Downregulated BMP-Smad1/5/8 signaling causes emphysema via dysfunction of alveolar type II epithelial cells.

Bone morphogenetic protein (BMP)-Smad1/5/8 signaling plays a crucial regulatory role in lung development and adult lung homeostasis. However, it remains elusive whether BMP-Smad1/5/8 signaling is involved in the pathogenesis of emphysema. In this study, we downregulated BMP-Smad1/5/8 signaling by overexpressing its antagonist Noggin in adult mouse alveolar type II epithelial cells (AT2s), resulting in an emphysematous phenotype mimicking the typical pathological features of human emphysema, including distal airspace enlargement, pulmonary inflammation, extracellular matrix remodeling, and impaired lung function. Dysregulation of BMP-Smad1/5/8 signaling in AT2s leads to inflammatory destruction dominated by macrophage infiltration, associated with reduced secretion of surfactant proteins and inhibition of AT2 proliferation and differentiation. Reactivation of BMP-Smad1/5/8 signaling by genetics or chemotherapy significantly attenuated the morphology and pathophysiology of emphysema and improved the lung function in Noggin-overexpressing lungs. We also found that BMP-Smad1/5/8 signaling was downregulated in cigarette smoke-induced emphysema, and that enhancing its activity in AT2s prevented or even reversed emphysema in the mouse model. Our data suggest that BMP-Smad1/5/8 signaling, located at the top of the signaling cascade that regulates lung homeostasis, represents a key molecular regulator of alveolar stem cell secretory and regenerative function, and could serve as a potential target for future prevention and treatment of pulmonary emphysema. © 2023 The Pathological Society of Great Britain and Ireland.

Long-term hypoxic hUCMSCs-derived extracellular vesicles alleviates allergic rhinitis through triggering immunotolerance of their VEGF-mediated inhibition of dendritic cells maturation.

BACKGROUND: Extensions of mesenchymal stem cells (MSCs) in vitro may lead to the loss of their biological functions. However, hypoxic culturation has been shown to enhance the proliferation, survival, and immunomodulatory capacity of MSCs. OBJECTIVE: We aimed to investigate the effects of long-term hypoxic cultivation on the properties of human umbilical cord-derived MSCs (hUCMSCs) and the therapeutic effects of their extracellular vesicles (EVs) in allergic rhinitis (AR). METHODS: Proliferation, senescence, telomerase activity and multipotent properties of hUCMSCs were analyzed under long-term culturation of hypoxia (1%) or normoxia (21%), and the therapeutic effects of their conditional medium (CM) and EVs were evaluated in OVA-induced AR mice. Effects of hypoxia-EVs (Hy-EVs) or normoxia-EVs (No-EVs) on human monocyte-derived dendritic cells (DCs) were investigated, and the possible mechanisms of Hy-EVs in induction of immunotolerance were further explored. RESULTS: Long-term hypoxia significantly promoted the proliferation, inhibited cell senescence, maintained the multipotent status of hUCMSCs. Hy-CM and Hy-EVs showed better therapeutic effects in AR mice compared to No-EVs, seen as improvement of AR-related behaviors such as rubbing and sneezing, and attenuation of inflammation in nasal tissues. In addition, Hy-EVs significantly reduced the expressions of HLA-DR, CD80, CD40, and CD83 induced by OVA plus LPS in DCs, inhibiting the maturation of DCs. Furthermore, we observed that VEGF was remarkably enriched in Hy-EVs, but not in No-EVs, and the inhibition of DCs maturation was markedly neutralized by VEGF antibodies, suggesting that VEGF derived from Hy-EVs was responsible for the inhibition of DCs maturation. CONCLUSION: Our results demonstrated that long-term hypoxia significantly promoted the proliferation, inhibited cell senescence, maintained the multipotent status of hUCMSCs, and hypoxia treated hUCMSCs-derived EVs enhanced their therapeutic effects in AR mice through VEGF-mediated inhibition of DCs maturation.

Case Report: A novel mixture of dose-fractioned radiation and immunotherapy for treatment of cholangiocarcinoma.

Malignant tumors of the biliary tract exhibit a high degree of malignancy and heterogeneity with a poor overall prognosis. Immunotherapy has limited benefits for patients with cholangiocarcinoma. Radiation therapy can change the tumor microenvironment, but its effect heavily depends on radiation dose and fraction. We report a case of advanced intrahepatic cholangiocarcinoma in a 43-year-old male patient, with a huge liver mass of 16.5 cm in diameter, with bone and liver metastases at the first diagnosis. First-line treatment with chemotherapy and PD1 inhibitor was sustained only for 8 months. In second-line treatment, radiotherapy was administered, with 5 Gy in 5 fractions administered to the entire tumor area and 25 Gy in 5 fractions to the solid lesions of the tumor. After the completion of radiotherapy, programmed cell death 1 inhibitor combined with tyrosine kinase inhibitor was maintained. The patient achieved a progression-free-survival time of 12 months and an overall survival time of 25 months. The success of our case suggests that mixed low- and high-dose radiation can significantly improve tumor control and survival time. In clinical practice, based on the characteristics of the tumor and existing treatment options, the rational combination of existing treatment regimens can improve the prognosis of cholangiocarcinoma.

Circulating tumour DNA biomarkers in savolitinib-treated patients with non-small cell lung cancer harbouring MET exon 14 skipping alterations: a post hoc analysis of a pivotal phase 2 study.

Background: Savolitinib, a selective MET inhibitor, showed efficacy in patients with non-small cell lung cancer (NSCLC), including pulmonary sarcomatoid carcinoma (PSC), harbouring MET exon 14 skipping alteration (METex14). Objective: To analyse post hoc, the association between circulating tumour DNA (ctDNA) biomarkers and clinical outcomes, including resistance, with savolitinib. Design: A multicentre, single-arm, open-label phase 2 study. Methods: All enrolled patients with baseline plasma samples were included. Outcomes were objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) by baseline METex14 and post-treatment clearance, coexisting gene alterations at baseline and disease progression. Results: Among 66 patients with baseline ctDNA sequencing, 46 (70%) had detectable METex14. Frequent coexisting baseline gene alterations included TP53 and POT1 mutations. Patients with detectable baseline METex14 exhibited worse PFS [hazard ratio (HR), 1.77; 95% confidence interval (CI), 0.88-3.57; p = 0.108] and OS (HR, 3.26; 95% CI, 1.35-7.89; p = 0.006) than those without, despite showing a numerically higher ORR. Among 24 patients with baseline detectable METex14 and evaluable postbaseline samples, 13 achieved METex14 clearance post-treatment. Median time to first clearance was 1.3 months (range, 0.7-1.5). METex14 post-treatment clearance was associated with better ORR (92.3%; 95% CI, 64.0-99.8 versus 36.4%; 95% CI, 10.9-69.2; p = 0.0078), PFS (HR, 0.44; 95% CI, 0.2-1.3; p = 0.1225) and OS (HR, 0.31; 95% CI, 0.1-1.0; p = 0.0397) versus non-clearance. Among 22 patients with disease progression, 10 acquired pathway alterations (e.g. in RAS/RAF and PI3K/PTEN) alone or with secondary MET mutations (D1228H/N and Y1230C/H/S). Conclusion: ctDNA biomarkers may allow for longitudinal monitoring of clinical outcomes with savolitinib in patients with METex14-positive PSC and other NSCLC subtypes. Specifically, undetectable baseline METex14 or post-treatment clearance may predict favourable clinical outcomes, while secondary MET mutations and other acquired gene alterations may explain resistance to savolitinib. Registration: The trial was registered with ClinicalTrials.gov (NCT02897479) on 13 September 2016.

CHD4 acts as a critical regulator in the survival of spermatogonial stem cells in mice†.

Spermatogenesis is sustained by homeostatic balance between the self-renewal and differentiation of spermatogonial stem cells, which is dependent on the strict regulation of transcription factor and chromatin modulator gene expression. Chromodomain helicase DNA-binding protein 4 is highly expressed in spermatogonial stem cells but roles in mouse spermatogenesis are not fully understood. Here, we report that the germ-cell-specific deletion of chromodomain helicase DNA-binding protein 4 resulted in complete infertility in male mice, with rapid loss of spermatogonial stem cells and excessive germ cell apoptosis. Chromodomain helicase DNA-binding protein 4-knockdown in cultured spermatogonial stem cells also promoted the expression of apoptosis-related genes and thereby activated the tumor necrosis factor signaling pathway. Mechanistically, chromodomain helicase DNA-binding protein 4 occupies the genomic regulatory region of key apoptosis-related genes, including Jun and Nfkb1. Together, our findings reveal the determinant role of chromodomain helicase DNA-binding protein 4 in spermatogonial stem cells survival in vivo, which will offer insight into the pathogenesis of male sterility and potential novel therapeutic targets.

RING finger 138 deregulation distorts NF-кB signaling and facilities colitis switch to aggressive malignancy.

Prolonged activation of nuclear factor (NF)-кB signaling significantly contributes to the development of colorectal cancer (CRC). New therapeutic opportunities are emerging from targeting this distorted cell signaling transduction. Here, we discovered the critical role of RING finger 138 (RNF138) in CRC tumorigenesis through regulating the NF-кB signaling, which is independent of its Ubiquitin-E3 ligase activity involved in DNA damage response. RNF138-/- mice were hyper-susceptible to the switch from colitis to aggressive malignancy, which coincided with sustained aberrant NF-кB signaling in the colonic cells. Furthermore, RNF138 suppresses the activation of NF-кB signaling pathway through preventing the translocation of NIK and IKK-Beta Binding Protein (NIBP) to the cytoplasm, which requires the ubiquitin interaction motif (UIM) domain. More importantly, we uncovered a significant correlation between poor prognosis and the downregulation of RNF138 associated with reinforced NF-кB signaling in clinical settings, raising the possibility of RNF138 dysregulation as an indicator for the therapeutic intervention targeting NF-кB signaling. Using the xenograft models built upon either RNF138-dificient CRC cells or the cells derived from the RNF138-dysregulated CRC patients, we demonstrated that the inhibition of NF-кB signaling effectively hampered tumor growth. Overall, our work defined the pathogenic role of aberrant NF-кB signaling due to RNF138 downregulation in the cascade events from the colitis switch to colonic neoplastic transformation and progression, and also highlights the possibility of targeting the NF-кB signaling in treating specific subtypes of CRC indicated by RNF138-ablation.

Curzerene suppresses progression of human glioblastoma through inhibition of glutathione S-transferase A4.

AIMS: Glioblastoma is the central nervous system tumor with the highest mortality rate, and the clinical effectiveness of chemotherapy is low. Curzerene can inhibit the progression of non-small-cell lung cancer, but its role in glioma has not been reported. The purpose of this study was to clarify the effect of curzerene on glioma progression and further explore its potential mechanism. METHODS: The expression of glutathione S-transferase A4 (GSTA4) in glioblastoma and the effect of curzerene on the expression of GSTA4 and matrix metalloproteinase 9 and the activation of the mTOR pathway were detected by Western blotting and RT-PCR, and the effects of curzerene treatment on glioma malignant character were detected by cell biological assays. The in vivo antitumor effects of curzerene were analyzed in a nude mouse xenograft model. RESULTS: Curzerene was found to inhibit the expression of GSTA4 mRNA and protein in U251 and U87 glioma cells, and this effect correlated with a downregulation of the proliferation of these cells in a time- and dose-dependent manner. Invasion and migration were also inhibited, and curzerene treatment correlated with induction of apoptosis. Curzerene inhibited the activation of the mTOR pathway and the expression of matrix metalloproteinase 9, and it correlated with increased 4-hydroxynonenal levels. In vivo, curzerene was found to significantly inhibit tumor growth in nude mice and to prolong the survival time of tumor-bearing nude mice. CONCLUSION: In conclusion, inhibition of GSTA4 correlates with positive outcomes in glioma models, and thus, this molecule is a candidate drug for the treatment of glioma.

A radiomics model predicts the response of patients with advanced gastric cancer to PD-1 inhibitor treatment.

Programmed cell death 1 (PD1) inhibitors have shown promising treatment effects in advanced gastric cancer, the beneficiary population not definite. This study aimed to construct an individualized radiomics model to predict the treatment benefits of PD-1 inhibitors in gastric cancer. Patients with advanced gastric cancer treated with PD-1 inhibitors were randomly divided into a training set (n = 58) and a validation set (n = 29). CT imaging data were extracted from medical records, and an individual radiomics nomogram was generated based on the imaging features and clinicopathological risk factors. Discrimination performance was evaluated by Harrell's c-index and receiver operator characteristic (ROC) curve analyses. The areas under the ROC curves (AUCs) were analyzed to predict anti-PD-1 efficacy and survival. We found that the radiomics nomogram could predict the response of gastric cancer to anti-PD-1 treatment. The AUC was 0.865 with a 95% CI of 0.812-0.828 in the training set, while the AUC was 0.778 with a 95% CI of 0.732-0.776 in the validation set. The diagnostic performance of the radiomics was significantly higher than that of the clinical factors (p < 0.01). Patients with a low risk of disease progression discriminated by the radiomics nomogram had longer progression-free survival than those with a high risk (6.5 vs. 3.2 months, HR 1.99, 95% CI: 1.19-3.31, p = 0.009). The radiomics nomogram based on CT imaging features and clinical risk factors could predict the treatment benefits of PD-1 inhibitors in advanced gastric cancer, enabling it to guide decision-making regarding clinical treatment.

Psychological states could affect postsurgical pain after hemorrhoidectomy: A prospective cohort study.

Background: Open hemorrhoidectomy is one of the standard procedures for grade IV hemorrhoids. Postsurgical pain is a common problem for patients. We aim to prospectively evaluate potential factors affecting postoperative pain among hemorrhoidectomy patients. Methods: An observational study was conducted on 360 patients who had undergone Milligan-Morgan open hemorrhoidectomy. Details of the surgery and baseline information were recorded. Preoperative anxiety and depression were analyzed via the self-rating anxiety scale 20 (SAS-20) and self-rating depression scales 20 (SDS-20), respectively. Postoperative pain score was performed daily after surgery until the patient was discharged. The numerical pain score was evaluated by the visual analogue scale (VAS). The association between preoperative psychological states (anxiety or depression) and postoperative pain was analyzed using a generalized additive mixed model. Results: A total of 340 patients eventually provided complete data and were included in our study. The average age was 43.3 ± 14.4 years, and 62.1% of patients were women. In total, 14.9% of patients had presurgical anxiety and 47.1% had presurgical depression. Postsurgical pain reached a peak point 1-2 days after surgery and went down to a very low level around 4-5 days after surgery. More excision of hemorrhoids could lead to more pain experience after surgery. Presurgical depression was associated with postsurgical pain. Patients who had presurgical depression had higher pain scores after surgery (2.3 ± 1.9 vs. 3.3 ± 1.9, p = 0.025). Conclusion: Preoperative depression and the amount of excisional hemorrhoids are positively related to postsurgical pain.

Exosomes Derived from Bone Mesenchymal Stem Cells Alleviate Compression-Induced Nucleus Pulposus Cell Apoptosis by Inhibiting Oxidative Stress.

Oxidative stress is relevant in compression-induced nucleus pulposus (NP) cell apoptosis and intervertebral disc (IVD) degeneration. Exosomes derived from bone mesenchymal stem cells (BMSCs-Exos) are key secretory products of MSCs, with important roles in tissue regeneration. This research is aimed at studying the protective impact of BMSCs-Exos on NP cell apoptosis caused by compression and investigating the underlying mechanisms. Our results indicated that we isolated BMSCs successfully. Exosomes were isolated from the BMSCs and found to alleviate the inhibitory effect that compression has on proliferation and viability in NP cells, decreasing the toxic effects of compression-induced NP cells. AnnexinV/PI double staining and TUNEL assays indicated that the BMSCs-Exos reduced compression-induced apoptosis. In addition, our research found that BMSCs-Exos suppressed compression-mediated NP oxidative stress by detecting the ROS and malondialdehyde level. Furthermore, BMSCs-Exos increased the mitochondrial membrane potential and alleviated compression-induced mitochondrial damage. These results indicate that BMSCs-Exos alleviate compression-mediated NP apoptosis by suppressing oxidative stress, which may provide a promising cell-free therapy for treating IVD degeneration.

Phase II, single-arm trial of preoperative short-course radiotherapy followed by chemotherapy and camrelizumab in locally advanced rectal cancer.

BACKGROUND: In locally advanced rectal cancer (LARC), preoperative short-course radiotherapy (SCRT) with delayed surgery has been shown to be as effective as long-course chemoradiotherapy, with only modest benefits. This study aimed to evaluate the efficacy and safety of preoperative SCRT combined with subsequent CAPOX (capecitabine and oxaliplatin) and the anti-PD-1 antibody camrelizumab in patients with LARC. METHODS: This was a prospective, single-arm, phase II trial. Treatment-naïve patients with histologically confirmed T3-4N0M0 or T1-4N+M0 rectal adenocarcinoma received 5×5 Gy SCRT with two subsequent 21-day cycles of CAPOX plus camrelizumab after 1 week, followed by radical surgery after 1 week. The primary endpoint was pathological complete response (pCR) rate. Biomarker analysis was performed to identify a potential predictor of pCR to treatment. RESULTS: From November 7, 2019 to September 14, 2020, 30 patients were enrolled, and 27 patients received at least one dose of CAPOX plus camrelizumab. Surgery was performed in 27 (100%) patients. The pCR (ypT0N0) rate was 48.1% (13/27), including 46.2% (12/26) for proficient mismatch repair (MMR) tumors and 100% (1/1) for deficient MMR tumors. Immune-related adverse events were all grade 1-2, with the most common being reactive cutaneous capillary endothelial proliferation (81.5%). No grade 4/5 adverse events occurred. Biomarker analysis showed patients without FGFR1-3 deletions had a better tendency for pCR. CONCLUSIONS: SCRT combined with subsequent CAPOX plus camrelizumab followed by delayed surgery showed a favorable pCR rate with good tolerance in patients with LARC, especially in the proficient MMR setting. A randomized controlled trial is ongoing to confirm these results. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov identifier: NCT04231552.

Once-daily savolitinib in Chinese patients with pulmonary sarcomatoid carcinomas and other non-small-cell lung cancers harbouring MET exon 14 skipping alterations: a multicentre, single-arm, open-label, phase 2 study.

BACKGROUND: Savolitinib is a selective MET tyrosine-kinase inhibitor. We investigated the activity and safety of savolitinib in patients with pulmonary sarcomatoid carcinoma and other non-small-cell lung cancer (NSCLC) subtypes positive for MET exon 14 skipping alterations (METex14-positive). METHODS: We did a multicentre, single-arm, open-label, phase 2 study across 32 hospitals in China. Eligible patients were 18 years or older with locally advanced or metastatic METex14-positive pulmonary sarcomatoid carcinoma or other NSCLC subtypes, had either presented with disease progression or toxicity intolerance towards one or more standard treatments or were deemed clinically unsuitable for standard treatment, were MET inhibitor-naive, and had measurable disease. Patients received either 600 mg (bodyweight ≥50 kg) or 400 mg (bodyweight <50 kg) of oral savolitinib once daily until disease progression, death, intolerable toxicity, initiation of other anti-tumour therapy, non-compliance, patient withdrawal, or patient discontinuation. Radiographic tumour evaluation was done at baseline, every 6 weeks within 1 year of the first dose, and every 12 weeks thereafter. The primary endpoint was objective response rate, defined as the proportion of patients with confirmed complete or partial responses by independent review committee (IRC) assessment. The primary endpoint was assessed in the tumour response evaluable set, which comprised all treated patients with a measurable lesion at baseline and at least one adequate scheduled post-baseline tumour assessment or the presence of radiological disease progression, with a sensitivity analysis done in the full analysis set, which comprised all patients who received at least one dose of savolitinib. Safety was also evaluated in the full analysis set. This study is registered with ClinicalTrials.gov, NCT02897479, and recruitment is complete, with treatment and follow-up ongoing. FINDINGS: From Nov 8, 2016, to Aug 3, 2020, 84 patients with METex14 skipping alterations were screened for eligibility, of whom 70 were enrolled, received savolitinib, and comprised the full analysis set. The IRC-assessed tumour response evaluable set comprised 61 patients. At a median follow-up of 17·6 months (IQR 14·2-24·4), the IRC-assessed objective response rate was 49·2% (36·1-62·3; 30 of 61 patients) in the tumour response evaluable set and 42·9% (95% CI 31·1-55·3; 30 of 70 patients) in the full analysis set. All 70 patients reported at least one treatment-related adverse event. Treatment-related adverse events of grade 3 or more occurred in 32 (46%) patients, the most frequent of which were increased aspartate aminotransferase (n=9), increased alanine aminotransferase (n=7), and peripheral oedema (n=6). Treatment-related serious adverse events occurred in 17 (24%) patients, the most common being abnormal hepatic function (n=3) and hypersensitivity (n=2). One death due to tumour lysis syndrome in a patient with pulmonary sarcomatoid carcinoma was assessed to be probably related to savolitinib by the investigator. INTERPRETATION: Savolitinib yielded promising activity and had an acceptable safety profile in patients with pulmonary sarcomatoid carcinoma and other NSCLC subtypes positive for METex14 skipping alterations. Savolitinib could therefore be a novel treatment option in this population. FUNDING: Hutchison MediPharma and AstraZeneca. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Fruquintinib with gefitinib as first-line therapy in patients carrying EGFR mutations with advanced non-small cell lung cancer: a single-arm, phase II study.

BACKGROUND: Fruquintinib is an oral vascular endothelial growth factor receptor inhibitor. Previous gefitinib studies with anti-angiogenics show promising efficacy. This phase II trial assessed efficacy and safety of fruquintinib in combination with gefitinib, in patients with advanced non-small cell lung cancer (NSCLC). METHODS: Fifty patients with stage IIIB/IV NSCLC and an epidermal growth factor receptor (EGFR) exon-19 deletion or exon-21 L858R mutation were enrolled between January 2017 and June 2019. Per protocol (version 1.0), patients received 4 mg fruquintinib once daily (qd) Days 1-21 of Cycle 1, using a 3-week-on/1-week-off schedule, plus continuous gefitinib 250 mg qd. If tolerated, patients proceeded to fruquintinib 5 mg qd (fruquintinib 5 mg group, n=26). Following protocol updates, dose escalation of fruquintinib from 4 mg qd to 5 mg qd was not allowed. The primary efficacy endpoint was objective response rate (ORR); secondary endpoints included progression-free survival (PFS), disease control rate (DCR), time to response, duration of response and adverse events (AEs). RESULTS: ORR was 73.5% (95% CI, 58.9-85.1) and DCR was 98.0% (95% CI, 89.2-100.0). Median PFS was 14.7 months for both groups; PFS was highest for patients with exon-19 deletion (16.5 months; 95% CI, 12.9-21.2). Grade ≥3 treatment-emergent AEs occurred in 17 (65.3%; fruquintinib 5 mg,) and 11 patients (45.8%; 4 mg). Serious AEs were recorded for nine patients (fruquintinib 5 mg, six patients; 4 mg, three). CONCLUSIONS: Fruquintinib and gefitinib treatment showed an acceptable safety profile and promising efficacy in patients with NSCLC.

PDCD6 cooperates with C-Raf to facilitate colorectal cancer progression via Raf/MEK/ERK activation.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies, and it's expected that the CRC burden will substantially increase in the next two decades. New biomarkers for targeted treatment and associated molecular mechanism of tumorigenesis remain to be explored. In this study, we investigated whether PDCD6 plays an oncogenic role in colorectal cancer and its underlying mechanism. METHODS: Programmed cell death protein 6 (PDCD6) expression in CRC samples were analyzed by immunohistochemistry and immunofluorescence. The prognosis between PDCD6 and clinical features were analyzed. The roles of PDCD6 in cellular proliferation and tumor growth were measured by using CCK8, colony formation, and tumor xenograft in nude mice. RNA-sequence (RNA-seq), Mass Spectrum (MS), Co-Immunoprecipitation (Co-IP) and Western blot were utilized to investigate the mechanism of tumor progression. Immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) were performed to determine the correlation of PDCD6 and MAPK pathway. RESULTS: Higher expression levels of PDCD6 in tumor tissues were associated with a poorer prognosis in patients with CRC. Furthermore, PDCD6 increased cell proliferation in vitro and tumor growth in vivo. Mechanistically, RNA-seq showed that PDCD6 could affect the activation of the MAPK signaling pathway. PDCD6 interacted with c-Raf, resulting in the activation of downstream c-Raf/MEK/ERK pathway and the upregulation of core cell proliferation genes such as MYC and JUN. CONCLUSIONS: These findings reveal the oncogenic effect of PDCD6 in CRC by activating c-Raf/MEK/ERK pathway and indicate that PDCD6 might be a potential prognostic indicator and therapeutic target for patients with colorectal cancer.

A rhodol-hemicyanine based ratiometric fluorescent probe for real-time monitoring of glutathione dynamics in living cells.

Glutathione (GSH) plays crucial roles in various physiological and pathological processes. The fluctuation of the GSH level is closely associated with a variety of diseases and cellular functions. Hence, it is important to real-time monitor the fluctuation of GSH in living cells. In this work, we presented a rhodol-hybridized hemicyanine fluorophore (RdH) as a selective, rapid-response, ratiometric, and reversible fluorescent probe for intracellular GSH (t1/2 = 89 ms, Kd = 1.42 mM). The imaging assays in living cells revealed that RdH could be used to real-time monitor GSH dynamics in A549 cells under a laser scanning confocal microscope by ratiometric fluorescence changes.

Response and acquired resistance to savolitinib in a patient with pulmonary sarcomatoid carcinoma harboring MET exon 14 skipping mutation: a case report.

BACKGROUND: Pulmonary sarcomatoid carcinoma (PSC) is a rare and poorly differentiated type of non-small cell lung cancer (NSCLC) with specific characteristics, which usually presents a challenge in clinical practice. Mesenchymal-epithelial transition (MET) gene has been identified as a promising target for treatments in the past few years. Here, we report a case of a patient with PSC harboring MET exon 14 mutation, who responded to a novel MET inhibitor - savolitinib. CASE PRESENTATION: A 75-year-old male patient with symptoms of cough, dyspnea and intermittent chest pain was diagnosed with sarcomatoid carcinoma. The tumor involved the right lung, the right hilum and multiple lesions in the right pleura, indicating a clinical disease stage IV. Next-generation sequencing of lung biopsy specimen indicated a MET exon 14 skipping mutation (NM_000245:c.3028+3A>G), with a variant allele frequency of 73.9%. The patient achieved a rapid and durable partial response with the initiation of savolitinib administration (600 mg, orally, once daily). The progression-free survival in this patient was 36 weeks. There were no ≥grade 3 adverse events reported and there was no dose reduction during treatment. Following savolitinib treatment, the allele frequency of MET exon 14 mutation in plasma circulating tumor DNA decreased with the reduction in tumor size. At the time of disease progression, fibroblast growth factor receptor 1 (FGFR1), EGFR and KRAS gene amplification were newly identified in tumor biopsy sample. CONCLUSION: This patient with PSC harboring MET exon 14 skipping mutation achieved significant clinical benefit with savolitinib treatment. Emergence of FGFR1, EGFR and KRAS gene amplification at the time of disease progression was likely responsible for the resistance to savolitinib in this case.

P53-R273H mutation enhances colorectal cancer stemness through regulating specific lncRNAs.

BACKGROUND: TP53 is one of the most frequently mutated genes among all cancer types, and TP53 mutants occur more than 60% in colorectal cancer (CRC). Among all mutants, there are three hot spots, including p53-R175H, p53-R248W and p53-R273H. Emerging evidence attributes cancer carcinogenesis to cancer stem cells (CSCs). Long noncoding RNAs (lncRNAs) play crucial roles in maintaining the stemness of CSCs. However, it is unknown if mutant p53-regulated lncRNAs are implicated in the maintenance of CSC stemness. METHODS: RNA-sequencing (RNA-seq) and ChIP-sequencing (ChIP-seq) were used to trace the lncRNA network regulated by p53-R273H in HCT116 endogenous p53 point mutant spheroid cells generated by the somatic cell knock-in method. RT-qPCR was used to detect lncRNA expression patterns, verifying the bioinformatics analysis. Transwell, spheroid formation, fluorescence activated cell sorter (FACS), xenograft nude mouse model, tumor frequency assessed by extreme limiting dilution analysis (ELDA), Western blot assays and chemoresistance analysis were performed to elucidate the functions and possible mechanism of lnc273-31 and lnc273-34 in cancer stem cells. RESULTS: p53-R273H exhibited more characteristics of CSC than p53-R175H and p53-R248W. RNA-seq profiling identified 37 up regulated and 4 down regulated differentially expressed lncRNAs regulated by p53-R273H. Combined with ChIP-seq profiling, we further verified two lncRNAs, named as lnc273-31 and lnc273-34, were essential in the maintenance of CSC stemness. Further investigation illustrated that lnc273-31 or lnc273-34 depletion dramatically diminished colorectal cancer migration, invasion, cancer stem cell self-renewal and chemoresistance in vitro. Moreover, the absence of lnc273-31 or lnc273-34 dramatically delayed cancer initiation and tumorigenic cell frequency in vivo. Also, lnc273-31 and lnc273-34 have an impact on epithelial-to mesenchymal transition (EMT). Finally, lnc273-31 and lnc273-34 were significantly highly expressed in CRC tissues with p53-R273H mutation compared to those with wildtype p53. CONCLUSIONS: The present study unveiled a high-confidence set of lncRNAs regulated by p53-R273H specific in colorectal CSCs. Furthermore, we demonstrated that two of them, lnc273-31 and lnc273-34, were required for colorectal CSC self-renewal, tumor propagation and chemoresistance. Also, the expression of these two lncRNAs augmented in colorectal cancer patient samples with p53-R273H mutation. These two lncRNAs may serve as promising predictors for patients with p53-R273H mutation and are vital for chemotherapy.

Pyocyanin induces NK92 cell apoptosis via mitochondrial damage and elevated intracellular Ca2.

Pseudomonas aeruginosa-derived pigment pyocyanin (PCN) has been proved to induce cell apoptosis mediated by the generation of reactive oxygen species (ROS), which has been studied mainly in epithelial cells and neutrophils. However, we previously found that the PCN-producing strain PA14 induces cell apoptosis in human NK cell line NK92 more effectively than in PCN-deficient strain PA14-phZ1/2 via a yet undetermined mechanism. In the current study, we found that PCN-induced NK92 cell apoptosis occurs through mitochondrial damage despite inhibiting intracellular ROS generation. Intracellular Ca2+ ([Ca2+]i) and Bcl-2 family proteins act as important "priming signals" for apoptosis. PCN treatment increased [Ca2+]i in NK92 cells more than twofold after 2 h stimulation, whereas the Ca2+-chelating agent ethylene glycol tetra-acetic acid (EGTA) inhibited apoptosis. PCN triggered the activation of Bim, Bid, Bik, Bak, and phospho-Bad in NK92 cells in a concentration-dependent manner, but these pro-apoptotic Bcl-2 family proteins were not inhibited by EGTA. In this study, we describe the function of PCN in NK92 cells and identify mitochondrial damage as the mechanism underlying the apoptosis. [Ca2+]i and pro-apoptotic Bcl-2 family proteins are novel targets for PCN-induced apoptosis. Clarification of the cytotoxic diversity of PCN provides a new therapeutic target for defense from P. aeruginosa-induced immune cell damage.

Rhomboid domain-containing protein 1 promotes breast cancer progression by regulating the p-Akt and CDK2 levels.

BACKGROUND: Our previous work revealed that rhomboid domain-containing protein 1 (RHBDD1) participates in the modulation of cell growth and apoptosis in colorectal cancer cells. This study aimed to investigate the function of RHBDD1 in regulating breast cancer progression and its underlying molecular basis. METHODS: Immunohistochemistry was performed to evaluate RHBDD1 expression in 116 breast cancer tissue and 39 adjacent normal tissue and expression of RHBDD1, phospho-Akt (p-Akt) and cyclin-dependent kinase 2 (CDK2) in the same 84 breast cancer specimens. RHBDD1-knock-out cells were established using breast cancer cell lines. In vitro studies were carried out to estimate the function of RHBDD1 in cell proliferation, migration and invasion. Fluorescence microscopy assay and flow cytometric analysis were used to measure apoptosis and cell cycle regulation. RNA sequencing and western blot analysis were used to investigate the molecular mechanisms of RHBDD1. RESULTS: RHBDD1 was highly up-regulated in breast cancer tissue compared with that in normal tissue and associated with pathological tumor (pT) stage, pathological tumor-node-metastasis (pTNM) stage and estrogen receptor (ER) expression. RHBDD1 up-regulation was associated with poor prognosis in several subtypes of breast cancer. Deletion of RHBDD1 promoted apoptosis and suppressed proliferation, migration and invasion in breast cancer cells. RHBDD1 deletion suppressed Akt activation and decreased CDK2 protein level via proteasome pathway, thus inhibited cell cycle progression and G1/S phase transition. Moreover, the protein level of RHBDD1, p-Akt and CDK2 was significantly positively correlated in breast cancer tissue. CONCLUSIONS: Our study reveals that RHBDD1 promotes breast cancer progression by regulating p-Akt and CDK2 protein levels, and might be a potential biomarker and prognostic indicator for breast cancer patients.

RNF138 confers cisplatin resistance in gastric cancer cells via activating Chk1 signaling pathway.

Chemotherapy resistance represents a major issue associated with gastric cancer (GC) treatment, and arises through multiple mechanisms, including modulation of the cell-cycle check point. Several ubiquitin kinases, including RING finger protein 138 (RNF138), have been reported to mediate the G2/M phase arrest. In this study, we investigated the role of RNF138 in the development of cisplatin resistance of two GC cell lines. We show that RNF138 levels are higher in cisplatin-resistant cell lines, compared with cisplatin-sensitive cells, and RNF138 expression was elevated during drug withdrawal following the cisplatin treatment. Using gene overexpression and silencing, we analyzed the impact of altering RNF138 level on GC cell viability, apoptosis, and cell cycle phenotypes in two isogenic cisplatin-sensitive and resistant cell lines. We show that RNF138 overexpression increased GC cell viability, decreased apoptosis and delayed cell cycle progression in the cisplatin-sensitive GC cells. Conversely, RNF138 silencing produced opposite phenotypes in the cisplatin-resistant cells. Moreover, RNF138-dependent phosphorylation of Chk1 was seen in GC cells, indicating a novel connection between cisplatin-induced DNA damage and apoptosis. Collectively, these data suggest that RNF138 modulates the cisplatin resistance in the GC cells, thus serving as a potential drug target to challenge chemotherapy failure. In addition, RNF138 can also be used as a marker to monitor the development of cisplatin resistance in GC treatment.