Transarterial chemoembolization (TACE) is the standard treatment for patients with intermediate-stage hepatocellular carcinoma (HCC), yet nearly half of treated patients fail to achieve durable benefit, and reliable biomarkers enabling early therapeutic stratification are still lacking. Treatment response is typically assessed by imaging one month after TACE and at three-month intervals, potentially delaying timely access to alternative therapies in non-responding patients. Circulating microRNAs (miRNAs) represent promising biomarkers due to their stability in body fluids and ease of detection. Here, we evaluated circulating miR-22 as an early predictor of TACE non-responder status and as a mechanistically relevant therapeutic target. Circulating miR-22 levels were measured by microarray and quantitative RT–PCR in three independent cohorts of early-to-intermediate-stage HCC patients undergoing TACE. Circulating miR-22 increased significantly in non-responders as early as 48 h after treatment, and fold changes consistently predicted treatment failure across two independent validation cohorts. Mechanistically, we identified the G2/M checkpoint kinase WEE1 as a direct functional target of miR-22. Modulation of the miR-22/WEE1 axis affected cell-cycle progression, proliferation, apoptosis, and DNA damage response in HCC cell lines and xenograft models. Under hypoxia-mimicking conditions combined with doxorubicin exposure, pharmacological inhibition of WEE1 induced mitotic catastrophe in highly proliferative miR-22-silenced cells. Collectively, these findings identify early post-TACE elevation of circulating miR-22 as a biomarker of non-response and highlight the miR-22/WEE1 axis as a potential target for precision treatment strategies in HCC.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide, but its cancerization and progression mechanisms are still poorly understood. Mitochondrial DNA (mtDNA) mutations have been involved in tumor progression by influencing metabolic rewiring and plasticity. In this work, we aimed to investigate the contribution of mtDNA mutations to the pathogenesis and prognosis of HCC. Whole exome sequencing data from the TCGA-LIHC project were used to reconstruct the mitochondrial genomes. Discovered variants were classified using the HmtVar pathogenicity scoring system and the ACMG/AMP standard guidelines. The validation of results was performed on a separate in-house cohort of selected HCC cases from our hospital. Pathogenic mtDNA mutations were present in 33.6% of patients. GSEA revealed pathogenic mtDNA mutations mainly targeting the reactive oxygen species (ROS) pathway, suggesting an increased ROS production in these tumors which may contribute to the survival, proliferation and metastatization capacity. Survival analysis revealed a significant decrease in the overall survival of patients harboring pathogenic mtDNA variants (p = 0.01). A similar trend was observed in the validation cohort. Overall, we showed that somatic mtDNA mutations occur in a significant proportion of HCC cases, expectedly acting as modifiers on the ROS pathway, and that their occurrence confers a worse prognosis.

Poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS)-based organic electrochemical transistors (OECTs) have been demonstrated as versatile biosensors in recent years. Owing to the biocompatibility and chemical stability of the organic mixed ionic and electronic conductor in cell media, they allow real-time, in vitro electrical monitoring of cell lines, providing quantitative outcomes of their health status. Here, we propose and validate a sensitive OECT device for direct in vitro monitoring of peripheral blood mononuclear cell (PBMC) activity in a coculture assay. PBMCs from patients with advanced hepatocellular carcinoma (HCC) responding and nonresponding to atezolizumab (anti-PD-L1) and bevacizumab (anti-VEGF) are cocultured with Huh7 and SNU449 HCC cell lines directly grown onto OECTs. A strong correlation between the OECT electrical response, standard cell growth, death assays, and IL6 levels is observed, which is confirmed for cocultures involving PBMCs from healthy controls and patients treated with immunosuppressive drugs (namely, positive and negative controls, respectively). Our low-cost and scalable device has the potential to detect PBMC activation induced by atezolizumab-bevacizumab in the very early stages of HCC treatment, allowing for nonresponders' inclusion in alternative treatments. This approach might be of great interest to several human cancers treated with immune checkpoint inhibitors (ICIs), providing sensitive, automated, and noninvasive tools to complement clinical practice.

Background: Immune checkpoint inhibitors (ICIs), including anti-programmed cell death protein 1 and its ligand (anti-PD-1/PD-L1), have transformed the treatment of advanced hepatocellular carcinoma (HCC). However, due to primary or acquired resistance, only a subset of patients achieves an objective response, highlighting the need for predictive biomarkers to allocate patients to the most promising therapeutic combination. In this regard, we investigated PD-1 expression in surgically resected HCCs and HCC-derived cell lines and its role in the response to anti-PD-1 blockade. Methods: Overall, tumour samples were obtained from 100 HCC patients who had been subjected to surgery. PD-1/CD8/CD3/CD68/Ki-67 were investigated by immunohistochemistry (IHC). PD-1 expression was also assayed in a DEN-induced HCC rat model. A subgroup of randomly selected HCCs were also studied by PD-1 immunofluorescence (IF) and RNAseq analysis. PD-1 expression was analysed in HepG2, Huh7, PLC/PR5 and SNU449 HCC cells, by western blotting, IF and flow cytometry. Cell lysates were treated with PNGase F to check PD1 glycosylation. Apoptosis, proliferation, and migration were assessed in cells after PD1 blockade obtained by using two different anti-PD-1 antibodies. Results: Our study provides evidence that PD-1 expression can be detected by IHC and even better by IF in neoplastic hepatocytes with subcellular distribution from the membrane to the nucleus and with a marked staining in steatotic cells. HCC cell lines express PD-1 with a similar subcellular localisation, dictating the response to PD-1 blockade. Through the regulation of AKT, ERK1/2 and mTOR downstream signalling pathways, PD-1 blockade acts as a tumour suppressor in Huh7 and in PLC/PRF5 characterised by PD-1 surface localisation and low PD-1 intracellular level, and as a tumour promoter in HepG2 cells characterised by the opposite PD-1 localisation pattern. Conclusion: Our study unveils a role of cancer cell-intrinsic PD-1 beside the well-known immune component in the response to PD-1 blockade. Interestingly, the response to treatment depends on PD-1 subcellular localisation and highlights immunofluorescence as the outperforming technique to be used in clinical practice as a putative predictive biomarker.

The approval of immunotherapy has revolutionized the management of hepatocellular carcinoma (HCC) patients. However, sorafenib remains a first-line therapeutic option for advanced patients and, in particular, for patients not eligible for immune checkpoint inhibitors, but its efficacy is limited by the onset of acquired resistance, highlighting the urgent need for predictive biomarkers. This study investigates the role of miR-22 in metabolic reprogramming and its potential as a biomarker in HCC. The analysis of miR-22 expression was performed in HCC patients and preclinical models by qPCR. Functional analyses in HCC cells evaluated GLUT1 as a direct miR-22 target. Cellular and metabolic assays evaluated the miR-22/GLUT1 axis’s role in metabolic changes, tumor aggressiveness, and sorafenib response. Circulating miR-22 was analyzed in sorafenib-treated HCC patients and rats. MiR-22 was downregulated in HCCs and associated with aggressive tumor features. Functionally, miR-22 modulated the HIF1A pathway, enhanced survival in stressful conditions, promoted a glycolytic shift, and enhanced cancer cell plasticity and sorafenib resistance via GLUT1 targeting. In addition, high serum miR-22 levels were associated with sorafenib resistance in HCC patients and rats. GLUT1 inhibition sensitized low miR-22-expressing HCC cells to sorafenib in preclinical models. These findings suggest that circulating miR-22 deserves attention as a predictive biomarker of sorafenib response. GLUT1 inhibition may represent a therapeutic strategy to combine with sorafenib, particularly in patients exhibiting high circulating miR-22 levels.

Small nucleolar RNAs are non-coding RNAs typically encoded within the introns of both protein-coding and non-coding genes. Interestingly, a significant fraction of snoRNA sequences is found as retained introns of specific mRNA isoforms expressed from their host gene. In the present study, we aimed to define the representation of small nucleolar RNA retaining transcripts across various human cell types and tissues including cancer. We found that these type of transcripts are widely represented in normal tissues and cancer-derived cell lines, appearing both in their full-length form and, frequently, in a shorter variant. We characterized the shortening position, which occurs at or very close to the retained small nucleolar RNA sequence at the 5' end. Interestingly, for some transcripts this shorter variant represents the only form detected. In addition, some of the small nucleolar RNA retaining transcripts can be localized into the cellular cytoplasmic fraction. Moreover, our findings point out that a variable but consistent proportion of small nucleolar RNA sequences in cells, tissues, and liquid biopsy samples is, in fact, present as small nucleolar RNA retaining transcripts, indicating that these elements should be carefully considered when snoRNA are evaluated as biomarkers. Considering that short reads and gene-based transcriptomic analysis completely overlooked these transcripts, potentially missing critical insights into their involvement in cancer and other diseases, our results strongly indicate that these type of transcripts should be further investigated in different contexts to better understand their biogenesis, sequence features, presence, and role within cells.

Immunotherapy has shown significant improvement in the survival of patients with hepatocellular carcinoma (HCC) compared to TKIs as first-line treatment. Unfortunately, approximately 30% of HCC exhibits intrinsic resistance to ICIs, making new therapeutic combinations urgently needed. The dysregulation of the Notch signaling pathway observed in HCC can affect immune cell response, reducing the efficacy of cancer immunotherapy. Here, we provide an overview of how Notch signaling regulates immune responses and present the therapeutic rationale for combining Notch signaling inhibition with ICIs to improve HCC treatment. Moreover, we propose using exosomes as non-invasive tools to assess Notch signaling activation in hepatic cancer cells, enabling accurate stratification of patients who can benefit from combined strategies.

The incidence of hepatocellular carcinoma (HCC) is increasing, and 40% of patients are diagnosed at advanced stages. Over the past 5 years, the number of clinically available treatments has dramatically increased for HCC, making patient management particularly complex. Immune checkpoint inhibitors (ICIs) have improved the overall survival of patients, showing a durable treatment benefit over time and a different response pattern with respect to tyrosine kinase inhibitors (TKIs). Although there is improved survival in responder cases, a sizeable group of patients are primary progressors or are ineligible for immunotherapy. Indeed, patients with nonviral etiologies, such as nonalcoholic steatohepatitis (NASH), and alterations in specific driver genes might be less responsive to immunotherapy. Therefore, improving the comprehension of mechanisms of drug resistance and identifying biomarkers that are informative of the best treatment approach are required actions to improve patient survival. Abundant evidence indicates that noncoding RNAs (ncRNAs) are pivotal players in cancer. Molecular mechanisms through which ncRNAs exert their effects in cancer progression and drug resistance have been widely investigated. Nevertheless, there are no studies summarizing the synergistic effect between ncRNA-based strategies and TKIs or ICIs in the preclinical setting. This review aims to provide up-to-date information regarding the possible use of ncRNAs as therapeutic targets in association with molecular-targeted agents and immunotherapies and as predictive tools for the selection of optimized treatment options in advanced HCCs.

Myelopoiesis provides for the formation and continued renewal of cells belonging primarily to the innate immune system. It is a highly plastic process that secures the response to external and internal stimuli to face acute and changing needs. Infections and chronic diseases including cancer can modulate it by producing several factors, impacting proliferation and differentiation programs. While the lymphocytic compartment has attracted major attention due to the role of adaptive immunity in anticancer immune response, in recent years, research has found convincing evidence that confirms the importance of innate immunity and the key function played by emergency myelopoiesis. Due to cancer’s ability to manipulate myelopoiesis to its own advantage, the purpose of this review is to outline myelopoiesis processes within the tumor microenvironment and suggest possible therapeutic lines of research to restore the physiological functioning of the host’s immune system, with a special outlook on hepatocellular carcinoma (HCC).

Supplementary Table 3 from Cyclin G1 Is a Target of miR-122a, a MicroRNA Frequently Down-regulated in Human Hepatocellular Carcinoma

Supplementary Table 1 from MiR-122/Cyclin G1 Interaction Modulates p53 Activity and Affects Doxorubicin Sensitivity of Human Hepatocarcinoma Cells

ST1: Discovery and validation surgical patient cohorts. ST2: Advanced HCC patient cohort. ST3: Primer sequences for luciferase assay. ST4: Primer sequences for PCR and qPCR. ST5: Antibodies. ST6: Probe sequences for EMSA. ST7: Deregulated miR-30 family members in rat model.

Supplementary Table 1 from Cyclin G1 Is a Target of miR-122a, a MicroRNA Frequently Down-regulated in Human Hepatocellular Carcinoma

Supplementary Table S1. Characteristics of surgical HCC patients analysed in this study. Supplementary Table S2. Patients assessed for circulating miR-221. Supplementary Table S3. Primer sequences and PCR conditions for the cloning experiment. Supplementary Table S4. Primer sequences and PCR conditions. Supplementary Table S5. Antibodies used in Western blot analysis.

Microarray analysis, cell transfection and infection, cell proliferation assay, clonogenic and sphere formation assays, TP53 hypothetical binding sites.

Figure S4. (A, E) Box plot graphs of serum AFP in HCC patients with high and low miR-221 or caspase-3 expression levels. High and low miR-221 or caspase-3 expression was identified on the basis of the median value.

Figure S2. Histopathological images of HCC nodules arisen in DEN-treated rats following Sorafenib administration. Nodules that responded to Sorafenib displayed large necrotic areas.