Study protocol of short-course radiotherapy combined with CAPOX and PD-1 inhibitor for locally advanced colon cancer: a randomised, prospective, multicentre, phase II trial (TORCH-C).

INTRODUCTION: The preliminary result of the TORCH trial has shown a promising complete response (CR) for managing locally advanced rectal cancer with neoadjuvant short-course radiotherapy (SCRT) combined with chemotherapy and PD-1 inhibitor. For locally advanced colon cancer (LACC) with bulky nodal disease and/or clinically T4, neoadjuvant chemotherapy followed by colectomy with en bloc removal of regional lymph nodes is the suggested treatment. However, the CR rate is less than 5%. TORCH-C will aim to investigate neoadjuvant SCRT combined with chemotherapy and PD-1 inhibitor in LACC. METHODS AND ANALYSIS: TORCH-C is a randomised, prospective, multicentre, double-arm, open, phase II trial of SCRT combined with chemotherapy and immunotherapy in LACC with microsatellite stable (MSS) patients and cT4 or bulky nodes. Eligible patients will be identified by the multidisciplinary team. 120 patients will be randomised 1:1 to the intervention or control arm. The patients in the control arm will receive four cycles of capecitabine plus oxaliplatin (CAPOX). The patients in the intervention arm will receive SCRT, followed by four cycles of CAPOX and PD-1 inhibitor (serplulimab). Both arms will receive curative surgery, followed by four cycles of CAPOX. The primary endpoint is pathological complete regression.TORCH-C (TORCH-colon) trial aims to investigate whether the combination of immunotherapy and chemoradiotherapy improves the treatment effect in LACC with MSS. TORCH-C will establish the TORCH platform, a key part of our long-term strategy to develop neoadjuvant treatment for colorectal cancer. ETHICS AND DISSEMINATION: This study was approved by the Ethics Committee of Fudan University Shanghai Cancer Center (approval number: 2211265-12). TRIAL REGISTRATION NUMBER: NCT05732493.

TORCH-R trial protocol: hypofractionated radiotherapy combined with chemotherapy and toripalimab for locally recurrent rectal cancer: a prospective, single-arm, two-cohort, phase II trial.

For patients with locally recurrent rectal cancer (LRRC), the response rate to chemoradiotherapy is 40%-50%. Additionally, only approximately 40%-50% of patients with recurrent rectal cancer are able to undergo R0 resection. Recent studies in locally advanced rectal cancer (LARC) have shown promising synergistic effects when combining immunotherapy (PD-1/PD-L1 antibodies) with neoadjuvant chemoradiotherapy (nCRT). Therefore, incorporating immunotherapy into the treatment regimen for LRRC patients has the potential to further improve response rates and prognosis. To investigate this, the TORCH-R trial was conducted. This prospective, single-arm, two-cohort, phase II trial focuses on the use of hypofractionated radiotherapy, chemotherapy, and immunotherapy in LRRC patients without or with oligometastases. The trial will include two cohorts: cohort A consists of rectal cancer patients who are treatment-naive for local recurrence, and cohort B includes patients with progressive disease after first-line chemotherapy. Cohort A and cohort B patients will receive 25-40 Gy/5 Fx irradiation or 15-30 Gy/5 Fx reirradiation for pelvic recurrence, respectively. Subsequently, they will undergo 18 weeks of chemotherapy, toripalimab, and stereotactic ablative radiotherapy (SABR) for all metastatic lesions between chemoimmunotherapy cycles. Decisions regarding follow-up of complete response (CR), radical surgery, sustained treatment of non-resection, or exiting the trial are made by a multidisciplinary team (MDT). The primary endpoint of this study is the local objective response rate (ORR). The secondary endpoints include the extrapelvic response rate, duration of response, local recurrence R0 resection rate, progression-free survival (PFS), overall survival (OS), and safety and tolerability. Notably, this trial represents the first clinical exploration of inducing hypofractionated radiotherapy, chemotherapy, and immunotherapy in LRRC patients. Clinical trial registration: https://clinicaltrials.gov/study/NCT05628038, identifier NCT05628038.

Short-course radiotherapy combined with chemotherapy and PD-1 inhibitor in low-lying early rectal cancer: study protocol for a single-arm, multicentre, prospective, phase II trial (TORCH-E).

INTRODUCTION: Current standard treatment for patients with early rectal cancer is radical surgical resection. Although radical surgery provides effective local tumour control, it also increases the mortality risk and considerable adverse effects, including bowel, bladder, sexual dysfunction and loss of anal function, especially in patients with low-lying rectal cancer. Recent studies have shown promising synergistic effects of the combination of programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) inhibitors and radiotherapy in improving tumour regression. For patients who reach a clinical complete response (cCR) after neoadjuvant therapy, a 'Watch & Wait' (W&W) approach can be adopted to preserve anorectal function and improve quality of life. Thus, this study aims to explore the efficacy and safety of radiotherapy combined with chemotherapy and PD-1 antibody in patients with low early rectal cancer. METHODS AND ANALYSIS: TORCH-E study is designed as a multicentre, prospective, phase II trial of short-course radiotherapy (SCRT) combined with chemotherapy and PD-1 inhibitor in patients with cT1-3bN0M0 low rectal cancer. The trial was initiated in December 2022 and is currently recruiting patients, with an anticipated completion of participant enrolment by June of the following year. The enrolled 34 patients will receive SCRT (25 Gy/5 Fx), followed by four cycles of capecitabine plus oxaliplatin chemotherapy and PD-1 antibody (toripalimab) and finally receive surgery or the W&W strategy. The primary endpoint is the complete response (CR) rate, that is, the rate of pathological complete response (pCR) plus cCR. The secondary endpoints include organ preservation rate, 3-year local recurrence-free survival rate, 3-year disease-free survival rate, 3-year overall survival rate, grade 3-4 adverse effects rate and patients' quality of life. ETHICS AND DISSEMINATION: This trial has been approved by the Ethics Committee of Fudan University Shanghai Cancer Center. Trial results will be disseminated via peer-reviewed journals and conference presentations. TRIAL REGISTRATION NUMBER: NCT05555888 (ClinicalTrials.gov).

Regorafenib alone or in combination with high/low-dose radiotherapy plus toripalimab as third-line treatment in patients with metastatic colorectal cancer: protocol for a prospective, randomized, controlled phase II clinical trial (SLOT).

Combination strategies to improve immunotherapy response in microsatellite stable metastatic colorectal cancer (MSS mCRC) remain an unmet need. Several single-arm clinical trials have shown promising synergistic effects between regorafenib and ICIs; however, some contradictory results have also been reported. Randomized controlled trials are needed to further validate the combination of regorafenib with ICIs. In addition, low-dose radiotherapy has been demonstrated to induce local immune responses by reprogramming the tumor microenvironment when combined with high-dose radiotherapy and ICIs. In this study, we designed a prospective, randomized, controlled phase II trial to investigate the efficacy and safety of regorafenib in combination with high/low-dose radiotherapy plus toripalimab in MSS mCRC compared to regorafenib alone. Patients with MSS metastatic adenocarcinoma of the colon or rectum will be enrolled and randomly assigned into two arms: a control arm and an experimental arm. Patients in the control arm will receive regorafenib monotherapy (120 mg once daily on days 1-21 of each 28 days cycle). Patients in the experimental arm will first receive one cycle of regorafenib (80 mg once daily on days 1-21 of each 28 days cycle) and toripalimab (240mg, q3w), followed by high-dose (4-8 fractions of 8-12Gy) and low-dose (1-10Gy at 0.5-2Gy/fraction) radiotherapy, and then continue regorafenib and toripalimab treatment. The primary endpoint is the objective response rate, and the secondary endpoints are disease control rate, duration of remission, median progress-free survival, median overall survival, and adverse events. Recruitment started in August 2023 and is ongoing. Clinical Trial Registration: https://clinicaltrials.gov/study/NCT05963490?cond=NCT05963490&rank=1, identifier NCT05963490.

RIFLE: a Phase II trial of stereotactic ablative radiotherapy combined with fruquintinib and tislelizumab in metastatic colorectal cancer.

Background: Currently, the prognosis for metastatic colorectal cancer (mCRC) still remains poor. The management of mCRC has become manifold because of the varied advances in the systemic and topical treatment approaches. For patients with limited number of metastases, radical local therapy plus systemic therapy can be a good choice to achieve long-term tumor control. In this study, we aimed to explore the efficacy and safety of the combination of fruquintinib, tislelizumab, and stereotactic ablative radiotherapy (SABR) in mCRC (RIFLE study). Methods: RIFLE was designed as a single-center, single-arm, prospective Phase II clinical trial. A total of 68 mCRC patients who have failed the first-line standard treatment will be recruited in the safety run-in phase (n = 6) and the expansion phase (n = 62), respectively. Eligible patients will receive SABR followed by fruquintinib (5 mg, d1-14, once every day) and tislelizumab (200 mg, d1, once every 3 weeks) within 2 weeks from completion of radiation. The expansion phase starts when the safety of the treatment is determined (dose limiting toxicity occur in no more than one-sixth of patients in the run-in phase). The primary end point is the objective response rate. The secondary end points include the disease control rate, duration of response, 3-year progression-free survival rate, 3-year overall survival rate, and toxicity. Conclusions: The results of this trial will provide a novel insight into SABR in combination with PD-1 antibody and vascular endothelial growth factor receptor inhibitor in the systematic treatment of metastatic colorectal cancer, which is expected to provide new therapeutic strategies and improve the prognosis for mCRC patients. Trial registration: NCT04948034 (ClinicalTrials.gov).

Long-term outcomes in a retrospective cohort of patients with rectal cancer with complete response after total neoadjuvant therapy: a propensity-score weighted analysis.

Background: The watch-and-wait (W&W) strategy is a novel treatment option for patients with rectal cancer who have a strong desire for organ preservation. The study aimed to explore the long-term outcomes of the W&W strategy in a large cohort of rectal cancer patients who achieved a clinical complete response (cCR) after consolidation total neoadjuvant therapy (TNT), and to compare with patients who achieved a pathological complete response (pCR) after radical surgery. Methods: The W&W group comprised patients who were assessed as having a cCR after consolidation TNT and adopted the W&W strategy. Patients who underwent standard resection and achieved a pCR were compared as a reference. Inverse probability of treatment weighting (IPTW)-adjusted Kaplan-Meier analysis with log-rank test was used to compare survival outcomes. Results: We included 89 and 171 patients in the W&W and pCR groups, respectively. The median follow-up period was 45 and 58 months for the W&W and pCR groups, respectively. After IPTW adjustment, the 2-year local regrowth/recurrence rate for the W&W and pCR groups were 9.9% and 2.0%, respectively (p < 0.001). The W&W and pCR groups had similar 5-year outcomes, including overall survival, disease-free survival, and distant metastasis-free survival (all p > 0.05). No significant difference was observed in the rates of distant metastasis between patients in the W&W group with local regrowth and those without local regrowth (25% versus 6.2%, p = 0.119). Conclusion: Patients who were managed with a W&W strategy after consolidation TNT had favorable survival outcomes, which were similar to those of patients with a pCR. The rate of local regrowth in W&W patients was lower in our study than in other studies as a result of the implementation of consolidation TNT.

Neoadjuvant chemoradiotherapy combined with immunotherapy for locally advanced rectal cancer: A new era for anal preservation.

For locally advanced (T3-4/N+M0) rectal cancer (LARC), neoadjuvant chemoradiotherapy (nCRT) followed by total mesorectal excision (TME) is the standard treatment. It was demonstrated to decrease the local recurrence rate and increase the tumor response grade. However, the distant metastasis remains an unresolved issue. And the demand for anus preservation and better quality of life increases in recent years. Radiotherapy and immunotherapy can be supplement to each other and the combination of the two treatments has a good theoretical basis. Recently, multiple clinical trials are ongoing in terms of the combination of nCRT and immunotherapy in LARC. It was reported that these trials achieved promising short-term efficacy in both MSI-H and MSS rectal cancers, which could further improve the rate of clinical complete response (cCR) and pathological complete response (pCR), so that increase the possibility of 'Watch and Wait (W&W)' approach. However, the cCR and pCR is not always consistent, which occurs more frequent when nCRT is combined with immunotherapy. Thus, the efficacy evaluation after neoadjuvant therapy is an important issue for patient selection of W&W approach. Evaluating the cCR accurately needs the combination of multiple traditional examinations, new detective methods, such as PET-CT, ctDNA-MRD and various omics studies. And finding accurate biomarkers can help guide the risk stratification and treatment decisions. And large-scale clinical trials need to be performed in the future to demonstrate the surprising efficacy and to explore the long-term prognosis.

Short-course radiotherapy combined with CAPOX and Toripalimab for the total neoadjuvant therapy of locally advanced rectal cancer: a randomized, prospective, multicentre, double-arm, phase II trial (TORCH).

BACKGROUND: For patients with locally advanced (T3-4/N +) rectal cancer (LARC), the standard treatment is neoadjuvant chemoradiotherapy combined with total mesorectal resection, which greatly decreases local recurrence but does not improve overall survival. For patients who achieve a complete clinical response (cCR) after nCRT, a "Watch & Wait" (W&W) approach can be received to improve quality of life. Currently, total neoadjuvant therapy (TNT) has been demonstrated to increase the complete response rate and achieve early control of distant metastasis. Recent studies have shown promising synergistic effects of the combination of immunotherapy (PD-1/PD-L1 antibodies) and radiotherapy. Thus, for LARC patients, the combination of immunotherapy and TNT is likely to further improve the rate of complete response and prognosis. The disparities between induction therapy and consolidation therapy need to be investigated. METHODS: TORCH is a randomized, prospective, multicentre, double-arm, phase II trial of short-course radiotherapy (SCRT) combined with chemotherapy and immunotherapy in LARC. 130 LARC patients will be treated with the TNT approach and assigned to the consolidation arm and induction arm. The consolidation arm will receive SCRT, followed by 6 cycles of capecitabine plus oxaliplatin (CAPOX) and Toripalimab. The induction arm will first receive 2 cycles of CAPOX and Toripalimab, then receive SCRT, followed by 4 cycles of CAPOX and Toripalimab. Both groups will receive curative surgery or the W&W strategy. The primary endpoint is the complete response rate (rate of pCR plus cCR). The secondary endpoints include the grade 3-4 acute adverse effects rate, 3-year disease-free survival (DFS) rate, 3-year local recurrence-free survival (LRFS) rate, 3-year OS rate, rate of surgical complications and quality of life (QoL) scores. The "pick the winner" method is used to investigate the better treatment regimen. The trial was opened on 13th April 2021, and the first patient was recruited on 6th May 2021. DISCUSSION: TORCH will investigate whether SCRT combined with chemotherapy and Toripalimab can achieve better complete response rates, good tolerance and prognosis in LARC patients. This is the first clinical trial to compare the efficacy of induced immunotherapy and consolidative immunotherapy based on the TNT strategy. TRIAL REGISTRATION: Trial Registration Number and Date of Registration: ClinicalTrials.gov NCT04518280 , August 15, 2020.

Immunotherapy and tumor mutational burden in cancer patients with liver metastases: A meta and real word cohort analysis.

Background: The predictive effects of liver metastases for immune-checkpoint inhibitors (ICIs) and the relationship between tumor mutational burden (TMB) and liver metastases (LM) remain unclear. Methods: A systematic review and meta-analysis were conducted to explore the heterogeneity of ICIs efficacy between patients with or without LM. A pan-cancer cohort of 1,661 patients who received ICIs was downloaded and analyzed to assess the association between TMB and LM. Results: Of 21053 studies identified in our search, eight single-arm studies and 24 randomized controlled trials were included. Overall, 17957 patients with advanced or metastatic cancers (4805 patients (26.8%) with LM and 13151 patients (73.2%) without LM) were enrolled. The pooled objective response rate (ORR) was 8.5% (95% CI 4%-13%) in the LM group versus 21% (95% CI 16%-21%) in the non-LM group. The pooled hazard ratio (HR) for death was 0.85 (95% CI 0.80-0.90) in the LM group treated with ICIs compared with the standard of care. In patients without LM who were treated with ICIs, the pooled HR for death was 0.78 (95% CI 0.73-0.82) compared with the standard of care. The difference in efficacy between patients with or without LM treated with ICIs was significant (p=0.04). Pan-cancer analysis revealed that the TMB-high rate was 10.8% in liver metastatic lesions versus 21.4% in other metastatic lesions (p=0.004). In addition, TMB was also significantly associated with OS as a binary cutoff (p=0.05) and was an independent prognostic variable (HR=0.98, P=0.047) as a continuous variable in patients with LM. Conclusions: In patients with LM, the efficacy of immunotherapy was attenuated, but TMB-high could predict better survival outcomes.

M5C regulator-mediated methylation modification patterns and tumor microenvironment infiltration characterization in lung adenocarcinoma.

BACKGROUND: In recent years, immunotherapy has made great progress, and the regulatory role of epigenetics has been verified. However, the role of 5-methylcytosine (m5C) in the tumor microenvironment (TME) and immunotherapy response remains unclear. METHODS: Based on 11 m5C regulators, we evaluated the m5C modification patterns of 572 lung adenocarcinoma (LUAD) patients. The m5C score was constructed by principal component analysis (PCA) algorithms in order to quantify the m5C modification pattern of individual LUAD patients. RESULTS: Two m5C methylation modification patterns were identified according to 11 m5C regulators. The two patterns had a remarkably distinct TME immune cell infiltration characterization. Next, 226 differentially expressed genes (DEGs) related to the m5C phenotype were screened. Patients were divided into three different gene cluster subtypes based on these genes, which had different TME immune cell infiltration and prognosis characteristics. The m5C score was constructed to quantify the m5C modification pattern of individual LUAD patients. We found that the high m5C score group had a better prognosis. The role of the m5C score in predicting prognosis was also verified in the dataset GSE31210. CONCLUSIONS: Our study revealed that m5C modification played a significant role in TME regulation of LUAD. Investigation of the m5C regulation mode may have some implications for tumor immunotherapy in the future.

CUL3 (cullin 3)-mediated ubiquitination and degradation of BECN1 (beclin 1) inhibit autophagy and promote tumor progression.

Macroautophagy/autophagy plays an important role during the development of human cancer. BECN1 (beclin 1), a core player in autophagy regulation, is downregulated in many kinds of malignancy. The underlying mechanism, however, has not been fully illuminated. Here, we found that CUL3 (cullin 3), an E3 ubiquitin ligase, could interact with BECN1 and promote the K48-linked ubiquitination and degradation of this protein; In addition, CUL3 led to a decrease in autophagic activity through downregulating BECN1. We also found that KLHL38 was a substrate adaptor of the CUL3 E3 ligase complex-mediated ubiquitination and degradation of BECN1. In breast and ovarian cancer, CUL3 could promote the proliferation of tumor cells, and the expression of CUL3 was related to poor prognosis in patients. Our study reveals the underlying mechanism of BECN1 ubiquitination and degradation that affects autophagic activity and subsequently leads to tumor progression, providing a novel therapeutic strategy that regulates autophagy to combat cancer.Abbreviations: ATG: autophagy-related BECN1: beclin 1 CHX: cycloheximide CoIP: co-immunoprecipitation CUL3: cullin 3 IP: immunoprecipitation MS: mass spectrometry PtdIns3K: phosphatidylinositol 3-kinase UPS: ubiquitin-proteasome system.

Choice of Treatment for Patients With Non-small-cell Lung Cancer >5 cm Between Surgery Alone and Surgery Plus Adjuvant Radiotherapy.

Background: According to the lung cancer staging project, T2b (>5-7 cm) and T3 (>7 cm) non-small cell lung cancers (NSCLC) should be reclassified into T3 and T4 groups. The objective of this study was to evaluate the effect of surgery alone or surgery plus adjuvant radiation (SART) on survival of node-negative patients with NSCLC >5 cm. Methods: We identified 4557 N0 patients with NSCLC >5 cm in the Surveillance, Epidemiology, and End Results database from 2004 to 2014. Overall survival (OS) and cancer-specific survival (CSS) were compared among patients who underwent surgery alone and SART. The proportional hazards model was applied to evaluate multiple prognostic factors. Results: 1,042 and 525 patients who underwent surgery alone and SART, respectively were enrolled after propensity-score matching. OS and CSS favored surgery alone rather than SART. Multivariate analysis showed that the number of lymph nodes examined more than six was associated with better OS and CSS for NSCLC >5 cm, especially in patients treated with surgery alone. Lobectomy should be recommended as the primary option for NSCLC >5 to 7 cm, whereas its superiority was not significant over sublobectomy for NSCLC >7 cm. Conclusion: Surgery alone should be recommended as the first choice for patients with NSCLC >5 cm. The number of examined lymph nodes should be more than six in patients with NSCLC >5 cm, especially for those who undergo surgery alone. For patients with NSCLC >7 cm who could not tolerate lobectomy, sublobectomy might be an alternative surgical procedure.

SOX2 promotes resistance of melanoma with PD-L1 high expression to T-cell-mediated cytotoxicity that can be reversed by SAHA.

BACKGROUND: Immune checkpoint inhibitors (ICIs) induce better tumor regression in melanoma with programmed cell death 1 ligand 1 (PD-L1) high expression, but there has been an upsurge of failed responses. In this study, we aimed to explore the additional mechanisms possibly accounting for ICIs resistance and interventional strategies to overcome the resistance in melanoma with PD-L1 high expression. METHODS: Melanoma xenografts and cytotoxicity assays were used to investigate function of SOX2 in regulating antitumor immunity. The activity of the janus kinase-signal transducer and activator of transcriptions (JAK-STAT) pathway was investigated by western blots, quantitative PCR and luciferase assay. Epigenetic compounds library screen was employed to identify inhibitors that could decrease SOX2 level. The effect of histone deacetylase inhibitor SAHA in antitumor immunity alone or in combination with immunotherapy was also determined in vitro and in vivo. Prognostic impact of SOX2 was analyzed using transcriptional profiles and clinical data download from the Gene Expression Omnibus and The Cancer Genome Atlas repository. RESULTS: We uncovered a role of SOX2 in attenuating the sensitivity of melanoma cells to CD8+ T-cell killing. Mechanistically, SOX2 inhibited phosphatases suppressor of cytokine signaling 3 (SOCS3) and protein tyrosine phosphatase non-receptor type 1 (PTPN1) transcription, induced duration activation of the JAK-STAT pathway and thereby overexpression of interferon stimulated genes resistance signature (ISG.RS). By targeting the SOX2-JAK-STAT signaling, SAHA promoted the antitumor efficacy of IFNγ or anti-PD-1 in vitro and in vivo. Moreover, SOX2 was an independent prognostic factor for poor survival and resistant to anti-PD-1 therapy in melanoma with PD-L1 high expression. CONCLUSIONS: Our data unveiled an additional function of SOX2 causing immune evasion of CD8+ T-cell killing through alleviating the JAK-STAT pathway and ISG.RS expression. We also provided a rationale to explore a novel combination of ICIs with SAHA clinically, especially in melanoma with PD-L1 and SOX2 high expression.

Clinical significance and function of RDH16 as a tumor-suppressing gene in hepatocellular carcinoma.

AIM: Our previous transcriptome sequencing analysis detected that retinol dehydrogenase 16 (RDH16) was dramatically downregulated in hepatocellular carcinoma (HCC). RDH16 belongs to the short-chain dehydrogenases/reductases super family, and its role in HCC remains unknown. This study aimed to investigate the expression and function of RDH16 in HCC. METHODS: The mRNA and protein level of RDH16 in HCC samples were detected by quantitative real-time polymerase chain reaction and immunohistochemistry analyses, respectively. The role of RDH16 in HCC was determined by in vitro and in vivo functional studies. RESULTS: Downregulation of RDH16 has been detected in approximately 90% of primary HCCs, which was significantly associated with high serum alpha-fetoprotein level, tumor size, microsatellite formation, thrombus, and poor overall survival of HCC patients. Compared with non-tumor tissues, higher density of methylation was identified in HCC samples. In addition, RDH16 increases the level of retinoic acid and blocks the de novo synthesis of fatty acid in HCC cells. Functional study shows that ectopic expression of RDH16 in HCC cells suppresses cell growth, clonogenicity, and cell motility. CONCLUSIONS: RDH16 might be a prognostic biomarker and intervention point for new therapeutic strategies in HCC.

Polo-Like Kinase 1 phosphorylates and stabilizes KLF4 to promote tumorigenesis in nasopharyngeal carcinoma.

Rationale: Advanced nasopharyngeal carcinoma (NPC) is an aggressive disease with no targeted therapies and poor outcomes. New innovative targets are urgently needed. KLF4 has been extensively studied in the context of tumors, and current data suggest that it can act as either a tissue-specific tumor-inhibiting or a tumor-promoting gene. Here, we found that KLF4 played as a tumor-promoting gene in NPC, and could be mediated by PLK1. Methods: Tissue immunohistochemistry (IHC) assay was performed to identify the role of KLF4 in NPC. Global gene expression experiments were performed to explore the molecular mechanisms underlying KLF4-dependent tumorigenesis. Small-molecule kinase inhibitor screening was performed to identify potential upstream kinases of KLF4. The pharmacologic activity of polo-like kinase inhibitor volasertib (BI6727) in vitro and in vivo was determined. Result: Our investigation showed that high expression of KLF4 was correlated with poor prognosis in NPC. Moreover, genome-wide profiling revealed that KLF4 directly activated oncogenic programmes, including gene sets associated with KRAS, VEGF, and MYC signalling. We further found that inhibition of polo-like kinase 1 could downregulate the expression of KLF4 and that PLK1 directly phosphorylated KLF4 at Ser234. Notably, phosphorylation of KLF4 by PLK1 caused the recruitment and binding of the E3 ligase TRAF6, which resulted in KLF4 K32 K63-linked ubiquitination and stabilization. Moreover, KLF4 could enhance TRAF6 expression at the transcriptional level, thus initiating a KLF4-TRAF6 feed-forward loop. Treatment with the PLK1 inhibitor volasertib (BI6727) significantly inhibited tumor growth in nude mice. Conclusion: Our study unveiled a new PLK1-TRAF6-KLF4 feed-forward loop. The resulting increase in KLF4 ubiquitination leads to stabilization and upregulation of KLF4, which leads to tumorigenesis in NPC. These results expand our understanding of the role of KLF4 in NPC and validate PLK1 inhibitors as potential therapeutic agents for NPC, especially cancer patients with KLF4 overexpression.

Regorafenib Promotes Antitumor Immunity via Inhibiting PD-L1 and IDO1 Expression in Melanoma.

PURPOSE: Immune checkpoint blockade (ICB) therapy induces durable tumor regressions in a minority of patients with cancer. In this study, we aimed to identify kinase inhibitors that were capable of increasing the antimelanoma immunity. EXPERIMENTAL DESIGN: Flow cytometry-based screening was performed to identify kinase inhibitors that can block the IFNγ-induced PD-L1 expression in melanoma cells. The pharmacologic activities of regorafenib alone or in combination with immunotherapy in vitro and in vivo were determined. The mechanisms of regorafenib were explored and analyzed in melanoma patients treated with or without anti-PD-1 using The Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets. RESULTS: Through screening of a kinase inhibitor library, we found approximately 20 agents that caused more than half reduction of cell surface PD-L1 level, and regorafenib was one of the most potent agents. Furthermore, our results showed that regorafenib, in vitro and in vivo, strongly promoted the antitumor efficacy when combined with IFNγ or ICB. By targeting the RET-Src axis, regorafenib potently inhibited JAK1/2-STAT1 and MAPK signaling and subsequently attenuated the IFNγ-induced PD-L1 and IDO1 expression without affecting MHC-I expression much. Moreover, RET and Src co-high expression was an independent unfavorable prognosis factor in melanoma patients with or without ICB through inhibiting the antitumor immune response. CONCLUSIONS: Our data unveiled a new mechanism of alleviating IFNγ-induced PD-L1 and IDO1 expression and provided a rationale to explore a novel combination of ICB with regorafenib clinically, especially in melanoma with RET/Src axis activation.

The role of m6A RNA methylation in cancer.

N6-methyladenosine (m6A), the most abundant internal modification of RNA in eukaryotic cells, has gained increasing attention in recent years. The m6A modification affects multiple aspects of RNA metabolism, ranging from RNA processing, nuclear export, RNA translation to decay. Emerging evidence suggests that m6A methylation plays a critical role in cancer through various mechanisms. Moreover, m6A methylation has provided more possibilities for the early diagnosis and treatment of cancers. In this review, we focus on m6A-associated mechanisms and functions in several major malignancies and summarize the dual role of m6A methylation as well as its prospects in cancer.

Regulation of glycolytic metabolism by autophagy in liver cancer involves selective autophagic degradation of HK2 (hexokinase 2).

Impaired macroautophagy/autophagy and high levels of glycolysis are prevalent in liver cancer. However, it remains unknown whether there is a regulatory relationship between autophagy and glycolytic metabolism. In this study, by utilizing cancer cells with basal or impaired autophagic flux, we demonstrated that glycolytic activity is negatively correlated with autophagy level. The autophagic degradation of HK2 (hexokinase 2), a crucial glycolytic enzyme catalyzing the conversion of glucose to glucose-6-phosphate, was found to be involved in the regulation of glycolysis by autophagy. The Lys63-linked ubiquitination of HK2 catalyzed by the E3 ligase TRAF6 was critical for the subsequent recognition of HK2 by the autophagy receptor protein SQSTM1/p62 for the process of selective autophagic degradation. In a tissue microarray of human liver cancer, the combination of high HK2 expression and high SQSTM1 expression was shown to have biological and prognostic significance. Furthermore, 3-BrPA, a pyruvate analog targeting HK2, significantly decreased the growth of autophagy-impaired tumors in vitro and in vivo (p < 0.05). By demonstrating the regulation of glycolysis by autophagy through the TRAF6- and SQSTM1-mediated ubiquitination system, our study may open an avenue for developing a glycolysis-targeting therapeutic intervention for treatment of autophagy-impaired liver cancer.

Overexpression of PTK6 predicts poor prognosis in bladder cancer patients.

Protein tyrosine kinase 6 (PTK6) is a non-receptor tyrosine kinase and works as an oncogene in various cancers. Recently, PTK6 has been used as a therapeutic target for breast cancer patients in a clinical study. However, the prognostic value of PTK6 in bladder cancer (BC) remains vague. Therefore, we retrieved 3 independent investigations of Oncomine database and found that PTK6 is highly expressed in BC tissues compared with corresponding normal controls. Similar results were also observed in clinical specimens at both mRNA and protein levels. Immunohistochemical analysis indicated that PTK6 overexpression was highly related to the T classification, N classification, grade, recurrence, and poor prognosis of BC patients. Furthermore, we demonstrated that when PTK6 expression was knocked down by siRNAs, cell proliferation and migration were considerably inhibited in BC cell lines T24 and EJ. By these approaches, we are intended to elucidate PTK6 may be a reliable therapeutic target in BC and might benefit from PTK6 inhibitors in the future.

Association between Mismatch-repair Genetic variation and the Risk of Multiple Primary Cancers: A Meta-Analysis.

Microsatellites instability (MSI) is a risk factor for multiple primary cancers (MPCs). However, a variety of studies focused on the risk in the hereditary non-polyposis colorectal cancer (HNPCC) not the sporadic colorectal cancer (CRC) patients. The aim of this meta-analysis was to comprehensive overview and quantitative summary the association between MSI and risk of MPCs. A comprehensive literature search in MEDLINE, EMBASE, Web of science, ScienceDirect, Weily and OVID was conducted. Up to May 2016, we identified 22 observational studies. We calculated the summary relative risk (RR) for the risk of MPCs in MSI patients compared with microsatellites stability (MSS) patients using fixed- or random-effects models. The RR of the association between mismatch-repair gene (MMR) genotype and MPCs was 2.59 (95% confidence interval [CI], 2.06 to 3.27); the RR was 2.14 (95% CI, 1.78 to 2.57) for sporadic CRC and 5.59 (95% CI, 2.69 to 11.59) for HNPCC for the MSI versus MSS category. The subgroup analyses showed different mutant gene, mutant locus, and mutant level of MMR with different influence on the patients susceptible to MPCs. In addition, MSI genotype increase the risk of MPC was not associated with an apparently specific in regard to site, timing, age and detection method. In conclusion, this meta-analysis indicates that MSI is associated with an increased risk of MPCs both in the HNPCC and sporadic CRC patients. Our findings will form the backbone of the treatment for MSI genotype may be an important valuable strategy for MPCs prevention.