Growing evidence has suggested that increased matrix stiffness can significantly strengthen the malignant characteristics of hepatocellular carcinoma (HCC) cells. However, whether and how increased matrix stiffness regulates the formation of invadopodia in HCC cells remain largely unknown. In the study, we developed different experimental systems in vitro and in vivo to explore the effects of matrix stiffness on the formation of invadopodia and its relevant molecular mechanism. Our results demonstrated that increased matrix stiffness remarkably augmented the migration and invasion abilities of HCC cells, upregulated the expressions of invadopodia-associated genes and enhanced the number of invadopodia. Two regulatory pathways contribute to matrix stiffness-driven invadopodia formation together in HCC cells, including direct triggering invadopodia formation through activating integrin β1 or Piezo1/ FAK/Src/Arg/cortactin pathway, and indirect stimulating invadopodia formation through improving EGF production to activate EGFR/Src/Arg/cortactin pathway. Src was identified as the common hub molecule of two synergistic regulatory pathways. Simultaneously, activation of integrin β1/RhoA/ROCK1/MLC2 and Piezo1/Ca2+/MLCK/MLC2 pathways mediate matrix stiffness-reinforced cell migration. This study uncovers a new mechanism by which mechanosensory pathway and biochemical signal pathway synergistically regulate the formation of invadopodia in HCC cells.

3543 Background: We previously have showed that fruquintinib combined with tislelizumab and HAIC had an ORR benefit (25.71%) consisting of 1 CR and 8 PR in advanced colorectal cancer liver metastases (CRLM) patients (pts) who had failed to standard therapy in this single-arm, open-label, single center phase II study (NCT05435313). Here, we aim to update the survival results. Methods: We enrolled CRLM pts who had failed to standard therapy. Eligible pts received fruquintinib (3mg, qd, po, D2-21, Q3W), combined with tislelizumab (200mg, ivgtt, D2,Q3W) and HAIC (TOMOX: raltitrexed 2mg/m2, D1, oxaliplatin 85mg/m2, D1, Q3W; or TOMIRI: raltitrexed 2mg/m2, D1, irinotecan 120mg/m2, D1, Q3W) for 4-6 cycles, followed by maintenance therapy with fruquintinib and tislelizumab until disease progression, death or unacceptable toxicity. The primary endpoint was ORR. The secondary endpoints included DCR, PFS, 6mo-PFS rate, OS and safety. In addition, subgroup analysis of ORR and PFS according to primary tumor site, RAS/BRAF status and history of bevacizumab (bev) were performed. Results: Between July 17, 2022 and June 15, 2023, 39 pts (median age, 59 years [range: 35--73], 25 [64.1%] male, 7 [17.95%] right-sided tumor, 38 [97.44%] MSS, 20 [51.28%] RAS/BRAF mutation) were enrolled. Among them, 23 (58.97%) pts had previously received bev. As of December 31, 2023, there were 4 pts drop out and 35 pts with MSS were included in the efficacy analysis. At a median follow-up of 12.52 mo, the median PFS was 7.43 mo (95%CI, 5.36-9.82) and 6mo-PFS rate was 60%, the median OS was 15.51mo (95%CI, 10.84-NA) and 1 year-OS rate was 61%. In the subgroup analysis, the median PFS was significantly longer with right-sided tumor pts than left-sided tumor pts (13.31 vs 6.90 mo, HR, 0.121 [95%CI, 0.046-0.320], p= 0.0062). Compared with pts who received bev in prior therapy, the median PFS of pts without bev was significantly improved (5.16 vs 8.31 mo, HR, 0.358 [95%CI, 0.133-0.965], p=0.0361). Favorable PFS was observed in RAS/BRAF mutation pts compared with RAS/BRAF wide-type pts (8.18 vs 7.43 mo). The results of PFS subgroups were consistent with ORR subgroup results (right-sided vs left-sided: 42.86% vs 21.43%, with bev vs without bev: 10% vs 46.67%) which was previously reported. Compared with safety profile reported previously, no additional safety signals were observed. Conclusions: The updated data on the survival analysis revealed that fruquintinib combined with tislelizumab and HAIC had a survival benefit in CRLM pts with MSS who had failed to standard therapy, especially for right-sided tumor pts. Based on these results, another study is ongoing to explore the efficacy for MSS CRLM pts with two cohorts (cohort 1: right-sided tumor, cohort 2: left-sided tumor), and the results will be presented in the future. Clinical trial information: NCT05435313 .

As a long-established chemotherapy drug, 5-fluorouracil (5-FU) is widely used to clinically manage colorectal cancer (CRC). However, a substantial portion of patients develop 5-FU resistance at some stage, which poses a great challenge. Therefore, revealing the mechanisms that could guide the development of effective strategies to overcome 5-FU resistance is required. Here, we report that the expression of PFKP was higher in HCT116/5-FU CRC. Furthermore, genetic suppression of PFKP suppresses glycolysis, NF-kappa B activation, and expression of GLUT1 and HK2 in HCT116/5-FU cells. PFKP overexpression promotes glycolysis and expression of GLUT1 and HK2 via the NF-kappa B signaling pathway in HCT116 cells. Our functional assays demonstrated that PFKP silencing could sensitize HCT116/5-FU cells to 5-FU with an elevated population of apoptotic cells. In contrast, forced expression of PFKP conferred 5-FU resistance in HCT116 cells. Furthermore, PFKP silencing significantly inhibited CRC xenograft tumor growth. Notably, the combination of PFKP silencing and 5-FU inhibited tumor growth. Therefore, our results demonstrated that PFKP enhances 5-FU resistance by promoting glycolysis, indicating that PFKP could be a novel candidate for targeted therapy for 5-FU-resistant CRC.

Microvascular invasion (MVI) is closely associated with high risk of recurrence and poor long-term survival in HCC patients. For these patients, active and effective adjuvant therapy is of great significance to improve the prognosis. Therefore, we explored the efficacy and safety of interferon-α (IFN-α) combined with PD-1 monoclonal antibody (tislelizumab, TIS) as adjuvant therapy for HCC patients with MVI. In this phase 2 study, histologically confirmed HCC patients who had undergone curative resection with MVI, Child-Pugh A, and an ECOG PS ≤1 were enrolled. Patients separately received TIS (200 mg IV Q3W) plus IFN-α (30 μg BIW) or active surveillance until disease progression, intolerable toxicity or withdrawal of consent. The primary endpoint was recurrence free survival (RFS). Secondary endpoints included overall survival (OS) and safety. From January 2021 to December 2021, TIS plus IFN-α group and active surveillance group enrolled 24 and 14 patients, respectively. Baseline characteristics were generally comparable between two groups, including MVI grade (M1: 90.0% vs 85.7%; M2: 10.0% vs 14.3%) and any tumours diameter > 5cm (65.0% vs 50.0%) in TIS plus IFN-α, respectively. At data cutoff (17 Apr 2023), RFS was not reached in TIS plus IFN-α group (95%CI: 9.8-NE), whereas in active surveillance group was 17.09 month (95%CI: 4.8-NE). 1-year RFS rate in TIS plus IFN-α and active surveillance group was 79.2% (95%CI: 0.5-0.9) and 50.0% (95%CI: 0.2-0.7), separately. Median OS was not reached in either group. No severe postoperative complications were observed. In TIS plus IFN-α group, Grade 1 or higher AEs were mainly rash (20.0%), pyrexia (15.0%), pruritus (10.0%) and diarrhea (10.0%). These results suggested that HCC patients with MVI may benefit from adjuvant tislelizumab plus IFN-α, which can be tested in a future confirmatory trial. We will continue follow-up and update the data further. In the meantime, related basic experiments are underway.

Background Gastric cancer (GC) is a cancer of the gastrointestinal tract that is highly malignant and has poor prognosis. Circular RNAs are a class of nonclassical RNA molecules that have been determined to be involved in GC malignancy in various ways. However, the underlying function and mechanism of circTDRD3 in gastric cancer remain largely unknown. Methods We analyzed circTDRD3 expression in databases and verified the findings in GC cell lines and tissue specimens. A series of functional gene overexpression and knockdown assays in vivo and in vitro were carried out to investigate the role of circTDRD3 in proliferation and metastasis. Here, we revealed the role of the miR-891b/ITGA2 axis by analyzing bioinformatics datasets. Furthermore, we performed dual-luciferase, fluorescence in situ hybridization, RNA pull-down, and functional rescue experiments to examine the relationships between circTDRD3 and its interacting molecules. Western blot confirmed the positive regulatory role of circTDRD3 in the AKT signaling pathway. A promoting effect of ATF4 on circTDRD3 was determined through chromatin immunoprecipitation. Results CircTDRD3 was significantly overexpressed in GC tissues compared with adjacent benign tissue, and its expression level was positively correlated with tumor volume and lymph node metastasis. CircTDRD3 promoted GC cell proliferation and migration in vitro and in vivo. Mechanistically, circTDRD3 exerted a tumor-promoting effect by regulating the miR-891b/ITGA2 axis and AKT signaling pathway in a positive feedback manner mediated by the transcription factor ATF4. Conclusions ATF4-mediated circTDRD3 overexpression modulates the proliferation and metastasis of GC cells through the miR-891b/ITGA2 axis in a positive feedback manner.

Abstract Intracellular transportation (IT) is dysregulated in tumors. Here, we identified N -ethylmaleimide-sensitive factor (NSF) as one of the IT-associated genes with upregulated expression in hepatocellular carcinoma (HCC). Furthermore, NSF expression is associated with malignant features and poor outcomes of HCC patients. NSF acts as an oncogenic driver that promotes HCC cell viability and metastasis by physically interacting with cell division cycle 5-like protein (CDC5L) and facilitating its nuclear accumulation in a vesicle-independent manner. In the nucleus, CDC5L transcriptionally activates CDK2-associated cullin 1 (CACUL1) expression via occupation of its promoter element. Thus, our findings provide novel insights into the mechanisms underlying the NSF-induced promotion of HCC progression and suggest potential therapeutic targets in the NSF-CDC5L-CACUL1 axis for HCC therapeutics.

AbstractBackgroundDespite integrin being highlighted as a stiffness‐sensor molecule in matrix stiffness‐driven angiogenesis, other stiffness‐sensor molecules and their mechanosensory pathways related to angiogenesis in hepatocellular carcinoma (HCC) remain obscure. Here, we explored the interplay between Piezo1 and integrin β1 in the mechanosensory pathway and their effects on HCC angiogenesis to better understand matrix stiffness‐induced angiogenesis.MethodsThe role of Piezo1 in matrix stiffness‐induced angiogenesis was investigated using orthotopic liver cancer SD rat models with high liver stiffness background, and its clinical significance was evaluated in human HCC tissues. Matrix stiffness‐mediated Piezo1 upregulation and activation were assayed using an in vitro fibronectin (FN)‐coated cell culture system with different stiffness, Western blotting and Ca2+ probe. The effects of shPiezo1‐conditioned medium (CM) on angiogenesis were examined by tube formation assay, wound healing assay and angiogenesis array. The underlying mechanism by which Piezo1 participated in matrix stiffness‐induced angiogenesis was analyzed by microRNA quantitative real‐time polymerase chain reaction (qRT‐PCR), matrix stiffness measurement, dual‐luciferase reporter assay, ubiquitination assay and co‐immunoprecipitation.ResultsIncreased matrix stiffness significantly upregulated Piezo1 expression at both cellular and tissue levels, and high expression of Piezo1 indicated an unfavorable prognosis. High matrix stiffness also noticeably enhanced the activation level of Piezo1, similar to its expression level. Piezo1 knockdown significantly suppressed tumor growth, angiogenesis, and lung metastasis of HCC rat models with high liver stiffness background. shPiezo1‐CM from HCC cells attenuated tube formation and migration abilities of vascular endothelial cells remarkably, and analysis of differentially expressed pro‐angiogenic factors revealed that Piezo1 promoted the expression and secretion of vascular endothelial growth factor (VEGF), CXC chemokine ligand 16 (CXCL16) and insulin‐like growth factor binding protein 2 (IGFBP2). Matrix stiffness‐caused Piezo1 upregulation/activation restrained hypoxia inducible factor‐1α (HIF‐1α) ubiquitination, subsequently enhanced the expression of downstream pro‐angiogenic factors to accelerate HCC angiogenesis. Besides, collagen 1 (COL1)‐reinforced tissue stiffening resulted in more expression of Piezo1 via miR‐625‐5p.ConclusionsThis study unravels a new mechanism by which the integrin β1/Piezo1 activation/Ca2+ influx/HIF‐1α ubiquitination/VEGF, CXCL16 and IGFBP2 pathway participates in matrix stiffness‐driven HCC angiogenesis. Simultaneously, a positive feedback regulation loop as stiff matrix/integrin β1/miR‐625‐5p/Piezo1 and COL1/stiffer matrix mediates matrix stiffness‐caused Piezo1 upregulation.

OBJECTIVE:The mechanisms underlying the contribution of primary tumour to pre-metastatic niche formation remains largely unknown in hepatocellular carcinoma (HCC). We previously reported that the released LOXL2 from HCC cells under higher stiffness stimulation facilitated the formation of lung pre-metastatic niche. Here, we further clarified the pathological role of LOXL2 in promoting lung pre-metastatic niche formation and lung metastasis occurrence in HCC and its relevant molecular mechanism.METHODS:Using two different animal models and an in vitro system of mechanically tuneable gel mirroring lung tissue stiffness, we explored the underlying mechanism of LOXL2 in pre-metastatic niche formation.RESULTS:We applied tail vein injection of CM-LV-LOXL2-OEsimulating tumour-released soluble factors to induce lung pre-metastatic niche formation and found that the injected LOXL2 remarkably enhanced CD11b+/CD45+ bone marrow-derived cells (BMDCs) recruitment and fibronectin expression in lung. Subsequently, LOXL2-overexpressed xenograft HCC models validated that tumour-secreted LOXL2 significantly promoted the occurrence of pulmonary metastasis. In vitro, LOXL2 and LOXL2-caused matrix stiffening not only obviously upregulated the expressions of MMP9 and fibronectin in lung fibroblasts, but also evidently increased the number of adherent HCC cells and the expression of chemokine CXCL12. The activation of PI3K-AKT pathway mediated LOXL2-upregulated fibronectin. HCC patients in High-LOXL2 group had higher ratio of tumour recurrence than HCC patients in Low-LOXL2 group, supporting a significance of LOXL2 in HCC progression and unfavourable outcome.CONCLUSION:Primary tumour-released LOXL2 promotes lung pre-metastatic niche formation and lung metastasis occurrence. LOXL2-caused matrix stiffening synergistically regulates lung pre-metastatic niche formation. Targeting LOXL2-induced lung pre-metastatic niche may be a novel intervention approach against HCC metastasis.

Surgery is becoming more practical and effective than conservative treatment in improving the poor outcomes of patients with breast cancer liver metastasis (BCLM). However, there is no generally acknowledged set of standards for identifying BCLM candidates who will benefit from surgery. Between January 2011 and September 2018, 67 female BCLM patients who underwent partial hepatectomy were selected for analysis in the present study. Prognostic factors after hepatectomy were determined. Univariate and multivariate analyses were performed to identify predictors of overall survival (OS) and intrahepatic recurrence-free survival (IHRFS). The 1-, 3- and 5-year OS of patients treated with surgery was 93.5%, 73.7% and 32.2%, respectively, with a median survival time of 57.59 months. The Pringle manoeuvre [hazard radio (HR) = 0.117, 95% CI0.015–0.942, p = 0.044] and an increased interval between breast surgery and BCLM diagnosis (HR0.178, 95% CI 0.037–0.869, p = 0.033) independently predicted improved overall survival for BCLM patients. The 1-, 2- and 3-year IHRFS of patients who underwent surgery was 62.8, 32.6% and 10.9%, respectively, with a median intrahepatic recurrence-free survival time of 13.47 months. Moderately differentiated tumours (HR  0.259, 95% CI 0.078–0.857, p = 0.027) and the development of liver metastasis more than 2 years after breast surgery (HR  0.270, 95% CI 0.108–0.675, p = 0.005) might be predictors of increased IHRFS. An interval of more than 2 years between breast cancer surgery and liver metastasis seems to be an indication of liver surgery in BCLM patients. The Pringle manoeuvre and moderately differentiated tumours are potential predictors associated with OS and IHRFS, respectively, as benefits from liver resection. Studies with increased sample sizes are warranted to validate our results.

BACKGROUND:Increased liver stiffness exerts a detrimental role in driving hepatocellular carcinoma (HCC) malignancy and progression, and indicates a high risk of unfavorable outcomes. However, it remains largely unknown how liver matrix stiffness as an independent cue triggers epithelial-mesenchymal transition (EMT) and facilitates HCC metastasis.METHODS:Buffalo rat HCC models with different liver stiffness backgrounds and an in vitro Col I-coated cell culture system with tunable stiffness were used in the study to explore the effects of matrix stiffness on EMT occurrence and its underlying molecular mechanism. Clinical significance of liver stiffness and key molecules required for stiffness-induced EMT were validated in HCC cohorts with different liver stiffness.RESULTS:HCC xenografts grown in higher stiffness liver exhibited worse malignant phenotypes and higher lung metastasis rate, suggesting that higher liver stiffness promotes HCC invasion and metastasis. Cell tests in vitro showed that higher matrix stiffness was able to strikingly strengthen malignant phenotypes and independently induce EMT occurrence in HCC cells, and three signaling pathways converging on Snail expression participated in stiffness-mediated effect on EMT including integrin-mediated S100A11 membrane translocation, eIF4E phosphorylation, and TGF β1 autocrine. Additionally, the key molecules required for stiffness-induced EMT were highly expressed in tumor tissues of HCC patients with higher liver stiffness and correlated with poor tumor differentiation and higher recurrence.CONCLUSIONS:Higher matrix stiffness as an initiator triggers epithelial-mesenchymal transition (EMT) in HCC cells independently, and three signaling pathways converging on Snail expression contribute to this pathological process. This work highlights a significant role of biomechanical signal in triggering EMT and facilitating HCC invasion and metastasis.

BACKGROUND:Higher matrix stiffness affects biological behavior of tumor cells, regulates tumor-associated gene/miRNA expression and stemness characteristic, and contributes to tumor invasion and metastasis. However, the linkage between higher matrix stiffness and pre-metastatic niche in hepatocellular carcinoma (HCC) is still largely unknown.METHODS:We comparatively analyzed the expressions of LOX family members in HCC cells grown on different stiffness substrates, and speculated that the secreted LOXL2 may mediate the linkage between higher matrix stiffness and pre-metastatic niche. Subsequently, we investigated the underlying molecular mechanism by which matrix stiffness induced LOXL2 expression in HCC cells, and explored the effects of LOXL2 on pre-metastatic niche formation, such as BMCs recruitment, fibronectin production, MMPs and CXCL12 expression, cell adhesion, etc. RESULTS: Higher matrix stiffness significantly upregulated LOXL2 expression in HCC cells, and activated JNK/c-JUN signaling pathway. Knockdown of integrin β1 and α5 suppressed LOXL2 expression and reversed the activation of above signaling pathway. Additionally, JNK inhibitor attenuated the expressions of p-JNK, p-c-JUN, c-JUN and LOXL2, and shRNA-c-JUN also decreased LOXL2 expression. CM-LV-LOXL2-OE and rhLOXL2 upregulated MMP9 expression and fibronectin production obviously in lung fibroblasts. Moreover, activation of Akt pathway contributed to LOXL2-induced fibronectin upregulation. LOXL2 in CM as chemoattractant increased motility and invasion of BMCs, implicating a significant role of LOXL2 in BMCs recruitment. Except that, CM-LV-LOXL2-OE as chemoattractant also increased the number of migrated HCC cells, and improved chemokine CXCL12 expression in lung fibroblasts. The number of HCC cells adhered to surface of lung fibroblasts treated with CM-LV-LOXL2-OE was remarkably higher than that of the control cells. These results indicated that the secreted LOXL2 facilitated the motility of HCC cells and strengthened CTCs settlement on the remodeled matrix "soil".CONCLUSION:Integrin β1/α5/JNK/c-JUN signaling pathway participates in higher matrix stiffness-induced LOXL2 upregulation in HCC cells. The secreted LOXL2 promotes fibronectin production, MMP9 and CXCL12 expression and BMDCs recruitment to assist pre-metastatic niche formation.