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Objective: Effective adjuvant therapeutic strategies are urgently needed to overcome MAPK inhibitor (MAPKi) resistance, which is one of the most common forms of resistance that has emerged in many types of cancers. Here, we aimed to systematically identify the genetic interactions underlying MAPKi resistance, and to further investigate the mechanisms that produce the genetic interactions that generate synergistic MAPKi resistance. Methods: We conducted a comprehensive pair-wise sgRNA-based high-throughput screening assay to identify synergistic interactions that sensitized cancer cells to MAPKi, and validated 3 genetic combinations through competitive growth, cell viability, and spheroid formation assays. We next conducted Kaplan-Meier survival analysis based on The Cancer Genome Atlas database and conducted immunohistochemistry to determine the clinical relevance of these synergistic combinations. We also investigated the MAPKi resistance mechanisms of these validated synergistic combinations by using co-immunoprecipitation, Western blot, qRTPCR, and immunofluorescence assays. Results: We constructed a systematic interaction network of MAPKi resistance and identified 3 novel synergistic combinations that effectively targeted MAPKi resistance (ITGB3 + IGF1R, ITGB3 + JNK, and HDGF + LGR5). We next analyzed their clinical relevance and the mechanisms by which they sensitized cancer cells to MAPKi exposure. Specifically, we discovered a novel protein complex, HDGF-LGR5, that adaptively responded to MAPKi to enhance cancer cell stemness, which was up- or downregulated by the inhibitors of ITGB3 + JNK or ITGB3 + IGF1R. Conclusions: Pair-wise sgRNA library screening provided systematic insights into elucidating MAPKi resistance in cancer cells. ITGB3- + IGF1R-targeting drugs (cilengitide + linsitinib) could be used as an effective therapy for suppressing the adaptive formation of the HDGF-LGR5 protein complex, which enhanced cancer stemness during MAPKi stress.

BackgroundWith the increasing incidence and prevalence of alcoholic liver disease, alcohol-related hepatocellular carcinoma has become a serious public health problem worthy of attention in China. However, there is currently no prognostic prediction model for alcohol-related hepatocellular carcinoma.MethodsThe retrospective analysis research of alcohol related hepatocellular carcinoma patients was conducted from January 2010 to December 2014. Independent prognostic factors of alcohol related hepatocellular carcinoma were identified by Lasso regression and multivariate COX proportional model analysis, and the nomogram model was constructed. The reliability and accuracy of the model were assessed using the concordance index(C-Index), receiver operating characteristic (ROC) curve and calibration curve. Evaluate the clinical benefit and application value of the model through clinical decision curve analysis (DCA). The prognosis was assessed by the Kaplan-Meier (KM) survival curve.ResultsIn sum, 383 patients were included in our study. Patients were stochastically assigned to training cohort (n=271) and validation cohort (n=112) according to 7:3 ratio. The predictors included in the nomogram were splenectomy, platelet count (PLT), creatinine (CRE), Prealbumin (PA), mean erythrocyte hemoglobin concentration (MCHC), red blood cell distribution width (RDW) and TNM. Our nomogram demonstrated excellent discriminatory power (C-index) and good calibration at 1-year, 3-year and 5- year overall survival (OS). Compared to TNM and Child-Pugh model, the nomogram had better discriminative ability and higher accuracy. DCA showed high clinical benefit and application value of the model.ConclusionThe nomogram model we established can precisely forcasting the prognosis of alcohol related hepatocellular carcinoma patients, which would be helpful for the early warning of alcohol related hepatocellular carcinoma and predict prognosis in patients with alcoholic hepatocellular carcinoma.

Objective: Hepatocellular carcinoma (HCC), the main type of liver cancer, has a high morbidity and mortality, and a poor prognosis. RNA helicase DDX5, which acts as a transcriptional co-regulator, is overexpressed in most malignant tumors and promotes cancer cell growth. Heat shock protein 90 (HSP90) is an important molecular chaperone in the conformational maturation and stabilization of numerous proteins involved in cell growth or survival. Methods: DDX5 mRNA and protein expression in surgically resected HCC tissues from 24 Asian patients were detected by quantitative real-time PCR and Western blot, respectively. The interaction of DDX5-HSP90 was determined by molecular docking, immunoprecipitation, and laser scanning confocal microscopy. The autophagy signal was detected by Western blot. The cell functions and signaling pathways of DDX5 were determined in 2 HCC cell lines. Two different murine HCC xenograft models were used to determine the function of DDX5 and the therapeutic effect of an HSP90 inhibitor. Results: HSP90 interacted directly with DDX5 and inhibited DDX5 protein degradation in the AMPK/ULK1-regulated autophagy pathway. The subsequent accumulation of DDX5 protein induced the malignant phenotype of HCC by activating the β-catenin signaling pathway. The silencing of DDX5 or treatment with HSP90 inhibitor both blocked in vivo tumor growth in a murine HCC xenograft model. High levels of HSP90 and DDX5 protein were associated with poor prognoses. Conclusions: HSP90 interacted with DDX5 protein and subsequently protected DDX5 protein from AMPK/ULK1-regulated autophagic degradation. DDX5 and HSP90 are therefore potential therapeutic targets for HCC.

Background Hepatocellular carcinoma (HCC) is a common cause of cancer mortality worldwide. Recent studies have shown that the polytopic enzyme membrane associated ring-CH-type finger 6 (MARCH6) participates in tumorigenesis, but its function in HCC development needs to be investigated. This study aimed to explore the role of MARCH6 in HCC. Methods Expression of MARCH6 in human HCC samples was checked by immunohistochemical staining assay. Clinical relevance of MARCH6 and activating transcription factor 2 (ATF2) was analyzed from TCGA database. CCK-8, EdU staining, colony formation and transwell were performed to assess cell proliferation, growth and migration. Xenografted tumorigenesis was used to examine in vivo role MARCH6. Immunoblotting was applied to detect protein abundance. Results We found that MARCH6 expression was elevated in human HCC samples. Over-expression of MARCH6 was associated with poor prognosis of HCC patients. Up-expression of MARCH6 promoted cell growth and migration of HCC cells. In contrast, the HCC cell growth and migration were suppressed by MARCH6 knockdown. Furthermore, the DNA synthesis was enhanced by MARCH6. The expression of ATF2 was potentiated by MARCH6 over-expression, while it was suppressed by MARCH6 silencing. TCGA database showed positive correlation between the expression of MARCH6 and ATF2. Importantly, ATF2 expression contributed to the oncogenic function of HCC cells. Conclusion Our findings suggest that MARCH6-mediated ATF2 up-regulation contributes to HCC development. MARCH6 may be a promising target for the diagnosis and treatment of HCC.

BackgroundGamma-aminobutyric acid transaminase (ABAT) catalyzes the conversion of gamma-aminobutyric acid (GABA) into succinic semialdehyde. Although some evidence supports a key role of ABAT in the progression of hepatocellular carcinoma (HCC), no systematic analysis is available. Thus, this study aimed to investigate the possible mechanisms related to low ABAT expression and the prognostic value and potential functions of ABAT in HCC.MethodsWe obtained relevant datasets from the Encyclopedia of RNA Interactomes, MethSurv, cBioPortal, TISIDB and The Cancer Genome Atlas and used bioinformatic methods to analyze DNA methylation, copy number variation, gene mutation, and upstream microRNAs (miRNAs) of ABAT, exploring the potential relationship between ABAT expression and the prognosis, glycolysis, and immune infiltration in HCC.ResultsThe results indicated that ABAT expression was lower in HCC tumor tissues than in normal tissues or adjacent tissues. Low ABAT expression was related to patient age, T stage classification, pathologic stage, histological grade, and alpha-fetoprotein level of HCC. Kaplan-Meier survival analyses indicated that low ABAT expression was correlated with poor HCC prognosis. ABAT was also verified as an independent risk factor in HCC via Cox multivariate analysis. Gene set enrichment analysis showed enrichment in various signaling pathways. Furthermore, DNA methylation, copy number variation, and gene mutation potentially induced low ABAT expression; miR-135a-5p was a potential upstream miRNA of ABAT. Additionally, ABAT expression was associated with glycolysis-related genes, infiltrated immune cells, immunoinhibitors, and immunostimulators in HCC.ConclusionsOur study reveals that deficient ABAT expression is correlated with disease progression and poor prognosis in HCC because of its role in tumorigenesis and tumor immunity.

570 Background: ORIENT-32 trial (NCT03794440) assessed sintilimab (anti-PD-1 antibody) plus a bevacizumab biosimilar (anti-VEGF antibody) versus sorafenib (Sor) as first-line treatment for unresectable HCC and demonstrated a significant improvement in both overall survival and progression-free survival. Here we report the updated results of objective response rate (ORR), time to response (TTR), duration of response (DoR) and depth of response (DpR). Methods: 571 eligible patients (pts) with unresectable HCC were enrolled and randomized (2:1) to receive sintilimab (200 mg IV Q3W) plus IBI305 (15 mg/kg IV Q3W) or Sor (400 mg orally, BID) until disease progression or unacceptable toxicity. Tumors were evaluated using Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1)and HCC-modified RECIST (mRECIST). ORR, TTR, DoR, and DpR were analyzed. The DpR was defined as the minimum percentage of (1) sum of longest diameter (SLD) change and (2) longest diameter (LD) change described as mean (standard deviation, SD). Results: At the data cutoff on Dec 30th, 2021, the median follow-up time was 26.7 months. The ORR in sintilimab plus IBI305 and Sor group was 21.0% (77/367) vs 4.7 (8/169) per RECIST 1.1 and 25.1% (92/367) vs 7.7% (13/169) per mRECIST. The median TTR in sintilimab plus IBI305 group was 2.8 (2.4–3.3) months per RECIST 1.1 and 2.6 (1.6–2.9) months per mRECIST. The median DoR in sintilimab plus IBI305gourp was 20.3 (12.3-NE) months per RECIST 1.1. The minimum percentage of SLD change was larger in the sintilimab plus IBI305 arm than in the Sor arm: (−13.4% (35.8) vs 3.2%(26.5) per RECIST 1.1). Similarly, the LD change in the largest liver lesion also favored sintilimab plus IBI305 arm (−27.6% (31.6) vs −11.5% (20.9)), including larger tumors (≥7 cm; −21.2% (30.4) vs −9.9% (23.7)) all per RECIST 1.1. Conclusions: Sintilimab plus IBI305 showed a significant improvement in ORR, TTR, DOR and DpR vs Sor in unresectable HCC. Clinical trial information: NCT03794440 .

Improving efficacy of oncolytic virotherapy remains challenging due to difficulty increasing specificity and immune responses against cancer and limited understanding of its population dynamics. Here, we construct programmable and modular synthetic gene circuits to control adenoviral replication and release of immune effectors selectively in hepatocellular carcinoma cells in response to multiple promoter and microRNA inputs. By performing mouse model experiments and computational simulations, we find that replicable adenovirus has a superior tumor-killing efficacy than non-replicable adenovirus. We observe a synergistic effect on promoting local lymphocyte cytotoxicity and systematic vaccination in immunocompetent mouse models by combining tumor lysis and secretion of immunomodulators. Furthermore, our computational simulations show that oncolytic virus which encodes immunomodulators can exert a more robust therapeutic efficacy than combinatorial treatment with oncolytic virus and immune effector. Our results provide an effective strategy to engineer oncolytic adenovirus, which may lead to innovative immunotherapies for a variety of cancers., It is difficult to improve the efficacy of oncolytic virotherapy due to immune system responses and limited understanding of population dynamics. Here the authors use synthetic biology gene circuits to control adenoviral replication and release of immunomodulators in hepatocellular carcinoma cells.

Aims SDF-1α induced chemotaxis plays an important role in hepatocellular carcinoma metastasis. CXCR4 stimulated by SDF-1α/CXCL12 triggers heterotrimeric G proteins activation, which regulate migration and chemotaxis of hepatocellular carcinoma cells. The pathways linking the chemokine GPCR/Gi signaling to actin polymerization for migration of cancer cells are not known. Materials and methods Through would healing assay, chemotaxis assay, F-actin polymerization assay, confocal assay, immunohistochemical assay, protein identification and coimmunoprecipitation assay, we detected the role and mechanisms of Annexin A2 in hepatocellular carcinoma. Key findings In the present study, we firstly investigated the role of Annexin A2 in HepG2 cell chemotaxis and metastasis. Immunohistochemical analysis showed that Annexin A2 was highly expressed in hepatocellular carcinoma tissues. Its expression was closely associated with lymph node and distant metastasis. Knockdown Annexin A2 impaired cancer cell chemotaxis. Co-immunoprecipitation results showed an interaction between Annexin A2 and ELMO1. CXCL12 triggers an ELMO1-dependent membrane translocation of Annexin A2. Significance Taken together, our results indicated an important role of Annexin A2 in hepatocellular carcinoma tissues metastasis and revealed a novel molecular mechanism of its activation.

PURPOSE Donafenib, a novel multikinase inhibitor and a deuterated sorafenib derivative, has shown efficacy in phase Ia and Ib hepatocellular carcinoma (HCC) studies. This study compared the efficacy and safety of donafenib versus sorafenib as first-line therapy for advanced HCC. PATIENTS AND METHODS This open-label, randomized, parallel-controlled, multicenter phase II-III trial enrolled patients with unresectable or metastatic HCC, a Child-Pugh score ≤ 7, and no prior systemic therapy from 37 sites across China. Patients were randomly assigned (1:1) to receive oral donafenib (0.2 g) or sorafenib (0.4 g) twice daily until intolerable toxicity or disease progression. The primary end point was overall survival (OS), tested for noninferiority and superiority. Efficacy was primarily assessed in the full analysis set (FAS), and safety was assessed in all treated patients. RESULTS Between March 21, 2016, and April 16, 2018, 668 patients (intention-to-treat) were randomly assigned to donafenib and sorafenib treatment arms; the FAS included 328 and 331 patients, respectively. Median OS was significantly longer with donafenib than sorafenib treatment (FAS; 12.1 v 10.3 months; hazard ratio, 0.831; 95% CI, 0.699 to 0.988; P = .0245); donafenib also exhibited superior OS outcomes versus sorafenib in the intention-to-treat population. The median progression-free survival was 3.7 v 3.6 months ( P = .0570). The objective response rate was 4.6% v 2.7% ( P = .2448), and the disease control rate was 30.8% v 28.7% (FAS; P = .5532). Drug-related grade ≥ 3 adverse events occurred in significantly fewer patients receiving donafenib than sorafenib (125 [38%] v 165 [50%]; P = .0018). CONCLUSION Donafenib showed superiority over sorafenib in improving OS and has favorable safety and tolerability in Chinese patients with advanced HCC, showing promise as a potential first-line monotherapy for these patients.

Jiamin Cheng,1,* Yinyin Li,1,* Xiaohui Wang,2,* Zheng Dong,1 Yan Chen,1 Rui Zhang,1 Jiagan Huang,1 Xueyuan Jin,1 Jianfei Yao,2 Aifang Ge,2 Lele Song,2,3,* Yinying Lu,1 Zhen Zeng1 1Comprehensive Liver Cancer Department, The Fifth Medical Center of the Chinese PLA General Hospital, Beijing, 100039, People’s Republic of China; 2HaploX Biotechnology, Shenzhen, Guangdong Province, People’s Republic of China; 3Department of Radiotherapy, The Eighth Medical Center of the Chinese PLA General Hospital, Beijing, 100091, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yinying Lu; Zhen ZengComprehensive Liver Cancer Department, The Fifth Medical Center of the Chinese PLA General Hospital, Beijing, 100039, People’s Republic of ChinaEmail luyinying1973@163.com; zengzhen1970@sina.comBackground: Immunotherapy combined with VEGF inhibitor has become the new first-line therapy for advanced or metastatic hepatocellular carcinoma (HCC). However, the biomarkers for response and prognosis stratification of HCC first-line combined immunotherapy have not been clarified.Methods: Here, we obtained the genomic alteration data from pre-therapeutic samples of 103 HCC patients using a 605-gene NGS test, and obtained the transcriptional and T cell receptor (TCR) diversity data from 18 patients who underwent the first-line combined immunotherapy using RNAseq and TCR sequencing, respectively. Patients received sorafenib/sintilimab or lenvatinib/sintilimab combined first-line therapy and the response was assessed at 3– 6 cycles of therapy.Results: No stratification of response was found in high-frequency key driver gene mutations, including TP53 and TERT. However, significantly higher ratio of progression (PD) was found in patients carrying MDM4 amplification. Similarly, FGF/3/4/19 amplifications could also result in high ratio of PD. The mRNA and lncRNA levels of eight genes related to hepatic metabolism and immune microenvironment exhibited significant differences between PR/SD and PD group, including DGKI, TNFSF14, CHST4, ACTIN1, PFKP, SLC51B, LCK and ERN1, suggesting stratification of response. Furthermore, moderate correlation was identified between the stratification genes (CHST4, SLC51B and ERN1) and immune factors (TIGIT, CD34, ICAM1, CCL5, CXCL9 and CXCL10), suggesting potential roles of these factors in immunoregulation. Strong linear correlation was found between any two of the three indexes for TCR CDR3 diversity, including Shannon–Wiener Index, Simpson index and evenness. However, no significant difference was found in the three indexes between the PR/SD and PD group, suggesting no stratification of response by these indexes.Conclusion: We identified several potential biomarkers for response stratification in the first-line combined immunotherapy. MDM4 was capable of predicting disease progression, and a panel mRNA and lncRNA of eight genes may also predict the response. Further validation is needed to verify these biomarkers.Keywords: hepatocellular carcinoma, HCC, immunotherapy, mRNA, lncRNA, TCR, sintilimab, sorafenib, lenvatinib

Background: Small nuclear ribonucleoprotein polypeptide A (SNRPA1) is a splicing factor (SF) responsible for the processing of pre−mRNA into mRNA. The purpose of this study was to explore the clinicopathological characteristics and prognostic significance of SNRPA1 mRNA expression in liver cancer and its potential mechanism as an SF.Methods: A total of 418 RNA−Seq and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database. Alternative splicing (AS) profiles were downloaded from TCGA SpliceSeq. Wilcoxon rank-sum tests were used to compare normal tissues with tumor tissues and analyze subgroups. We used the Kaplan−Meier analysis method to draw the survival curves. Univariate and multivariate Cox analyses were employed to estimate the prognostic value of SNRPA1. Gene set enrichment analysis (GSEA) was performed to identify the signaling pathways. Then we used univariate and Pearson's correlation tests to analyze the correlation between SFs and exon skip (ES) events. Wilcoxon rank-sum tests were applied to analyze the relationships between different spliceosome and cancers. We also analyzed the relationship between the expression of SCP2 (related to SNRPA1) and clinical symptoms. We then evaluated the expression levels of these genes with clinical samples and The Clinical Proteomic Tumor Analysis Consortium (CPTAC). Results: The level of SNRPA1 mRNA expression in liver cancer was significantly up−regulated in tumor tissues compared with normal tissues (p = 1.411e−27) in liver cancer and was positively correlated with survival status (p = 0.035). In addition, we found that SNRPA1 mRNA levels can reflect the prognosis of liver cancer (hazard ratio [HR] = 1.08, 95% confidence interval [CI]: 1.02–1.14, p = 0.005). The enriched KEGG pathway by GESA revealed a splicesome as the main pathway of SNRPA1. Alternative splicing events of SNRPA1 was related to the expression of SCP2. SNRPA1 exon 6 skip and SCP2 exon 12 skip correlated with many cancer types. Finally, SNRPA1 and SCP2 percent−spliced−in (PSI) values positively correlated with survival status (p = 3.022e−04 and p = 2.932e−03). Conclusions: Splicing Factor SNRPA1 is superior to its mRNA expression in predicting the prognosis of liver cancer and correlates with the SCP2 spliceosome, which is consistent with that of SNRPA1 in clinical samples.

e16112 Background: Recently, atezolizumab plus bevacizumab (A+T) has shown promising effects on hepatocellular carcinoma (HCC). So far, no study explored the effectiveness and safety of A+T combined with interventional therapy in patients with HCC. This study aimed to explore short-term efficacy and safety of A+T combined with interventional therapy in first-line treatment of advanced HCC. Methods: In this retrospective study, 13 advanced HCC patients received first-line A+T combined with interventional therapy from September 2020 to September 2021 were enrolled. Liver function was evaluated by Child-Pugh score. Objective response rate (ORR), disease control rate (DCR), progression free survival (PFS) and adverse events (AEs) were recorded in accordance with RECIST v1.1 and CTCAE v5.0. The PFS was defined as the time from first dosing of A+T or first time of interventional therapy until disease progressive or death. Results: A total of 13 HCC patients with Barcelona Clinic Liver Cancer (BCLC) stage C were reviewed. The liver function of 8 patients was Child-Pugh A while the other 5 patients was Child-Pugh B. Median time of following up was 8.9 (5.2-12.6) months. The ORR and DCR were 30.8% and 84.6%, respectively. One patient was able to conduct hepatectomy after treatment. And CR was confirmed by pathological report after surgery. The median PFS was 9.7 (7.3-12.1) months. The 3-month PFS rate was 84.6%. Nearly all patients suffered from AEs. The most frequency AE were hyperthyroidism (6 of 15), following hypertension (4 of 15), weight loss (4 of 15). While platelet-cell- decrease (3 of 15) turn out to be the most frequent serious AE, but it could be well managed. No AE-related death happened. Total two patients died, of progression of tumor and septic shock respectively. Conclusions: A+T combined with interventional therapy showed potential short-term anti-tumor effect and tolerance on advanced HCC.[Table: see text]

e16115 Background: The efficacy of first-line ICIs combination therapy patients with advanced hepatocellular carcinoma (HCC) was better than that of TKIs alone. This study was conducted to explore whether regorafenib combined with ICIs has better efficacy in second-line therapy. Methods: This retrospective study included 101 patients who have received regorafenib after the failure of at least one molecular-targeted therapy evaluated between March 2017 to November 2021. The patients were divided into two groups based on receiving regorafenib (cohort A) or regorafenib combined with ICIs(cohort B). The primary endpoint was Progression-Free Survival (PFS), and the secondary endpoints included the Objective Response Rate (ORR), Disease Control Rate (DCR), Overall Survival (OS), and safety. Results: 29 patients were in the cohort A and 72 were in the cohort B. There were no significant differences in age, gender, weight, ECOG performance status, Child-Pugh, BCLC staging, and the proportion of previous therapy regimens between the two cohorts. Among them, the first-line therapy mainly includes sorafenib, lenvatinib with or without ICIs. The median PFS (4.0 months), 6 months PFS rate (25.66%) and DCR(65.52%) of cohort A were poor than that of cohort B (8.7 months, 83.33%, 74.13%), (P＜0.05). OS data was not yet available. First-line therapy, use of TKI with ICIs (n = 31) or TKI without ICIs(n = 27) (PFS 8.70m VS 8.37m，P = 0.84), use of sorafenib with or without ICIs (n = 22), or lenvatinib with or without ICIs (n = 33)(PFS 8.87m VS 7.60m，P = 0.77), didn’t affect the PFS and ORR of the later line therapeutic regimens. In the cohort B, the ORR of the second-line (29.31%) was better than that of the third-line (0), (P = 0.03). Adverse events (AEs) of all grades were statistically different between the two groups (P = 0.03), but there was no significant difference in the incidence of grade 3-4 AE. Conclusions: In the later-line therapy, regorafenib combined with ICIs therapy showed better curative effect, especially in the second-line. Regorafenib combined with ICIs is recommended as early as second-line therapy to benefit patients. Different regimens of first-line therapy have no effect on PFS in second-line therapy.[Table: see text]

e16155 Background: Combining lenvatinib (a multikinase inhibitor) with programmed cell death protein-1 (PD-1) inhibitor has been explored in Unresectable hepatocellular carcinoma (uHCC) in phase 1b trials. This study aimed to investigate the real-word efficacy and safety of lenvatinib plus PD-1 inhibitor for large Chinese patients uHCC cohorts. Methods: BCLC stage B or C uHCC patients were included. Data was collected from Oct 2017 to Nov 2021 retrospectively. Patients were treated with lenvatinib and PD-1 inhibitor (pembrolizumab or nivolumab or camrelizumab or sintilimab or toripalimab or tislelizumab). The primary endpoint was overall survival (OS) and secondary endpoints were progression-free survival (PFS) and objective response rate (ORR) evaluated by RECIST v1.1 criteria. Adverse events (AEs) were assessed using CTCAE v5.0. Prognostic factors of survival were also analyzed. Results: 378 uHCC patients of BCLC stage B (12.7%) and C (87.3%) from two medical centers were included in China. The median age was 55 (range 18-89) years and 86.5% patients were male. ECOG scores were 0 (43.7%), 1 (43.4%) and 2 (13.0%), while Child-pugh grade were A (77.5%) and B (22.5%) respectively. The median OS was 17.8 (95% CI 14.0-21.6) months. The median PFS was 6.9 (95% CI 6.0-7.9) months. The best objective response rate (ORR) and disease control rate (DCR) was 74/378 (19.6%) and 278/378 (73.5%) respectively. Child-pugh grade (A vs. B), ECOG score (0 vs. 1-2), BCLC stage (B vs. C) and achieve response (yes vs. no) were the prognostic factors for both OS and PFS. All (100%) experienced all-grade adverse events (AE). The most frequent AEs were fatigue, diarrhea, hypertension, decreased appetite. The most common ≥Grade 3 AEs were hypertension (13.2%), fatigue (7.1%), decreased platelet count (6.3%), increased blood bilirubin (6.1%), diarrhea (6.1%) and gastrointestinal hemorrhage (4.8%). Conclusions: This real-world study of lenvatinib plus PD-1 inhibitors achieved long survival and considerable ORR for uHCC patients in China. The tolerability of combination therapy was acceptable but should be still monitored closely. Clinical trial information: NCT03892577. [Table: see text]

Aim: To investigate the quantitative relationship between the positive detection rate (PDR) in colorectal tumor detection and the mSEPT9 level. Materials & methods: The level of blood mSEPT9 in various colorectal diseases was quantified by the Epi proColon 2.0 assay. ΔΔCt values were calculated representing the mSEPT9 level. A total of 1347 subjects were recruited in this quantitative study. Results: PDR or sensitivity was positively correlated with the progression of colorectal tumors and the mSEPT9 level in an exponential relationship. The mSEPT9 level of CRC exhibited a distinct pattern of distribution. Strong correlation was found between mSEPT9 level and PDR or sensitivity in various tumor differentiation, pathological types or metastasis. Conclusion: The quantitative profiling of blood mSEPT9 determines the detection performance on colorectal tumors.

Yan Chen,1,2,* Qinglei Zeng,3,* Xiufang Liu,4 Junliang Fu,1 Zhen Zeng,1 Zhiqin Zhao,1 Ze Liu,1 Wenlin Bai,1 Zheng Dong,1 Hongjin Liu,1 Xiaoxia Lu,1 Yunfeng Zhu,2,5 Yinying Lu1 1Comprehensive Liver Cancer Center, Beijing 302 Hospital, Beijing 100039, P.R. China; 2College of Life Sciences and Bio-Engineering, Beijing Jiaotong University, Beijing 100044, P.R. China; 3Department of Infectious Disease, The First Affiliated Hospital of Zhengzhou University, Zheng Zhou 450052, Henan Province, P.R. China; 4Department of Oncology, Chinese PLA 251 Hospital, Zhangjiakou 075000, P.R. China; 5Cancer Center in Division of Internal Medicine, Chinese PLA General Hospital, Beijing 100853, P.R. China *These authors contributed equally to thiswork Background: LINE-1 ORF-1p is encoded by the human pro-oncogene LINE-1. Our previous work showed that LINE-1 ORF-1p could enhance the resistance of hepatocellular carcinoma (HCC) cells to antitumor agents. However, the mechanisms involved in LINE-1 ORF-1p-mediated drug resistance remain largely unknown.Materials and methods: The endogenous mRNA level of LINE-1 ORF-1p in clinical HCC specimens was examined using quantitative PCR (qPCR). The prognosis of HCC patients was assessed using time to progression and overall survival. The transcription factor activity of pregnenolone X receptor (PXR) was examined using luciferase gene reporter assays, qPCR, chromatin immunoprecipitation assays and cellular subfraction assays. Protein interaction between LINE-1 ORF-1p and PXR was detected by co-immunoprecipitation. The effect of LINE-1 ORF-1p on sorafenib resistance in HCC cells was studied using in vitro and in vivo models.Results: A high level of LINE-1 ORF-1p in clinical specimens was related to poor prognosis in patients who received sorafenib treatment. LINE-1 ORF-1p increased the transcription factor activity of PXR by interacting with PXR and enhancing its cytoplasmic/nuclear translocation, and recruiting PXR to its downstream gene promoter, in turn enhancing the expression of the sorafenib resistance-related genes, CYP3A4 and mdr-1. LINE-1 ORF-1p enhanced the resistance to and clearance of sorafenib in HCC cells.Conclusion: LINE-1 ORF-1p enhances the transcription factor activation of PXR and promotes the clearance of and resistance to sorafenib in HCC cells. Keywords: LINE-1 ORF-1p, sorafenib resistance, pregnenolone X receptor, hepatocellular carcinoma, transcription factor activation

Recent studies suggest that Src family kinase (SFK) plays important roles in systemic sclerosis and pulmonary fibrosis. However, how SFKs contributed to the pathogenesis of liver fibrosis remains largely unknown. Here, we investigated the role of Fyn, a member of SFK, in hepatic stellate cell (HSC) activation and liver fibrosis, and evaluated the anti-fibrotic effects of Saracatinib, a clinically proven safe Fyn inhibitor. Fyn activation was examined in human normal and fibrotic liver tissues. The roles of Fyn in HSC activation and liver fibrosis were evaluated in HSC cell lines by using Fyn siRNA and in Fyn knockout mice. The effects of Saracatinib on HSC activation and liver fibrosis were determined in primary HSCs and CCl4 induced liver fibrosis model. We showed that the Fyn was activated in the liver of human fibrosis patients. TGF-β induced the activation of Fyn in HSC cell lines. Knockdown of Fyn significantly blocked HSC activation, proliferation, and migration. Fyn deficient mice were resistant to CCl4 induced liver fibrosis. Saracatinib treatment abolished the activation of Fyn, downregulated the Fyn/FAK/N-WASP signaling in HSCs, and subsequently prevented the activation of HSCs. Saracatinib treatment significantly reduced the severity liver fibrosis induced by CCl4 in mice. In conclusions, our findings supported the critical role of Fyn in HSC activation and development of liver fibrosis. Fyn could serve as a promising drug target for liver fibrosis treatment. Fyn inhibitor Saracatinib significantly inhibited HSC activation and attenuated liver fibrosis in mouse model.

Background & Aims: Hepatocellular carcinoma (HCC), the main type of liver cancer, has a high morbidity and mortality rate and poor prognosis. RNA helicase DDX5, which acts as a transcriptional co-regulator, is overexpressed in most malignant tumors and promotes cancer cell activities. Heat shock protein 90 (HSP90) is an important molecular chaperone in the conformational maturation and stabilization of numerous proteins involved in cell growth or survival. Methods: DDX5 mRNA and protein expression in surgically resected HCC tissues from 24 Asian patients were detected by quantitative real-time PCR and western blotting, respectively. The interaction of DDX5-HSP90 was testified by molecular docking, immunoprecipitation and laser scanning confocal microscopy (LSCM). The signal of autophagy was detected by western blotting. The cells functions and signalling pathway of DDX5 was examined in two HCC cell lines. Two different murine HCC xenograft models were used to determine the function of DDX5 and the therapeutic effect of HSP90 inhibitor. Results: High levels of DDX5 protein were associated with poor prognosis. HSP90 interacted directly with DDX5 and protected DDX5 protein from AMPK/ULK1-regulated autophagy degradation. The subsequent accumulation of DDX5 protein promoted the malignant phenotype of HCC by activating the β-catenin signaling pathway. Silence of DDX5 or treatment with HSP90 inhibitor both blocks in vivo tumor growth in murine HCC xenograft model. Conclusions: HSP90 interacts with DDX5 protein and subsequently protects DDX5 protein from AMPK/ULK1-regulated autophagic degradation. DDX5 or HSP90 is the potential therapeutic target for HCC. Funding Statement: National Natural Science Foundation of China (81672467, 81702773, 81702389), Major National R&D Project (2018ZX10723204, 2018ZX10302205) and Natural Science Foundation of Beijing (7172207). Declaration of Interests: The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Ethics Approval Statement: This study was approved by the Ethics Committee at each hospital and all patients provided informed consent prior to participation in the study.

e16127 Background: To evaluate the efficacy and safety of sorafenib or lenvatinib combined with sintilimab in patients with advanced hepatocellular carcinoma. Methods: This retrospective study included patients with advanced/metastatic hepatocellular carcinoma who received sintilimab (iv. 200 mg, Q3W) combined with sorafenib (oral, 400 mg twice-daily) (cohort A) or lenvatinib (oral, 12 mg/day for bodyweight≥60 kg or 8 mg/day for bodyweight < 60 kg) (cohort B) as first line therapy between March 2019 to December 2020 in the 5th medical center of the PLA general hospital of China. The primary endpoint was Progression-Free Survival (PFS), and the secondary endpoints included the Objective Response Rate (ORR), Disease Control Rate (DCR), Overall Survival (OS), Time to Progression (TTP) and safety. Results: 45 patients were enrolled, of which 29 were in the cohort A and 16 were in the cohort B. Except for the extrahepatic metastasis (62.1% vs 25.0%, P= 0.029), there were no significant differences in age, gender, weight, ECOG performance status, Child-Pugh, BCLC staging, and the proportion of previous treatment regimens between the two cohorts. The mean (±SD) exposure cycles in the two cohorts were 7.2±6.7 vs 7.1±4.7 ( P= 0.584). The median PFS of cohort A (8.2 months, 95%CI 3.1-19.5) was longer than that of cohort B (5.2 months, 95%CI 2.0-10.8), but there was no significant difference (HR 0.55, 95%CI 0.24-1.29, P = 0.161). However, there was no significant difference between the two cohorts in ORR (24.1% vs 6.3%, P= 0.226), DCR (82.8% vs 75.0%, P= 0.700) and median TTF (8.2 vs 4.6months, HR 0.49, 95%CI 0.21- 1.10, P= 0.074). OS data were not yet mature. There was no significant difference in the incidence of all grades and grade 3-4 adverse events between the two cohorts. The most common grade 3-4 adverse events in the two cohorts were hand-foot syndrome (17.2%, 5/29) and lung infection (12.5%, 2/16). There was 1 patient (immune hepatitis) in cohort A and 2 patients (pulmonary infection) in cohort B leading to treatment interruption due to adverse events, and no deaths due to treatment. Conclusions: Sorafenib or lenvatinib combined with sintilimab showed a good efficacy and safety in patients with advanced hepatocellular carcinoma, the safety and tolerability profiles were consistent with those previously observed. Sorafenib plus sintilimab provided an improved PFS versus lenvatinib, which requires a large sample of randomized controlled trial to confirm.[Table: see text]

e16135 Background: Recently, tyrosine kinase inhibitor plus PD-1 inhibitor regimen has shown promising effects on hepatocellular carcinoma (HCC). So far, no study explored the effectiveness of upfront versus add-on combination therapy in patients with systemic treatment-naïve and treatment-experienced HCCThis study aimed to explore the effectiveness and safety of sintilimab combined sorafenib in patients with untreated or sorafenib monotherapy-refractory unresectable HCC. Methods: In this retrospective study, unresectable HCC patients received sintilimab plus sorafenib from January 2018 to December 2020 were enrolled. According to the prior treatment, patients were grouped as first-line and second-line (received sorafenib combined sintilimab after progression on sorafenib alone). Objective response rate (ORR), disease control rate (DCR), progression free survival (PFS), safety, and the change of Child-Pugh score from baseline to progression were recorded. The PFS was defined as the time from first dosing of sintilimab until disease progressive or death. Results: A total of 31 patients were reviewed, including 19 patients in first-line group and 12 patients in second-line group. There were two patients with Barcelona Clinic Liver Cancer (BCLC) stage B and 17 patients with BCLC stage C in first-line group, and one patient with BCLC stage A and 11 patients with BCLC stage C in second-line group, respectively. The ORR and DCR were 27.8% and 77.8% in first-line group, and 16.7% and 75.0% in second-line group, respectively. Fourteen cases in first-line group and 10 cases in second-line group showed PD during the follow-up. The 6-month PFS rate of first-line group and second-line group were 47.1% and 64.8%, respectively. Thirteen and 9 patients experienced AE in two groups. Four and 2 grade 3 AEs occurred in two groups, respectively. The most frequency AE were hand-foot-skin reaction in both group (58.0% and 50.0%, respectively). One patient discontinued sintilimab due to AE in second-line group. Among PD patients, the Child-Pugh score improved in 2 cases, maintained in 5 cases, and deteriorated in 7 cases in the first-line group; and improved in 3 cases, miantained in 3 cases, and deteriorated in 4 cases in the second-line group. Conclusions: Sorafenib plus sintilimab showed potential anti-tumor effect and tolerance on unresectable HCC, either as first-line therapy or after progression with sorafenib monotherapy. Outcomes.[Table: see text]

4506 Background: Sorafenib is still the standard first-line therapy for advanced hepatocellular carcinoma (HCC). Donafenib, a novel multikinase inhibitor, showed potential benefits in a previous phase Ib study in HCC. Methods: In this open-label, randomized phase II/III trial (ZGDH3), patients with unresectable or metastatic HCC, a Child-Pugh liver function score ≤ 7, and no prior systemic therapy were enrolled from 37 sites across China and randomized (1:1) to receive oral donafenib (0.2 g) or sorafenib (0.4 g) twice daily until intolerable toxicity or disease progression. The primary endpoint was overall survival (OS). Efficacy analysis was primarily based on the full analysis set (FAS). Results: Between March 2016 and April 2018, 668 patients were randomized (donafenib, 334; sorafenib, 334) and included in the intention-to-treat (ITT) set, of whom 659 were analysed by FAS (328 vs 331). Donafenib was associated with a significantly longer median OS than sorafenib in both FAS (12.1 months vs 10.3 months, hazard ratio 0.831, 95% confidence interval 0.699–0.988, p = 0.0363) and ITT (12.0 months vs 10.1 months, 0.839, 0.706–0.996, p = 0.0446). There were no significant differences in median progression-free survival (3.7 months vs 3.6 months, p = 0.2824), objective response rate (4.6% vs 2.7%, p = 0.2448), and disease control rate (30.8% vs 28.7%, p = 0.5532). Grade 3 or worse adverse events (AEs) occurred in 191 (57.4%) and 224 (67.5%) patients ( p = 0.0082), respectively, and AEs of special interest and those leading to treatment interruption occurred in 287 (86.2%) vs 309 (93.1%, p = 0.0049) and 101 (30.3%) vs 141 (42.5%, p = 0.0013). A numerically lower number of patients reported serious AEs (55 [16.5%] vs 67 [20.2%], p = 0.2307) with donafenib. Common AEs with donafenib included hand-foot skin reaction (50.5%), aspartate aminotransferase increased (40.5%), blood bilirubin increased (39.0%), platelet count decreased (37.8%), and diarrhea (36.6%). Conclusions: Donafenib significantly improves OS over sorafenib with favourable safety and tolerability. Donafenib is a promising superior first-line therapy for advanced HCC. Funding: Zelgen. Clinical trial information: NCT02645981 .

We performed genomic and transcriptomic sequencing of 133 combined hepatocellular and intrahepatic cholangiocarcinoma (cHCC-ICC) cases, including separate, combined, and mixed subtypes. Integrative comparison of cHCC-ICC with hepatocellular carcinoma and intrahepatic cholangiocarcinoma revealed that combined and mixed type cHCC-ICCs are distinct subtypes with different clinical and molecular features. Integrating laser microdissection, cancer cell fraction analysis, and single nucleus sequencing, we revealed both mono- and multiclonal origins in the separate type cHCC-ICCs, whereas combined and mixed type cHCC-ICCs were all monoclonal origin. Notably, cHCC-ICCs showed significantly higher expression of Nestin, suggesting Nestin may serve as a biomarker for diagnosing cHCC-ICC. Our results provide important biological and clinical insights into cHCC-ICC.

Background Epigenetic alterations are well documented in hepatocarcinogenesis. However, hypomethylation of long interspersed nuclear element 1(LINE-1) promoter and its relationship with clinicopathological features in hepatocellular carcinoma (HCC) remain unknown. Methods The bisulfite-specific PCR and DNA sequencing analysis was performed to assess the methylation status of LINE-1 promoter in a pilot cohort of 71 patients with HCC. Additionally, methylation levels of two hot CpG sites of LINE-1 promoter, site 7 and 18 were measured by real-time PCR and compared with clinicopathological parameters in a cohort of 172 HCC. All the patients included were in BCLC stage A or B. Results Most patients with HCC (87.3%) showed hypomethylation of LINE-1 promoter compared with HBV-related cirrhosis and normal controls (P

Widespread interest in the mechanism of transcriptional regulation by the androgen receptor (AR) has been stimulated by the finding that AR signaling is critically important in the progression of human prostate cancers. Co-factors, the co-repressors, or the co-activators are responsible for the regulation of AR activation. The pro-oncogene human Long Interspersed Nucleotide acid Element-1 (LINE-1) encodes LINE-1 ORF-1p and plays important roles in the development and progression of several human carcinomas. In this study, the results showed that LINE-1 ORF-1p increased the AR transcriptional activity and in turn enhanced the expression of prostate specific antigen (PSA) in the presence of R1881. A physical protein-protein interaction between the AR signaling and the LINE-1 ORF-1p was identified by the immunoprecipitation assays and GST pull-down assays. Furthermore, LINE-1 ORF-1p would function as a novel AR positive co-regulator through modulating its cytoplasm/nucleus translocation and the recruitment to the androgen response element in the PSA gene promoter. Our date also showed that the LINE-1 ORF-1p promoted the proliferation and anchor-independent growth of LNCaP (ligand dependent) and PC-3 (ligand independent) human prostatic carcinoma cells. By investigating a novel role of the LINE-1 ORF-1p in the androgen/androgen receptor signaling pathway regulation, our study identifies that LINE-1 ORF-1p may be a novel AR co-regulator and molecular target for human prostate carcinoma therapy.

Hepatocellular carcinoma (HCC) is a common digestive malignancy. MiR-223, a well-identified miRNA, exhibits diverse properties in different cancers. In this study, we demonstrated that miR-223 could suppress cell growth and promote apoptosis in HepG2 and Bel-7402 HCC cell lines. We screened and identified a novel miR-223 target, Ras-related protein Rab-1(Rab1). Upregulation of miR-223 would specifically and markedly down-regulate Rab1 expression. In addition, miR-223-overexpressing subclones showed significant cell growth inhibition by increasing cell apoptosis in HepG2 and Bel-7402 cells. To identify the mechanisms, we firstly investigated the mTOR pathway and found that pmTOR, p70S6K and Bcl-2 were dramatically down-regulated after miR-223 transfection, while no changes in the level of Bax was visualized. Furthermore, our data showed that the anti-tumor effects arising from miR-223 transfection in HCC cells may be due to the deactivation of mTOR pathway caused by the suppression of Rab1 expression when miR-223 is overexpressed. In summary, our results indicate that miR-223 functions as a tumor suppressor and plays a critical role in inhibiting the tumorigenesis and promoting the apoptosis of HCC through the mTOR signaling pathway in vitro. By targeting Rab1, miR-223 efficiently mediates the mTOR pathway. Given these, miR-223 may be a potential therapeutic target for treating HCC.

Sorafenib may prolong survival in patients with advanced hepatocellular carcinoma (HCC), but with limited efficacy. The present study aimed to prospectively investigate the efficacy and analyze the prognostic factors for survival in sorafenib-treated patients with advanced HCC. The baseline characteristics and clinical outcomes of 110 patients with advanced hepatitis B virus-related HCC treated with sorafenib with/without local therapy (transarterial chemoembolization with/without cryoablation) at a single liver cancer center were recorded. Predictors of progression-free survival (PFS) and overall survival (OS) were determined by multivariate analysis. A total of 14 (12.7%) patients achieved complete response (CR), 16 (14.5%) achieved partial response (PR) and 40 (36.4%) achieved stable disease (SD) lasting longer than 8 weeks. The median OS and PFS for the whole cohort were 10.5 [95% confidence interval (CI), 8.7–12.3] and 5.0 months (95% CI, 3.7–6.3), respectively. Sorafenib in combination with local therapy was an independent predictor for longer PFS, whereas Eastern Cooperative Group (ECOG) performance status (PS) and Child-Pugh class were associated with reduced PFS. Local therapy was associated with longer OS while ECOG PS and α-fetoprotein were associated with reduced OS. In a subset of patients with radiological progressive disease, a significant difference was found in OS between patients who continued taking sorafenib and those who discontinued therapy (11 vs. 7.5 months, P

MicroRNA-153 (miR-153) is considered to be a tumor regulator. Silencing of miR-153 expression induced apoptosis in breast cancer cells. Data on mechanism suggest that up-regulation of miR- 153 level promotes cell proliferation via the down regulation of the expression of PTEN or FOXO1, which attenuates the proliferation of cancerous cells. This study aims to identify the effect of miR-153 on the activity of chemotherapeutic and targeted agents in HCC cells and to investigate the mechanisms involved. MTT, soft agar, trans-well and flow cytometry assays were performed to examine whether miR-153 down-regulated the activity of the chemotherapeutic and targeted drugs, Sorafenib, Etoposide and Paclitaxel in HCC cells. The rate of proliferation inhibition, relative survival rates and IC 50 values of each drug were calculated. Western blot and luciferase assays were performed to assess whether miR-153 modulates the expression of important genes related to cell proliferation, apoptosis or survival. Results showed that miR-153 attenuated the effect of Etoposide, Paclitaxel and Sorafenib on HepG2 cells; the IC 50 value increased from 0.25±0.01μmol/L to 1.02±0.14μmol/L, 0.05±0.01μmol/L to 0.14±0.02μmol/L and from 1.09±0.15μmol/L to 5.18±0.99μmol/L, respectively. In addition, miR-153 also reduced the effect of these drugs on MHCC- 97H, MHCC-97 L and L-02 cells; and it also reduced the effects of Sorafenib, Etoposide and Paclitaxel on anchorindependent growth of HepG2 cells. Over-expression of miR-153 down-regulated the activity of Etoposide and Paclitaxel on cell cycle arrest of HepG2 cells and the effect of Sorafenib on the invasion and migration of HepG2 cells. Furthermore, overexpression of miR-153 also enhanced the growth of HepG2, MHCC-97H, MHCC-97 L and L-02 cells. Mechanisms data showed that overexpression of miR-153 down regulated the activity of luciferase reporters, p15-Luc and p21-Luc; and enhanced the protein level of pro-survival or anti-apoptosis proteins Survivin and BCL-2. These results show that overexpression of miR-153 protects HepG2 cells against the effects of these drugs via multiple mechanisms, and miR-153 may be a novel target for HCC in future diagnostic and therapeutic interventions.

Triptolide is a traditional Chinese medicinal herb-derived antineoplastic agent. However, its antitumor activity against gynecologic carcinomas has not yet been well described. It is the purpose of this article to investigate the effect and mechanism of triptolide in human ovarian cancer using both A2780 (p53 wild) and OVCAR-3 (p53 mutated) cells. Our results showed that triptolide exerted a potent inhibitory effect on the growth and proliferation of both cell lines in a dose- and time-dependent manner and that the effect was independent of the expression of p53. In contrast, triptolide had only a marginal cytotoxicity in noncancerous ovary cells, lung fibroblast cells, and macrophage cells, indicating differential inhibitory effects of the drug on cell growth between ovarian cancer cells and normal tissue cells. Exposure of the ovarian cancer cells to triptolide induced apoptosis, as evaluated by annexin V/propidium iodide-labeled flow cytometry. Triptolide-induced apoptosis was accompanied by cytochrome c release and caspase-3 activation and was associated with downregulation of Bcl-2 and upregulation of Bax. Cell cycle analysis demonstrated that treatment with triptolide induced cell cycle S phase arrest in A2780 cells and G2/M phase arrest in OVCAR-3 cells. Further detection by Western blotting revealed that the cell cycle arrest by triptolide in both cell lines occurred in concert with increased expression of p21(CIP1/WAF1). This study shows that triptolide selectively kills ovarian cancer cells with different p53 status predominantly through regulating the coordinate and dynamic cellular processes of proliferation and apoptosis, thereby making it a promising chemotherapeutic agent against a broad spectrum of ovarian carcinomas.

Long interspersed nucleotide element (LINE)-1 ORF-1p is encoded by the human pro-oncogene LINE-1. It is involved in the development and progression of several human carcinomas, such as hepatocellular carcinoma and lung and breast cancers. The hepatocyte growth factor (HGF)/ETS-1 signaling pathway is involved in regulation of cancer cell proliferation, metastasis and invasion. The biological function of the interaction between LINE-1 ORF-1p and the HGF/ETS-1 signaling pathway in regulation of human breast cancer proliferation remains largely unknown. Here, we showed that LINE-1 ORF-1p enhanced ETS-1 transcriptional activity and increased expression of downstream genes of ETS-1. Interaction between ETS-1 and LINE-1 ORF-1p was identified by immunoprecipitation assays. LINE-1 ORF-1p modulated ETS-1 activity through cytoplasm/nucleus translocation and recruitment to the ETS-1 binding element in the MMP1 gene promoter. We also showed that LINE-1 ORF-1p promoted proliferation and anchorage-independent growth of MDA-MB-231 breast cancer cells. By investigating a novel role of the LINE-1 ORF-1p in the HGF/ETS-1 signaling pathway and MDA-MB-231 cells, we demonstrated that LINE-1 ORF-1p may be a novel ETS-1 coactivator and molecular target for therapy of human triple negative breast cancer.

Sorafenib may prolong survival in patients with advanced hepatocellular carcinoma (HCC), but with limited efficacy. The present study aimed to assess the safety and efficacy of sorafenib combined with cryotherapy (cryoRx) for the treatment of advanced HCC. A total of 104 patients met the following criteria: advanced HCC without distant metastasis, presence of portal vein thrombosis, Child-Pugh class A or B and life expectancy of at least 12 weeks. All patients were randomly assigned to sorafenib and cryoRx (n=52) or sorafenib-alone (n=52) treatment groups. The primary end-point of the study was overall survival (OS). The secondary end-points included time to progression (TTP) and tolerability. Microvessel density (MVD) was assessed following immunostaining for CD34. In a median of 10.5 (4-26) months follow-up, the median OS was 12.5 months (95% CI 10.6-16.4) in the combination therapy vs. 8.6 months (7.3-10.4) in the sorafenib-alone (P=0.01) group. The median TTP was 9.5 months (8.4-13.5) in the combination therapy vs. 5.3 months (3.8-6.9) in the sorafenib alone (P=0.02) group. CryoRx was an independent factor associated with improved clinical outcomes of sorafenib for the treatment of advanced HCC. Patients with low intratumoral MVD receiving the combination therapy exhibited a significantly longer median TTP and OS compared to those receiving sorafenib. High intratumoral MVD was an independent predictor of poor responses to sorafenib for advanced HCC. Compared with previous reports of sorafenib-related adverse drug reactions (ADRs), cryoRx did not further increase the frequency and degree of sorafenib-related ADRs. In conclusion, compared to sorafenib alone, the addition of cryoRx to sorafenib significantly improves the clinical outcomes of sorafenib for the treatment of advanced HCC with acceptable tolerance and similar safety profiles as previously reported. High intratumoral MVD is predictive of poor responses to sorafenib in advanced HCC patients.