Chemotherapy For Hepatocellular Carcinoma Research Articles

Abstract LB303: Suppression of stress granule formation is a novel vulnerability imposed by mutant p53

Abstract Mutation of the tumor suppressor p53 is the most common cancer specific event. Most of these mutations are missense mutations in the DNA-binding domain of p53, causing loss of wild-type p53 (wtp53) transcriptional activity. Missense mutant p53 (mutp53) also enhances cancer malignancy in a manner independent of wtp53. Hence, mutp53 is an ideal therapeutic target due to its uniqueness and high expression levels in cancers; however, directly targeting mutp53 protein itself has been challenging. To better understand the mechanism of oncogenic function of mutp53, we performed in silico TCGA database analysis and found that patients with missense mutp53-carrying hepatocellular carcinoma (HCC), but not other types of cancer, had unexpectedly better overall survival than patients with p53-null HCC. Since sorafenib (SOR) has historically been used as a single agent chemotherapy for HCC, unlike other cancer types, we hypothesized that SOR treatment may have yet unappreciated advantages for treating mutp53-expressing tumors. Indeed, knockdown of mutp53 rendered HCC cells and xenografts more resistant to SOR. Consistent with a report showing that SOR induces ER stress-mediated pro-survival stress granule (SG) formation, mutp53-expressing HCC cells, treated with SOR or other ER stress inducers, formed significantly fewer SGs with increased apoptosis, as compared with mutp53-knockdown cells. This result was partly due to mutp53’s ability to interact with an eIF2α kinase PERK and a key SG core protein, G3BP1. Moreover, the oligomerization domain of mutp53 was required for the interaction of mutp53 with G3BP1 and subsequent suppression of ER-stress-mediated SG formation and survival. These results suggest that inhibition of SG formation is a novel vulnerability imposed by mutp53, leading to sensitization of cancer cells to ER stress. Our results also emphasize the use of ER stress inducers to treat mutp53-expressing tumors and mutp53 as a biomarker of SOR sensitivity. Citation Format: Elizabeth Thoenen, Atul Ranjan, Alejandro Parrales, Shigeto Nishikawa, Dan Dixon, Sugako Oka, Tomoo Iwakuma. Suppression of stress granule formation is a novel vulnerability imposed by mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB303.

Development of an efficient liposomal DOX delivery formulation for HCC therapy by targeting CK2α.

Hepatocellular carcinoma (HCC) is a digestive tract cancer with high mortality and poor prognosis, especially in China. Current chemotherapeutic drugs lead to poor prognosis, low efficacy, and high side effects due to weak targeting specificity and rapidly formed multidrug resistance (MDR). Based on the previous studies on the doxorubicin (DOX) formulation for cancer targeting therapy, we developed a novel DOX delivery formulation for the targeting chemotherapy of HCC and DOX resistant HCC. HCSP4 was previously screened and casein kinase 2α (CK2α) was predicted as its specific target on HCC cells in our lab. In the study, miR125a-5p was firstly predicted as an MDR inhibiting miRNA, and then CK2α was validated as the target of HCSP4 and miR125a-5p using CK2α-/-HepG2 cells. Based on the above, an HCC targeting and MDR inhibiting DOX delivery liposomal formulation, HCSP4/Lipo-DOX/miR125a-5p was synthesized and tested for its HCC therapeutic efficacy in vitro. The results showed that the liposomal DOX delivery formulation targeted to HCC cells specifically and sensitively, and presented the satisfied therapeutic efficacy for HCC, particularly for DOX resistant HCC. The potential therapeutic mechanism of the DOX delivery formulation was explored, and the formulation inhibited the expression of MDR-relevant genes including ATP-binding cassette subfamily B member 1 (ABCB1, also known as P-glycoprotein), ATP-binding cassette subfamily C member 5 (ABCC5), enhancer of zeste homolog 2 (EZH2), and ATPase Na+/K+ transporting subunit beta 1 (ATP1B1). Our study presents a novel targeting chemotherapeutic drug formulation for the therapy of HCC, especially for drug resistant HCC, although it is primarily and needs further study in vivo, but provided a new strategy for the development of novel anticancer drugs.

A Novel Cholesterol Metabolism-Related lncRNA Signature Predicts the Prognosis of Patients with Hepatocellular Carcinoma and Their Response to Immunotherapy.

The survival rate of hepatocellular carcinoma (HCC) is low and the prognosis is poor. Metabolic reprogramming is still an emerging hallmark of cancer, and reprogramming of cholesterol metabolism plays a crucial action in tumor pathogenesis. Increasing evidence suggests that cholesterol metabolism affects the cell proliferation, invasion, migration, and resistance to chemotherapy of HCC. To date, no long noncoding RNA (lncRNA) signature associated with cholesterol metabolism has been developed to predict the outcome of patients with HCC. The RNA-seq data as well as the prognostic and clinical data were obtained from The Cancer Genome Atlas (TCGA) database. We conducted univariate and multivariate analyses to assess cholesterol metabolism-related lncRNAs correlated with the prognosis of patients with HCC in order to construct a prognostic signature. Functional differences between low- and high-risk groups were investigated using genomic enrichment analysis (GSEA). Kaplan-Meier (KM) curves were applied to explore the overall survival (OS) of the low- and high-risk groups. Single-sample genomic enrichment analysis (ssGSEA) was applied to investigate the association between this predictive signature and immune function. We subsequently examined how this signature relates to treatment response in HCC patients. A prognostic signature comprising six lncRNAs related to cholesterol metabolism was constructed (AC124798.1, AL031985.3, AC103760.1, NRAV, WAC-AS1 and AC022613.1). We found that low-risk groups showed a better prognosis than high-risk groups. In HCC patients, the cholesterol metabolism-related lncRNA signature may be served as an independent prognostic factor. Cholesterol metabolism-related lncRNA signature had higher diagnostic efficiency compared to clinicopathologic variables. After stratifying patients according to different clinicopathological variables, patients with low-risk had a longer OS compared with high-risk patients. The ssGSEA demonstrated that this signature was closely related to the immune status of HCC patients. GSEA analysis demonstrated that immune- and tumor-related pathways were predominantly enriched in the high-risk group. High-risk patients were more responsive to immune checkpoint inhibitors (ICIs) and conventional chemotherapeutic agents. This cholesterol metabolism-related lncRNA signature can predict the prognosis of HCC patients and guide the clinical management of HCC patients, including immunotherapy.

Sorafenib-Encapsulated Liposomes to Activate Hypoxia-Sensitive Tirapazamine for Synergistic Chemotherapy of Hepatocellular Carcinoma.

Combination therapy with the synergistic effect is an effective way in cancer chemotherapy. Herein, an antiangiogenic sorafenib (SOR) and hypoxia-activated prodrug tirapazamine (TPZ)-coencapsulated liposome (LipTPZ/SOR) is prepared for chemotherapy of hepatocellular carcinoma (HCC). SOR is a multi-target tyrosine kinase inhibitor that can inhibit tumor cell proliferation and angiogenesis. The antiangiogenesis effect of SOR can reduce oxygen supply and aggravate tumor hypoxia, which is able to activate hypoxia-sensitive prodrug TPZ, exhibiting the synergistic antitumor effect. LipTPZ/SOR at different molar ratios of TPZ and SOR can significantly inhibit the proliferation of hepatocellular carcinoma cells. The mole ratio of TPZ and SOR was optimized to 2:1, which exhibited the best synergetic antitumor effect. The synergistic antitumor mechanism of SOR and TPZ was also investigated in vivo. After treated with SOR, the number of vessels was decreased, and the degree of hypoxia was aggravated in tumor tissues. What is more, in the presence of SOR, TPZ could be activated to inhibit tumor growth. The combination of TPZ and SOR exhibited an excellent synergistic antitumor effect. This research not only provides an innovative strategy to aggravate tumor hypoxia to promote TPZ activation but also paints a blueprint about a new nanochemotherapy regimen for the synergistic chemotherapy of HCC, which has excellent biosafety and bright clinical application prospects.

A new chemotherapy strategy for advanced hepatocellular carcinoma with exrahepatic metastasis: predictors of long-term survival

The prognosis of hepatocellular carcinoma (HCC) with extrahepatic metastasis (EHM) is extremely poor. This study aimed to identify prognostic factors for systemic chemotherapy of HCC with EHM. Eighty-five patients who received systemic chemotherapy for HCC with EHM between May 2014 and October 2021 were retrospectively evaluated. Patient demographic data and characteristics of hepatic tumors and EHM were assessed to identify factors that were significantly associated with prognosis. Of the 85 patients, 68 (80.0%) had pulmonary metastasis, 11 (12.9%) had abdominal lymph node metastasis, 7 (8.2%) had abdominal metastasis, and 4 (4.7%) had bone metastasis. The median overall survival (OS) was 17.0 months, and the median progression-free survival (PFS) was 5.1 months. Univariate analysis of OS showed that synchronous EHM-HCC, serum albumin level<35 g/l and number of hepatic tumors>1 were significantly associated with poorer OS. The results of the multivariate analysis indicated that the serum albumin level and number of hepatic tumors were independent prognostic factors. Subgroup analysis of patients with 0, 1, or 2 of these independent prognostic factors showed that the median OS was 24.0 months, 16.2 months and 7.7 months and that the ORR was 38.3%, 22.6% and 0, respectively. Systemic chemotherapy is beneficial for well-selected HCC patients with EHM. The number of hepatic tumors and serum albumin level were independent risk factors for prognosis, and the number of risk factors significantly influenced OS. Therefore, these factors need to be considered before administering systemic chemotherapy for HCC patients with EHM.

SPINK1-induced tumor plasticity provides a therapeutic window for chemotherapy in hepatocellular carcinoma

Tumor lineage plasticity, considered a hallmark of cancer, denotes the phenomenon in which tumor cells co-opt developmental pathways to attain cellular plasticity, enabling them to evade targeted therapeutic interventions. However, the underlying molecular events remain largely elusive. Our recent study identified CD133/Prom1 in hepatocellular carcinoma (HCC) tumors to mark proliferative tumor-propagating cells with cancer stem cell-like properties, that follow a dedifferentiation trajectory towards a more embryonic state. Here we show SPINK1 to strongly associate with CD133 + HCC, and tumor dedifferentiation. Enhanced transcriptional activity of SPINK1 is mediated by promoter binding of ELF3, which like CD133, is found to increase following 5-FU and cisplatin treatment; while targeted depletion of CD133 will reduce both ELF3 and SPINK1. Functionally, SPINK1 overexpression promotes tumor initiation, self-renewal, and chemoresistance by driving a deregulated EGFR-ERK-CDK4/6-E2F2 signaling axis to induce dedifferentiation of HCC cells into their ancestral lineages. Depleting SPINK1 function by neutralizing antibody treatment or in vivo lentivirus-mediated Spink1 knockdown dampens HCC cancer growth and their ability to resist chemotherapy. Targeting oncofetal SPINK1 may represent a promising therapeutic option for HCC treatment.

Synergistic impacts of rifampicin and doxorubicin against thioacetamide-induced hepatocellular carcinoma in rats

Background and aimsCombination therapy is a promising new strategy that has been proposed to increase the efficacy of cancer treatment. We aimed to investigate the anti-cancer activity of rifampicin monotherapy and its combination with doxorubicin against hepatocellular carcinoma (HCC). Materials and methodsThe in vitro half maximal inhibitory concentration (IC50) and selectivity index (SI) of the drugs under investigation against HepG2 and human lung fibroblast (WI38) cell lines were determined. For the in vivo experiment, male Sprague-Dawley albino rats were injected with thioacetamide at 200 mg/kg twice a week for 90 days; HCC development was confirmed histopathologically. Following HCC induction, the rats were treated with intraperitoneal doxorubicin, rifampicin, or their combination for 45 or 90 days. After sacrifice, the livers were examined histopathologically. The levels of aminotransferases, albumin, bilirubin, malondialdehyde, superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (TAC), and nitric oxide were measured by spectrophotometry. Alpha-fetoprotein, cancer antigen 19-9, tumor necrosis factor-alpha, interleukin-6, Bcl-2-associated X protein, caspase 3, caspase 8, and p53 were estimated using ELISA. ResultsIn vitro, the combination of doxorubicin and rifampicin showed the highest SI of 3.43. In vivo, among the measured markers, the levels of TAC, CAT, SOD, and p53 decreased (P < 0.001) and the rest of the measured marker levels increased (P < 0.001) in the HCC-bearing rats; after treatment in all groups, all these changes improved toward normal in a time-dependent manner. The combination of doxorubicin and rifampicin optimized the effects of the two individual drugs and exerted the best antioxidant effects. ConclusionsIn general, compared with rifampicin or doxorubicin alone, combination therapy has favorable outcomes. Based on our results, the combination of rifampicin and doxorubicin might be applicable for HCC chemotherapy.

Vessels encapsulating tumor clusters: a novel efficacy predictor of hepatic arterial infusion chemotherapy in unresectable hepatocellular carcinoma.

Vessels encapsulating tumor clusters (VETC) is a novel vascular pattern structurally and functionally distinct from microvascular invasion (MVI) in hepatocellular carcinoma (HCC). This study aims to explore the prognostic value of VETC in patients receiving hepatic arterial infusion chemotherapy (HAIC) for unresectable HCC. From January 2016 to December 2017, 145 patients receiving HAIC as the initial treatment for unresectable HCC were enrolled and stratified into two groups according to their VETC status. Overall survival (OS), progression-free survival (PFS), overall response rate (ORR), and disease control rate (DCR) were evaluated. The patients were divided into two groups: VETC+ (n = 31, 21.8%) and VETC- (n = 114, 78.2%). The patients in the VETC+ group had worse ORR and DCR than those in the VETC- group (RECIST: ORR: 25.8% vs. 47.4%, P = 0.031; DCR: 56.1% vs. 76.3%, P = 0.007; mRECIST: ORR: 41.0% vs. 52.6%, P = 0.008; DCR: 56.1% vs. 76.3%, P = 0.007). Patients with VETC+ had significantly shorter OS and PFS than those with VETC- (median OS: 10.2 vs. 21.6months, P < 0.001; median PFS: 3.3 vs. 7.2months, P < 0.001). Multivariate analysis revealed VETC status as an independent prognostic factor for OS (HR: 2.40; 95% CI: 1.46-3.94; P = 0.001) and PFS (HR: 1.97; 95% CI: 1.20-3.22; P = 0.007). VETC status correlates remarkably well with the tumor response and long-term survival in patients undergoing HAIC. It may be a promising efficacy predictor and help identify patients who will benefit from HAIC.

Survival benefit of neoadjuvant hepatic arterial infusion chemotherapy followed by hepatectomy for hepatocellular carcinoma with portal vein tumor thrombus.

Background/purpose: The prognosis of hepatocellular carcinoma (HCC) patients with portal vein tumor thrombus (PVTT) is generally poor and hepatectomy is optional for these patients. This study aims to explore the survival benefits of neoadjuvant hepatic arterial infusion chemotherapy (HAIC) for resectable HCC with PVTT. Methods: This retrospective study included 120 resectable HCC patients with PVTT who underwent hepatectomy, from January 2017 to January 2021 at Sun Yat-sen University Cancer Center. Of these patients, the overall survival (OS) and recurrence-free survival (RFS) of 55 patients who received hepatectomy alone (Surgery group) and 65 patients who received neoadjuvant HAIC followed by hepatectomy (HAIC-Surgery group) were compared. Logistic regression analysis was conducted to develop a model predicting the response to neoadjuvant HAIC. Results: The OS rates for the HAIC-Surgery group at 1, 3, and 5years were 94.9%, 78%, and 66.4%, respectively, compared with 84.6%, 47.6%, and 37.2% in the Surgery group (p < 0.001). The RFS rates were 88.7%, 56.2%, and 38.6% versus 84.9%, 38.3%, and 22.6% (p = 0.002). The subgroup analysis revealed that the survival benefit of neoadjuvant HAIC was limited to patients who responded to it. The logistic model, consisting of AFP and CRP, that predicted the response to neoadjuvant HAIC performed well, with an area under the ROC curve (AUC) of 0.756. Conclusion: Neoadjuvant HAIC followed by hepatectomy is associated with a longer survival outcome than hepatectomy alone for HCC patients with PVTT and the survival benefit is limited to patients who respond to neoadjuvant FOLFOX-HAIC.

Deep dissection of stemness-related hierarchies in hepatocellular carcinoma

BackgroundIncreasing evidence suggests that hepatocellular carcinoma (HCC) stem cells (LCSCs) play an essential part in HCC recurrence, metastasis, and chemotherapy and radiotherapy resistance. Multiple studies have demonstrated that stemness-related genes facilitate the progression of tumors. However, the mechanism by which stemness-related genes contribute to HCC is not well understood. Here, we aim to construct a stemness-related score (SRscores) model for deeper analysis of stemness-related genes, assisting with the prognosis and individualized treatment of HCC patients.Further, we found that the gene LPCAT1 was highly expressed in tumor tissues by immunohistochemistry, and sphere-forming assay revealed that knockdown of LPCAT1 inhibited the sphere-forming ability of hepatocellular carcinoma cells.MethodsWe used the TCGA-LIHC dataset to screen stemness-related genes of HCC from the MSigDB database. Prognosis, tumor microenvironment, immunological checkpoints, tumor immune dysfunction, rejection, treatment sensitivity, and putative biological pathways were examined. Random forest created the SRscores model. The anti-PD-1/anti-CTLA4 immunotherapy, tumor mutational burden, medication sensitivity, and cancer stem cell index were compared between the high- and low-risk score groups. We also examined risk scores for different cell types using single-cell RNA sequencing data and correlated transcription factor activity in cancer stem cells with SRscores genes. Finally, we tested core marker expression and biological functions.ResultsPatients can be divided into two subtypes (Cluster1 and Cluster2) based on the TCGA-LIHC dataset's identification of 11 stemness-related genes. Additionally, a SRscores was developed based on subtypes. Cluster2 and the group with the lowest SRscores had superior survival and immunotherapy response than Cluster1 and the group with the highest SRscores. The group with a high SRscores was significantly more enriched in classical tumor pathways than the group with a low SRscores. Multiple transcription factors and SRscores genes are correlated. The core gene LPCAT1 is highly expressed in rat liver cancer tissues and promotes tumor cell sphere formation.ConclusionA SRscores model can be utilized to predict the prognosis of HCC patients as well as their response to immunotherapy.

Identification and validation of a m7G-related lncRNA signature for predicting the prognosis and therapy response in hepatocellular carcinoma.

N7-methylguanosine (m7G) is one of the most common RNA posttranscriptional modifications; however, its potential role in hepatocellular carcinoma (HCC) remains unknown. We developed a prediction signature based on m7G-related long noncoding RNAs (lncRNAs) to predict HCC prognosis and provide a reference for immunotherapy and chemotherapy. RNA-seq data from The Cancer Genome Atlas (TCGA) database and relevant clinical data were used. Univariate and multivariate Cox regression analyses were conducted to identify m7G-related lncRNAs with prognostic value to build a predictive signature. We evaluated the prognostic value and clinical relevance of this signature and explored the correlation between the predictive signature and the chemotherapy treatment response of HCC. Moreover, an in vitro study to validate the function of CASC19 was performed. Six m7G-related lncRNAs were identified to create a signature. This signature was considered an independent risk factor for the prognosis of patients with HCC. TIDE analyses showed that the high-risk group might be more sensitive to immunotherapy. ssGSEA indicated that the predictive signature was strongly related to the immune activities of HCC. HCC in high-risk patients was more sensitive to the common chemotherapy drugs bleomycin, doxorubicin, gemcitabine, and lenalidomide. In vitro knockdown of CASC19 inhibited the proliferation, migration and invasion of HCC cells. We established a 6 m7G-related lncRNA signature that may assist in predicting the prognosis and response to chemotherapy and immunotherapy of HCC.

Identification of PANoptosis-Based Prognostic Signature for Predicting Efficacy of Immunotherapy and Chemotherapy in Hepatocellular Carcinoma.

PANoptosis has been a research hotspot, but the role of PANoptosis in hepatocellular carcinoma (HCC) remains widely unknown. Drug resistance and low response rate are the main limitations of chemotherapy and immunotherapy in HCC. Thus, construction of a prognostic signature to predict prognosis and recognize ideal patients for corresponding chemotherapy and immunotherapy is necessary. The mRNA expression data of HCC patients was collected from TCGA database. Through LASSO and Cox regression, we developed a prognostic signature based on PANoptosis-related genes. KM analysis and ROC curve were implemented to evaluate the prognostic efficacy of this signature, and ICGC and GEO database were used as external validation cohorts. The immune cell infiltration, immune status, and IC50 of chemotherapeutic drugs were compared among different risk subgroups. The relationships between the signature and the efficacy of ICI therapy, sorafenib treatment, and transcatheter arterial chemoembolization (TACE) therapy were investigated. A 3-gene prognostic signature was constructed which divided the patients into low- and high-risk subgroups. Low-risk patients had better prognosis, and the risk score was proved to be an independent predictor of overall survival (OS), which had a well predictive effect. Patients in high-risk population had more immunosuppressive cells (Tregs, M0 macrophages, and MDSCs), higher TIDE score and TP53 mutation rate, and elevated activity of base excision repair (BER) pathways. Patients with low risk benefited more from ICI, TACE, and sorafenib therapy. The predictive value of the risk score was comparable with TIDE and MSI for OS under ICI therapy. The risk score could be a biomarker to predict the response to ICI, TACE, and sorafenib therapy. The novel signature based on PANoptosis is a promising biomarker to distinguish the prognosis predict the benefit of ICI, TACE, and sorafenib therapy, and forecast the response to them.

NeoTACE: A multicenter, randomized study evaluating the efficacy and safety of neoadjuvant HAIC for TACE plus donafenib in BCLC B stage hepatocellular carcinoma outside of up-to-seven.

TPS4186 Background: Transarterial chemoembolization (TACE) is standard treatment for hepatocellular carcinoma (HCC) patients in BCLC B stage, while a number of studies demonstrated compromised effect of TACE for patients with large HCC or tumor burden outside of up-to-7. Recently, two phase 3 studies showed the survival benefit of Hepatic arterial infusion chemotherapy (HAIC) for large HCC and as neoadjuvant treatment for HCC outside of Milan criteria. A Phase 3 study also showed the survival benefit of donafenib for unresectable HCC compared with sorafenib. Here, we propose a prospective randomized study to investigate the benefit of HAIC as a neoadjuvant therapy for TACE plus donafenib in unresectable BCLC B stage HCC outside of up-to seven. Methods: This is a multicenter, open-label, randomized study designed to evaluate the efficacy and safety of neoadjuvant HAIC for TACE plus donafenib compared with TACE plus donafenib in BCLC B stage unresectable HCC outside of up-to-seven. Participants are randomized in 1:1 ratio to either Arm A, receiving 2-4 cycles neoadjuvant HAIC treatment plus donafenib initially, then TACE plus donafenib sequentially, or Arm B, just receiving TACE treatment plus donafenib. HAIC consisted of infusions of oxaliplatin (35 mg/m2 for 2 hours), followed by 5-fluorouracil (600 mg/m2 for 22 hours) on day1-3 every 4 weeks. The primary endpoint is PFS, and the secondary endpoints are OS, ORR, DCR, and adverse events. We hypothesize that neoadjuvant HAIC for TACE plus donafenib will improve PFS from 6 months to 11 months, with the hazard ratio of 0.55. With one-sided significance level of 0.05 and power of 0.8, the sample size for randomization will be 156. The study, registered with clinical trial ID of NCT05171166, started enrolment in Feb 2022. As of Oct 2022, 65 patients have been enrolled and 11 patients have been randomized. Clinical trial information: NCT05171166 .

Machine learning-based prognostic modeling of lysosome-related genes for predicting prognosis and immune status of patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. Lysosomes are organelles that play an important role in cancer progression by breaking down biomolecules. However, the molecular mechanisms of lysosome-related genes in HCC are not fully understood. We downloaded HCC datasets from TCGA and GEO as well as lysosome-related gene sets from AIMGO. After univariate Cox screening of the set of lysosome-associated genes differentially expressed in HCC and normal tissues, risk models were built by machine learning. Model effects were assessed using the concordance index (C-index), Kaplan-Meier (K-M) and receiver operating characteristic curves (ROC). Additionally, we explored the biological function and immune microenvironment between the high- and low-risk groups, and analyzed the response of the high- and low-risk groups to immunotherapy responsiveness and chemotherapeutic agents. Finally, we explored the function of a key gene (RAMP3) at the cellular level. Univariate Cox yielded 46 differentially and prognostically significant lysosome-related genes, and risk models were constructed using eight genes (RAMP3, GPLD1, FABP5, CD68, CSPG4, SORT1, CSPG5, CSF3R) derived from machine learning. The risk model was a better predictor of clinical outcomes, with the higher risk group having worse clinical outcomes. There were significant differences in biological function, immune microenvironment, and responsiveness to immunotherapy and drug sensitivity between the high and low-risk groups. Finally, we found that RAMP3 inhibited the proliferation, migration, and invasion of HCC cells and correlated with the sensitivity of HCC cells to Idarubicin. Lysosome-associated gene risk models built by machine learning can effectively predict patient prognosis and offer new prospects for chemotherapy and immunotherapy in HCC. In addition, cellular-level experiments suggest that RAMP3 may be a new target for the treatment of HCC.

A novel cuproptosis-related lncRNA signature predicts the prognosis and immunotherapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most serious malignant tumors with a poor prognosis worldwide. Cuproptosis is a novel copper-dependent cell death form, involving mitochondrial respiration and lipoylated components of the tricarboxylic acid (TCA) cycle. Long non-coding RNAs (lncRNAs) have been demonstrated to affect the tumorigenesis, growth, and metastasis of HCC. We explored the potential roles of cuproptosis-related lncRNAs in predicting the prognosis for HCC. The RNA-seq transcriptome data, mutation data, and clinical information data of HCC patients were downloaded from The Cancer Genome Atlas (TCGA) database. The least absolute shrinkage and selection operator (LASSO) algorithm and Cox regression analyses were performed to identify a prognostic cuproptosis-related lncRNA signature. The receiver operating characteristic (ROC) analysis was used to evaluate the predictive value of the lncRNA signature for HCC. The enrichment pathways, immune functions, immune cell infiltration, tumor mutation burden, and drug sensitivity were also analyzed. We constructed a prognostic model consisting of 8 cuproptosis-related lncRNAs for HCC. The patients were divided into high-risk group and low-risk group according to the riskscore calculated using the model. Kaplan-Meier analysis revealed that the high-risk lncRNA signature was correlated with poor overall survival [hazard ratio (HR) =1.009, 95% confidence interval (CI) = 1.002-1.015; p= 0.010)] of HCC. A prognostic nomogram incorporated the lncRNA signature and clinicopathological features were constructed and showed favorable performance for predicting prognosis of HCC patients. In addition, the most immune-related functions were significantly different between the high-risk and low-risk groups. Tumor mutation burden (TMB) and immune checkpoints were also expressed differently between the two risk groups. Finally, HCC patients with low-risk score were more sensitive to several chemotherapy drugs. The novel cuproptosis-related lncRNA signature could be used to predict prognosis and evaluate the effect of chemotherapy for HCC.

Abstract 5799: SPINK1-induced tumor plasticity provides a therapeutic window for chemotherapy in hepatocellular carcinoma

Abstract Intratumor molecular heterogeneity of hepatocellular carcinoma (HCC) is partly attributed to the presence of cancer stem cells (CSCs), which we now know represents a critical root of tumor recurrence and chemotherapy resistance. CD133 is known to represent an important functional marker of liver CSCs. We have demonstrated CD133 to enrich following chemotherapy treatment, while CD133/Prom1-depletion would enhance sensitivity of HCC tumors to chemotherapy. Yet unfortunately, CD133 is not specific to HCC, but is also expressed in normal regenerating liver. Identifying critical factors expressed specifically in liver CD133+ CSCs, but not in liver normal CD133+ stem/progenitor cells may offer important therapeutic opportunities overcoming chemoresistance in HCC. RNA-seq profiling comparing sorted CD133+ and CD133- subsets of normal regenerating liver induced by DDC diet and HCC induced by either N-nitrosodiethylamine (DEN)/carbon tetrachloride (CCl4) or hydrodynamic tail vein injection of oncogenic plasmids AKT and NRAS identified Serine Peptidase Inhibitor Kazal Type I (SPINK1) to be distinctly expressed in the HCC CD133+ subpopulation but not in normal regenerating liver CD133+ subpopulation. SPINK1 overexpression in HCC clinical samples is correlated with a poor prognosis. Expression of SPINK1 increased during early liver progenitor development, peaked during the premature hepatocyte stage, decreased during hepatocyte maturation and increased progressively from well-differentiated to poorly differentiated HCCs. Enhanced transcriptional activity of SPINK1 was mediated by promoter binding of the epithelial cell-specific transcription factor ELF3, which like CD133, were both increased following chemotherapy treatment. SPINK1 inhibition by lentiviral-based knockdown or a specific monoclonal antibody (mAb) mitigated tumor initiation, self-renewal and chemoresistance. Mechanistically, secretory SPINK1 bound to epidermal growth factor receptor (EGFR) activating MEK/ERK signaling and consequently promoting the formation of CDK4/6-cyclin D1 complex to phosphorylate Rb and release E2F2, allowing the cells to overcome G1/S checkpoint promoting cell cycle progression as well as transcribing stemness, oncogenic dedifferentiation and chemoresistance-related genes. Collectively, our findings suggest SPINK1 to play a critical role in hepatocarcinogenesis and that SPINK1 mAb may represent a novel therapeutic option for the treatment of HCC, targeting at the CD133+ CSC tumor roots and overcoming chemoresistance. Citation Format: Ki-Fong Man, Lei Zhou, Ying-Tung Lam, Jun Yu, Jing-Ping Yun, Xin-Yuan Guan, Ming Liu, Stephanie Ma. SPINK1-induced tumor plasticity provides a therapeutic window for chemotherapy in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5799.

LncRNA model predicts liver cancer drug resistance and validate in vitro experiments.

Introduction: Hepatocellular carcinoma (HCC) patients may benefit from chemotherapy, but drug resistance is an important obstacle to favorable prognoses. Overcoming drug resistance is an urgent problem to be solved. Methods: Differential expression analysis was used to identify long non-coding RNAs (LncRNAs) that differed in chemotherapy-sensitive and chemotherapy-resistant patients. Machine learning algorithms including random forest (RF), lasso regression (LR), and support vector machines (SVMs) were used to identify important chemotherapy-related LncRNAs. A back propagation (BP) network was then used to validate the predictive capacity of important LncRNAs. The molecular functions of hub LncRNAs were investigated via qRT-PCR and cell proliferation assay. Molecular-docking technique was used to explore candidate drug of targets of hub LncRNA in the model. Results: A total of 125 differentially expressed LncRNAs between sensitive and resistant patients. Seventeen important LncRNAs were identified via RF, and seven factors were identified via LR. With respect to SVM, the top 15 LncRNAs of AvgRank were selected. Five merge chemotherapy-related LncRNAs were used to predict chemotherapy resistance with high accuracy. CAHM was a hub LncRNA of model and expression high in sorafenib resistance cell lines. In addition, the results of CCK8 showed that the sensitivity of HepG2-sorafenib cells to sorafenib was significantly lower than that of HepG2; and the sensitivity of HepG2-sorafenib cells transfected with sh-CAHM was significantly higher than that of Sorafenib. In the non-transfection group, the results of clone formation experiments showed that the number of clones formed by HepG2-sorafenib cells treated with sorafenib was significantly more than that of HepG2; after HepG2-sorafenib cells were transfected with sh-CAHM, the number of clones formed by Sorafenib treatment was significantly higher than that of HepG2 cells. The number was significantly less than that of HepG2-s + sh-NC group. Molecular Docking results indicate that Moschus was candidate drug for target protein of CAHM. Conclusion: Five chemotherapy-related LncRNAs could predict drug resistance in HCC with high accuracy, and the hub LncRNA CAHM has potential as a new biomarker for HCC chemotherapy resistance.

The Impact of a Liver Transplant Program on the Outcomes of Hepatocellular Carcinoma.

We sought to evaluate the impact of liver transplantation (LT) programs on the prognosis of hepatocellular carcinoma (HCC) patients who underwent liver resection (LR) and noncurative intent treatment. LT programs have an array of resources and services that would positively affect the prognosis of patients with HCC. Patients who underwent LT, LR, radiotherapy (RT), or chemotherapy (CTx) for HCC between 2004 and 2018 were included in the National Cancer Database. Institutions with LT programs were defined as those that performed 1 or more LT for at least 5 years. Centers were stratified by hospital volume. The impact of LT programs was assessed after propensity score matching to achieve covariate balance. A total of 71,735 patients were identified, of which 7997 received LT (11.1%), 12,683 LR (17.7%), 15,675 RT (21.9%), and 35,380 CTx (49.3%). Among a total of 1267 distinct institutions, 94 (7.4%) were categorized as LT programs. Designation as an LT program was also associated with a high volume of LR and noncurative intent treatment (both P <0.001). After propensity score matching, LT programs were associated with better survival among LR and noncurative intent treatment patients. Although hospital volume was also associated with improved prognosis, LT programs were associated with additional survival benefits in noncurative intent treatment. On the other hand, no such benefit was noted in patients who underwent LR. The presence of an LT program was associated with a higher volume of LR and noncurative intent treatment. Furthermore, designation as an LT program had a "halo effect" on the prognosis of patients undergoing RT/CTx that went beyond the procedure-volume effect.

Prediction of Cytotoxicity Against HepG2 by Quantitative Structure-Activity Relation (QSAR) Modelling

Hepatocellular carcinoma (HCC) is the dominant subtype of liver cancer with very low survival rate but the chemotherapy for HCC is still in grey zone due to the limited efficacy and high toxicity profile of approved drugs raising the heavy demand on drug development for HCC. The study aimed to establish a desirability based quantitative structure activity relation (QSAR) model to predict the activity of chemical compounds against one of HCC cell line (HepG2). Different support vector machine (SVM) models were constructed and ensembled to 10 virtual screening protocols. These protocols were validated by an external dataset in combination with decoys as interference. Results showed that ensemble models exhibited improved area under the Receiver Operating Characteristic Curve (ROC), sensitivity, and specificity compared to base models in training and test set. When being validated for virtual screening to recover known active molecules in the mixture with known inactive and decoy compounds, all virtual screening protocols have good performance with good Boltzmann-Enhanced Discrimination of ROC (BEDROC) and enrichment factor (EF). The best protocol with BEDROC of 0.63 and EF of 29.55 was suitable for further screening of active compounds against HepG2 cell line. HIGHLIGHTS Ensemble quantitative structure-activity relationship (QSAR) models improved the performance of single models Cytotoxicity prediction was transformed to desirability score as a general output for easily integration in multi-objective optimization Virtual models for screening of HepG2 cytotoxicity compounds were well validated GRAPHICAL ABSTRACT

Synergistic Effect of Lenvatinib and Chemotherapy in Hepatocellular Carcinoma Using Preclinical Models.

The current study aimed to evaluate the synergistic efficacy of lenvatinib and FOLFOX (infusional fluorouracil (FU), folinic acid, and oxaliplatin) in hepatocellular carcinoma (HCC) using patient-derived xenograft (PDX) and PDX-derived organotypic spheroid (XDOTS) models in vivo and in vitro. PDX and matched XDOTS models originating from three patients with HCC were established. All models were divided into four groups and treated with drugs alone or in combination. Tumor growth in the PDX models was measured and recorded, and angiogenesis and phosphorylation of the vascular endothelial growth factor receptor (VEGFR2), rearranged during transfection (RET), and extracellular signal-regulated kinase (ERK) were detected using immunohistochemistry and Western blot assays. The proliferative ability of XDOTS was evaluated through active staining and immunofluorescence staining, and the effect of the combined medication was evaluated using the Celltiter-Glo luminescent cell viability assay. Three PDX models with genetic characteristics similar to those of the original tumors were successfully established. Combining lenvatinib with FOLFOX led to a higher tumor growth inhibition rate than individual therapies (P < 0.01). Immunohistochemical analysis demonstrated that the combined treatment significantly inhibited the proliferation and angiogenesis of PDX tissues (P < 0.05), and Western blot analysis showed that the combined treatment significantly inhibited the phosphorylation of VEGFR2, RET, and ERK compared with single-agent treatment. Additionally, all three matched XDOTS models were successfully cultured with satisfactory activity and proliferation, and the combined therapies led to better suppression of XDOTS growth compared with individual therapy (P < 0.05). Lenvatinib combined with FOLFOX had a synergistic antitumor effect in HCC PDX and XDOTS models by inhibiting the phosphorylation of VEGFR, RET, and ERK.