A positive feedback between PDIA3P1 and OCT4 promotes the cancer stem cell properties of esophageal squamous cell carcinoma.

BACKGROUND: Increasing evidence has indicated that long non-coding RNAs (lncRNAs) have been proven to regulate esophageal cancer progression. The lncRNA protein disulfide isomerase family A member 3 pseudogene 1 (PDIA3P1) has been shown to promote cancer stem cell properties; however, its mechanism of action remains unclear. In this study, we investigated the regulation of esophageal cancer stem cell properties by the interaction of PDIA3P1 with proteins. METHODS: The GEPIA2 and Gene Expression Omnibus databases were used to analyze gene expression. PDIA3P1 expression in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Loss-of-function experiments were performed to determine the effects of PDIA3P1 on ESCC cell proliferation, migration, and invasion. The sphere formation assay, number of side population cells, and CD271 + /CD44 + cells were detected by flow cytometry to identify the cancer stem cell properties. RNA immunoprecipitation (RIP), RNA pull-down, co-immunoprecipitation (co-IP), dual luciferase reporter, and cleavage under targets and tagmentation (CUT&Tag) assays were performed to elucidate the underlying molecular mechanisms. RESULTS: PDIA3P1 expression was upregulated in ESCC cell lines and tissues. Functionally, higher PDIA3P1 expression promoted cell proliferation, invasion, and metastasis and inhibited apoptosis in esophageal cancer. Importantly, PDIA3P1 promoted cancer stem cell properties in ESCC. Mechanistically, PDIA3P1 interacted with and stabilized octamer-binding transcription factor 4 (OCT4) by eliminating its ubiquitination by the ubiquitinating enzyme WW domain-containing protein 2 (WWP2). Moreover, as a transcription factor, OCT4 bound to the PDIA3P1 promoter and promoted its transcription. CONCLUSIONS: Our research revealed a novel mechanism by which a positive feedback loop exists between PDIA3P1 and OCT4. It also demonstrated that the PDIA3P1-WWP2-OCT4 loop is beneficial for promoting the cancer stem cell properties of ESCC. Owing to this regulatory relationship, the PDIA3P1-WWP2-OCT4-positive feedback loop might be used in the diagnosis and prognosis, as well as in the development of novel therapeutics for esophageal cancer.

Case report: Response to tepotinib in Chinese non-small cell lung cancer patients harboring METex14 skipping with varying features.

MET exon 14 (METex14) skipping is the most reported MET mutation in non-small cell lung cancer (NSCLC) and has been confirmed to respond to MET tyrosine kinase inhibitors (TKI) in clinical trials. While MET TKI tepotinib was recently approved for METex14 skipping NSCLC in China, real-world evidence is limited. We report our experience treating NSCLC patients referred from oncology sites across China with tepotinib in the Boao Lecheng Pilot Zone. Four patients have been prescribed the drug with a median age of 67 years (range, 61-71 years). One patient has concomitant BRAF V600E mutation, and another patient had savolitinib as first line of therapy but discontinued due to hepatotoxicity. Till the end of follow-up, four patients were all on tepotinib therapy, with a median duration of therapy of 19 months. One patient achieved partial response and three achieved stable disease. Three patients had peripheral edema, but all were mild. Our experience showed in real clinical setting, tepotinib had robust and durable clinical activity and a favorable toxicity profile in Chinese patients with METex14 skipping NSCLC. It is the first report on the effectiveness of tepotinib in a patient with both METex14 skipping and BRAF V600E mutations and successful MET inhibitor switch after MET inhibitor-induced liver injury.

Intracellular expression of Vitreoscilla hemoglobin improves production of Yarrowia lipolytica lipase LIP2 in a recombinant Pichia pastoris.

The Yarrowia lipolytica lipase LIP2 (YlLIP2) gene lip2 and Vitreoscilla hemoglobin gene vgb were co-expressed in Pichia pastoris, both under the control of AOX1 promoter, in order to alleviate respiration limitation under conditions of high cell-density fermentation and enhance YlLIP2 production. The results showed that recombinant P. pastoris strains harboring the lip2 and vgb genes (VHb(+)) displayed higher biomass and YlLIP2 activity than control strains (VHb(-)). Compared with VHb(-) cells, the expression levels of YlLIP2 in VHb-expressing cells when oxygen was not a limiting factor were improved 31.5% in shake-flask culture and 22% in a 10-L fermentor. Under non-limiting dissolved oxygen (DO) conditions, the maximum YlLIP2 activity of VHb(+) in a 10-L fermentor reached 33,000 U/mL. Oxygen limitation had a more negative effect on YlLIP2 productivity in VHb(-) cells than in VHb(+) cells. The highest YlLIP2 activity of VHb(+) cells was approximately 1.84-fold higher than that of VHb(-) cells at lower DO levels. Moreover, the recombinant strain VHb(+) exhibited a higher specific oxygen uptake rate and achieved higher cell viability under oxygen limiting and non-limiting conditions compared with VHb(-) cells. Therefore, the above results suggest that intracellular expression of VHb in recombinant P. pastoris has the potential to improve cell growth and industrial enzyme production.

[Expression of Vitreoscilla hemoglobin improves recombinant lipase production in Pichia pastoris].

Yarrowia lipolytica lipase Lip2 (YlLip2) is an important industrial enzyme with many potential applications. To alleviate the dissolved oxygen (DO) limitation and improve YlLip2 production during high-cell density fermentation, the YlLip2 gene lip2 and Vitreoscilla hemoglobin (VHb) gene vgb were co-expressed in Pichiapastoris under the control of AOX1 and PsADH2 promoter, respectively. The PsADH2 promoter from Pichia stipitis could be activated under oxygen limitation. The SDS-PAGE and CO-difference spectrum analysis indicated that VHb and YlLip2 had successfully co-expressed in recombinant strains. Compared with the control cells (VHb-, GS115/9Klip2), the expression levels of YlLip2 in VHb-expressing cells (VHb+, GS115/9Klip2-pZPVT) under oxygen limitation were improved 25% in shake-flask culture and 83% in a 10 L fermentor. Moreover, the VHb+ cells displayed higher biomass than VHb- cells at lower DO levels in a 10 L fermentor. In this study, we also achieved a VHb-expressing clone harboring multicopy lip2 gene (GS115/9Klip2-pZPVTlip2 49#), which showed the maximum lipolytic activity of 33 900 U/mL in a 10 L fermentor under lower DO conditions. Therefore, it can be seen that expression of VHb with PsADH2 promoter in P. pastoris combined with increasing copies of lip2 gene is an effective strategy to improve YlLip2 production.