discovery of novel biomarkers and an AI-powered clinical decision support system (CDSS) can turn liquid biopsy into an important tool for personalized management of HCC. Despite the current challenges, the integration of AI shows promise to significantly improve patient outcomes and revolutionize the field of oncology

Multidrug resistance in hepatic cancer stem cells: the emerging role of miRNAs

There is a fast growing body of evidence that shows several miRNAs are essential to the key features of cancer stem cells (CSC) in hepatocellular carcinoma. However, the function of the miRNAs in different hepatic CSCs and their role in multidrug resistance mechanisms, in particular in those related to the CSC marker ABCG2, is still poorly understood. This limitation is mainly due to the heterogeneity of hepatocellular carcinoma tissues, different CSC markers and high number of deregulated miRNAs, both in primary tumor sites as well as in the circulation. The identification of CSC-related miRNAs and the modulation of hepatocellular carcinoma multidrug resistance would provide a systemic approach in the management of the disease

Weighted miRNA co-expression networks analysis identifies circulating miRNA predicting overall survival in hepatocellular carcinoma patients

The weighted gene co-expression network analysis (WGCNA) has been used to explore gene expression datasets by constructing biological networks based on the likelihood expression profile among genes. In recent years, WGCNA found application in biomarker discovery studies, including miRNA. Serum samples from 20 patients with hepatocellular carcinoma (HCC) were profiled through miRNA 3.0 gene array and miRNAs biomarker candidates were identified through WGCNA. Results were validated by qRT-PCR in 102 HCC serum samples collected at diagnosis. WGCNA identified 16 miRNA modules, nine of them were significantly associated with the clinical characteristics of the patient. The Red module had a significant negative correlation with patients Survival (-\u20090.59, p\u2009=\u20090.007) and albumin (-\u20090.52, p\u2009=\u20090.02), and a positive correlation with PCR (0.61, p\u2009=\u20090.004) and alpha-fetoprotein (0.51, p\u2009=\u20090.02). In the red module, 16 circulating miRNAs were significantly associated with patient survival. MiR-3185 and miR-4507 were identified as predictors of patient survival after the validation phase. At diagnosis, high expression of circulating miR-3185 and miR-4507 identifies patients with longer survival (HR 2.02, 95% CI 1.10-3.73, p\u2009=\u20090.0086, and HR of 1.75, 95% CI 1.02-3.02, p\u2009=\u20090.037, respectively). Thought a WGCNA we identified miR-3185 and miR-4507 as promising candidate biomarkers predicting a longer survival in HCC patients

Circulatory miRNA as a Biomarker for Therapy Response and Disease-Free Survival in Hepatocellular Carcinoma

The clinical outcome of hepatocellular carcinoma (HCC) treatment remains unsatisfactory, contributing to the high mortality of HCC worldwide. Circulating miRNAs have the potential to be a predictor of therapy response. Microarray profiling was performed in serum samples of 20 HCC patients before treatment. Circulating miRNAs associated with treatment response were validated in 86 serum HCC samples using the qRT-PCR system. Patients were treated either with curative treatments (resection or radiofrequency) or trans-arterial chemoembolization (TACE), and grouped according to therapy response in complete responders (CR) and partial responders or progressive disease (PRPD), following mRECIST criteria. Four miRNA candidates from the discovery phase (miR-4443, miR-4454, miR-4492, and miR-4530) were validated. Before therapy, miR-4454 and miR-4530 were up-regulated in CR to curative treatments (2.83 fold, p = 0.02 and 2.33 fold, p = 0.008, respectively) and were able to differentiate CR from PRPD (area under the curve (AUC) = 0.74, sens/spec 79/63% and AUC = 0.77, sens/spec 72/73%). On the contrary, miR-4443 was 1.95 times down-regulated in CR (p = 0.05) with an AUC of 0.72 (sens = 70%, spec = 60%) in distinguishing CR vs. PRPD. The combination of the three miRNAs was able to predict the response to curative treatment with an AUC of 0.84 (sens = 72%, spec = 75%). The higher levels of miR-4454 and miR-4530 in were associated to longer overall survival (HR = 2.79, p = 0.029 and HR = 2.97, p = 0.011, respectively). Before TACE, miR-4492 was significantly up-regulated in CR patients (FC = 2.67, p = 0.01) and able to differentiate CR from PRPD (AUC = 0.84, sens/spec 84.6/71%). We demonstrated that different miRNAs predictors can be used as potential prognostic circulating biomarkers according to the selected treatment for HCC