PHF12 regulates HDAC1 to promote tumorigenesis via EGFR/AKT signaling pathway in non-small cell lung cancer.

BACKGROUND: Lung cancer stands as the second most prevalent malignant neoplasm worldwide. Addressing the underlying mechanisms propelling the progression of non-small cell lung cancer is of paramount importance. In this study, we have elucidated the pivotal role of PHF12 in this context. MATERIALS AND METHODS: We harnessed clinical lung cancer tissue samples and non-small cell lung cancer cell lines to discern the expression pattern of PHF12. In vitro assays probing cell proliferation were conducted to substantiate the functional impact of PHF12. Furthermore, an in vivo Xenograft model was employed to dissect the role of PHF12. Employing ChIP assays and qRT-PCR, we delved into the intricate binding dynamics between PHF12 and HDAC1. Mechanistic insights into the PHF12-HDAC1 axis in lung cancer progression were pursued via RNA-seq and GSEA analyses. RESULTS: Notably, PHF12 exhibited a substantial upregulation within tumor tissue, concomitant with its correlation to HDAC1. The trilogy of cell proliferation assays, transwell assays, and the Xenograft model collectively underscored the promoting influence of PHF12 on lung cancer proliferation, both in vitro and in vivo. The ChIP assay unveiled the transcriptional regulatory role of PHF12 in governing HDAC1 expression. This correlation extended to both mRNA and protein levels. PHF12 promotes NSCLC progression through regulating HDCA1 expression. Intriguingly, the rescue of function within NSCLC cell lines post PHF12 knockdown was achievable through HDAC1 overexpression. Additionally, our findings unveiled the capacity of the PHF12-HDAC1 axis to activate the EGFR/AKT signaling pathway, thereby further corroborating its significance in lung cancer progression. CONCLUSION: Our study identified PHF12 as an oncogenic role in lung cancer proliferation and migration for the first time. PHF12 transcriptionally regulate HDAC1 and activate EGFR/AKT signaling pathway in NSCLC progression. PHF12 may serve as an important target in lung cancer therapy.

Correlation between immunotherapy biomarker PD-L1 expression and genetic alteration in patients with non-small cell lung cancer.

Programmed death-ligand 1 (PD-L1) has been widely used in immunotherapy evaluation of patients with non-small cell lung cancer (NSCLC). However, the effect is not particularly ideal, and the association between PD-L1 and genetic alterations requires more exploration. Here, we performed targeted next-generation sequencing and PD-L1 immunohistochemistry (IHC) testing for PD-L1 expression on both tumor cells (TCs) and tumor-infiltrating immune cells (ICs) in 1549 patients. Our studies showed that surgical method of resection was positively correlated with IC+, and a low tumor mutation burden (TMB) was negatively correlated with TC+. Furthermore, we found that EGFR was mutually exclusive with both ALK and STK11. In addition, the features between PD-L1 expression status and genomic alterations were characterized. These results suggest that clinical characteristics and molecular phenotypes are associated with PD-L1 expression signatures, which may provide novel insights for improving the efficiency of immune checkpoint inhibitors (ICIs) in immunotherapy.

Unexpected curative effect of PD-1 inhibitor in gastric cancer with brain metastasis: A case report.

Background: Gastric cancer (GC) is the third most common cause of cancer-related death in the world. Several clinical trials have proven that the use of PD-1/PD-L1 inhibitors can improve the survival of late-stage GC patients and is suggested in NCCN and CSCO guidelines. However, the correlation between PD-L1 expression and the response to PD-1/PD-L1 inhibitors is still controversial. GC rarely develops brain metastasis (BrM) and currently there is no therapeutic protocol for GC BrMs. Case presentation: We report a case of a 46-year-old male suffering from GC with PD-L1 negative BrMs 12 years after GC resection and 5 cycles of chemotherapy. We treated the patient with the immune checkpoint inhibitor (ICI) pembrolizumab and all metastatic tumors achieved a complete response (CR). A durable remission of the tumors is confirmed after 4 years of follow-up. Conclusion: We shared a rare case with PD-L1 negative GC BrM responsive to PD-1/PD-L1 inhibitors, the mechanism of which is still unclear. The protocol of therapeutic choice for late-stage GC with BrM is urgently needed. And we are expecting biomarkers other than PD-L1 expressions to predict the efficacy of ICI treatment.

VEGF single nucleotide polymorphisms predict improved outcome in advanced non-small cell lung cancer patients treated with platinum-based chemotherapy.

We aimed to investigate the prognostic role of genetic variants of VEGF in advanced NSCLC patients treated with platinum-based chemotherapy. A total of 196 patients with advanced NSCLC treated with first-line platinum-based chemotherapy were enrolled. We evaluated the relationship between VEGF polymorphisms and efficacy outcomes and chemotherapy toxicity. We found that rs699947, rs833061 and rs1005230 were in full linkage disequilibrium. Patients with CC genotype of rs833061 had a significant longer PFS than TT genotype (CC vs TT, HR = 1.67, 95%CI = 1.01-2.76, P = 0.043). Patients harbouring CC genotype had longer PFS compared with CT genotype (P < 0.001). Moreover, CC genotypes conferred a significantly increased PFS compared to CT and TT genotype in dominant model (CC vs CT + TT, HR = 1.95, 95%CI = 1.23-3.10, P = 0.005). Patients carrying TT genotype of rs833061 had improved both ORR (HR = 0.54, 95%CI = 0.30-0.98, P = 0.041) and DCR (HR = 0.37, 95%CI = 0.20-0.66, P = 0.001) than non-TT patients. Furthermore, no association was found between any rs833061 alleles and adverse events (P = 0.425), but patients carrying rs1570360 AA genotype were more likely to experience grade 3-4 toxicities (P = 0.004) (GG vs AA, HR = 3.16, 95%CI = 1.26-7.94, P = 0.015). In conclusion, the variant homozygote CC of rs833061 exhibited a better prognosis based on association analysis. The present study provides reference for the future study of platinum-based chemotherapy response and toxicity.

Clinical Outcomes of Patients with Epidermal Growth Factor Receptor-Mutated Non-Small-Cell Lung Cancer with Leptomeningeal Metastasis in the Modern Target Therapy Era.

BACKGROUND: Leptomeningeal metastasis (LM) is a severe complication in patients with non-small-cell lung cancer (NSCLC) and the optimal treatment strategy remains a challenge. This study aimed to investigate the treatment strategies and clinical outcomes in these patients. METHODS: We retrospectively reviewed the data of 44 patients with epidermal growth factor receptor (EGFR)-mutated NSCLC with LM between 2014 and 2020 at our institute. The patient characteristics, treatment approaches, LM progression-free survival (LMPFS) and overall survival (OS) after the diagnosis of LM (OSLM) were analyzed. RESULTS: The median OSLM was 16.0 months and the 3-year OS rate was 22.5%. The PFSLM in EGFR T790M-positive NSCLC patients with leptomeingeal disease was significantly improved by initiation of third-generation tyrosine kinase inhibitors (TKIs) compared with that of patients who were T790M negative (14.0 vs. 7.0 months; P = 0.030). A significantly higher LM disease control rate was shown in patients who received third-generation TKIs compared with previous generations of TKIs (90.1% vs. 60.0%; P = 0.024). Better Eastern Cooperative Oncology Group performance status, EGFR exon 19del, and clinical improvement of LM after therapy were independently associated with better OS. CONCLUSIONS: The survival of patients with NSCLC with LM has improved in the target therapy era. Our study provided real-world clinical evidence that patients with EGFR-mutated NSCLC who developed LM from previous TKIs can be benefit from third-generation EGFR-TKIs, especially for patients with EGFR T790M-positive.

Digital PCR-Based Detection of EGFR Mutations in Paired Plasma and CSF Samples of Lung Adenocarcinoma Patients with Central Nervous System Metastases.

BACKGROUND: The presence of specific mutations in the EGFR gene informs the clinical pathway of therapy for patients with lung adenocarcinoma (LAC), including those with central nervous system (CNS) metastases. Plasma circulating cell-free DNA (cfDNA) has been demonstrated to carry the mutational information of LACs, which serves as a biomarker to guide treatment. However, whether the cerebrospinal fluid (CSF) enriches circulating tumor DNA (ctDNA) released from CNS metastatic lesions of LAC, and whether the CSF ctDNA can be used to characterize these lesions remains unknown. OBJECTIVE: To explore the EGFR status in CNS metastases of LAC patients, and to guide the treatment of intra- and extracranial tumors in these patients. PATIENTS AND METHODS: The EGFR mutational status in the cfDNA from paired CSF and plasma samples from LAC patients with CNS metastases, including 20 brain metastases (BM) and 15 leptomeningeal metastases (LM), was assessed by droplet digital polymerase chain reaction (ddPCR). The clinical outcomes of the EGFR status-based intervention were investigated. RESULTS: EGFR mutations were detected in 23/35 LAC patients (65.7%). EGFR mutations in the plasma or CSF were detected in 6/11 (54.5%) and 5/10 (50%) BM patients, and in 4/11 (36.4%) and 9/12(75%) LM patients, respectively. The prevalence of the T790M mutation was significantly higher in plasma (9/23) than in CSF (3/23) samples. The sensitivity and specificity of the ddPCR-based EGFR mutation test in CSF or plasma samples versus the primary tumor samples were 56% and 89% versus 46% and 100%, respectively. Twelve patients received a first-generation EGFR TKI (tyrosine kinase inhibitor) after the detection of sensitive EGFR mutations in their CSF or plasma, and five patients were switched from a first-generation EGFR TKI to osimertinib after the detection of the T790M mutation. CONCLUSIONS: The EGFR T790M mutation in plasma cfDNA is a sensitive marker for EGFR TKI resistance when CNS metastases progressed. CSF ctDNA increases the diagnostic validity for EGFR genotyping of lung cancer brain metastasis. ddPCR in CSF and plasma samples could provide less invasive and close monitoring of the EGFR status of LAC patients with CNS metastases.

Upregulation of phosphoserine phosphatase contributes to tumor progression and predicts poor prognosis in non-small cell lung cancer patients.

BACKGROUND: Growing evidence indicates that high phosphoserine phosphatase (PSPH) expression is associated with tumor prognosis in many types of cancers. However, the role of PSPH in non-small cell lung cancer (NSCLC) is unclear. The purpose of this study was to investigate the clinical significance of PSPH in NSCLC. METHODS: One hundred forty-three patients with histologically confirmed NSCLC who underwent surgery were included. Quantitative real-time PCR and Western blot were used to assess PSPH expression in paired tumor and corresponding adjacent non-tumorous tissues. The role of PSPH in invasion and cell growth was investigated in vitro. RESULTS: Compared to adjacent normal lung tissues, PSPH messenger RNA and protein levels were significantly higher in NSCLC tissues, and the PSPH expression level was positively related to clinical stage, metastasis, and recurrence. High PSPH expression was predictive of poor overall survival. A549 cells transfected with small interfering-PSPH showed inhibited cell migration, invasion, and proliferation. We further demonstrated that PSPH might promote the invasive capabilities of NSCLC cells through the AKT/AMPK signaling pathway. CONCLUSION: Our results indicate that PSPH may act as a putative oncogene in NSCLC, and may be a vital molecular marker for the metastasis and proliferation of NSCLC cells by regulating the AKT/AMPK signaling pathway.

Different next-generation sequencing pipelines based detection of tumor DNA in cerebrospinal fluid of lung adenocarcinoma cancer patients with leptomeningeal metastases.

BACKGROUND: The nucleic acid mutation status in intracranial metastasis is markedly significant clinically. The goal of the current study was to explore whether the tumor-associated mutations can be detected by different next-generation sequencing (NGS) pipelines in paired cerebrospinal fluid (CSF) and plasma samples from lung adenocarcinoma (LAC) patients with leptomeningeal metastases (LM). METHODS: Paired CSF cell free DNA (cfDNA), CSF cells, plasma and formalin-fixed and paraffin-embedded (FFPE) samples of primary tumors were collected from 29 LAC patients with LM to detect the mutations by different NGS pipelines. RESULTS: DNA libraries were generated successfully for 79 various samples in total for NGS sequencing, of which mutations were detected in 7 plasma samples (24.14%), 12 CSF cfDNA samples (66.67%), and 10 CSF cells (76.9%) samples. For the 26 patients with detected mutations, 8/26(30.77%) had mutations in plasma, which was significantly lower than that those from CSF cfDNA (12/15, 80.00%), CSF cells (10/11, 90.91%) and FFPE samples (13/17, 76.47%). When the input DNA of CSF cells was less than 20 ng, the cHOPE pipeline of NGS identified the most mutations for epidermal growth factor receptor (EGFR). CONCLUSIONS: NGS-based detection of mutations in cfDNA or cells from CSF provided more information than from plasma samples from LAC patients with LM. In addition, the cHOPE pipeline performed better than the other three NGS pipelines when input DNA from CSF cells was low.

TBL1XR1 as a potential therapeutic target that promotes epithelial-mesenchymal transition in lung squamous cell carcinoma.

Transducin (ß)-like 1 X-linked receptor 1 (TBL1XR1) has been demonstrated to serve a vital role in tumor progression. However, the biological role and molecular mechanisms of TBL1XR1 in lung squamous cell carcinoma (SCC) remain largely unknown. The purpose of the present study was to investigate the biological role of TBL1XR1 and its mechanism in lung SCC. TBL1XR1 was expressed in a human bronchial epithelial cell line and in lung SCC cell lines. The present study analyzed TBL1XR1-induced proliferation, invasion and migration abilities in vitro using the cell counting kit-8 assay, cell invasion assay and wound healing assay, respectively. This study examined the effects of TBL1XR1 on epithelial-mesenchymal transition (EMT) in lung SCC cells and activation of the transforming growth factor (TGF)-ß/mothers against decapentaplegic homolog (Smad) signaling pathway by western blotting. The results indicated that TBL1XR1 was upregulated in lung SCC cells. Overexpression of TBL1XR1 increased the rate of cell proliferation compared with the control group. In vitro, overexpression of TBL1XR1 promoted cell invasion and migration ability compared with the control group. In addition, overexpression of TBL1XR1 produced a mesenchymal phenotype, while cells with downregulated TBL1XR1 produced an epithelial phenotype. Overexpression of TBL1XR1 significantly increased E-cadherin protein expression whilst snail family transcriptional repressor 1 (SNAI1), zinc finger E-box binding homebox 1 (ZEB1), p-Smad2/3, Smad2 and Smad3 protein expression was significantly reduced, compared with the control group. Downregulation of TBL1XR1 produced the opposite results. The present study indicated that TBL1XR1 contributed to lung SCC development and progression, and therefore TBL1XR1 may be a potential therapeutic target. TBL1XR1 may induce EMT of lung SCC cells through activation of the TGF-ß/Smad signaling pathway.

PSPH Mediates the Metastasis and Proliferation of Non-small Cell Lung Cancer through MAPK Signaling Pathways.

Growing evidence indicates that phosphoserine phosphatase (PSPH) is up-regulated and correlates with prognosis in multiple types of cancer. However, little is known about the roles of PSPH in NSCLC. Thus, the aim of the present study was to demonstrate the expression of PSPH in human NSCLC and reveal its biological functions and the underlying mechanisms. qRT-PCR, western blot and immunohistochemistry were used to assess the expression of NSCLC patient specimens and NSCLC cell lines. The functions of PSPH in migration and invasion were determined using trans-well and wound-healing assays. Cell proliferation capacity was performed by cell counting kit-8 (CCK-8), colony formation assays and cell cycle analysis. We demonstrated that PSPH was overexpressed in NSCLC specimens compared with the adjacent non-tumorous specimens, and high expression of PSPH was associated with clinical stage, metastasis and gender in NSCLC. Decreased expression of PSPH inhibited NSCLC cells migration, invasion and proliferation. Most importantly, further experiments demonstrated that PSPH might regulate NSCLC progress through MAPK signaling pathways. Lastly, immunohistochemistry (IHC) revealed that the PSPH expression level was positively correlated with the clinical stage in NSCLC patients. These results suggest that PSPH may act as a putative oncogene and a potential therapeutic target in NSCLC.

Efficacy and safety of bevacizumab combined with chemotherapy in symptomatic brain metastases from lung adenocarcinoma: a retrospective analysis.

BACKGROUND: Currently, the treatment of symptomatic brain metastases from lung adenocarcinoma has remained difficult. Bevacizumab combined with chemotherapy is one of the standard treatments of lung adenocarcinoma. This study was designed to investigate the efficacy and safety of bevacizumab combined with chemotherapy in symptomatic brain metastases from lung adenocarcinoma that are not suitable for local treatments, and to explore the predictive value of baseline serum vascular endothelial growth factor (VEGF) for the treatment. METHODS: We retrospectively reviewed 14 consecutive patients, between Jan 2015 and Jul 2017, with brain metastases from lung adenocarcinoma who received bevacizumab and chemotherapy to determine efficacy and toxicity. Kaplan-Meier method was used to estimate survival curves, and univariate and multivariate analyses were performed by Cox proportional hazard model. The primary endpoints were objective response rate (ORR) and intracranial ORR (iORR). The secondary endpoints were progression-free survival (PFS), intracranial PFS (iPFS), overall survival (OS) and disease control rate (DCR). RESULTS: The efficacy of 12 patient was evaluated. Overall ORR was 25% (3/12) and the iORR of brain lesions was 33.3% (4/12). DCR was 75% (9/12). The median OS was 18.3 months, the median PFS was 6.7 months, and the median iPFS was 12 months. After 2 cycles of bevacizumab, 10 patients showed improved symptoms of central nervous system (CNS), and the symptom control rate was 83.3% (10/12). Head MRI showed that edema in the brain was greatly reduced in 6 patients, resulting in the lessened usage of dexamethasone. iPFS was significantly shorter in high VEGF group (3.6 vs. 8.0 m, P=0.02), and multivariate analysis showed a significant correlation between iPFS and serum baseline VEGF level (P=0.023). The most commonly adverse events of bevacizumab included leukopenia [5 (35.7%)], fatigue [3 (21.4%)], thrombocytopenia [3 (21.4%)], anemia [2 (14.3%)], which were mostly degree I and II. CONCLUSIONS: This study showed bevacizumab combined with chemotherapy could effectively control intracranial lesions, relieve symptoms, and improve the quality of life and survival of patients with brain metastases from lung adenocarcinoma. Serum baseline VEGF may be a predictor of efficacy of bevacizumab plus chemotherapy in the treatment of brain metastases from lung adenocarcinoma.

Epidermal Growth Factor Receptor Mutation Detection in Cerebrospinal Fluid of Lung Adenocarcinoma Patients with Leptomeningeal Metastasis.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutations are associated with leptomeningeal metastases (LM) of nonsmall cell lung cancer and sensitivity to tyrosine kinase inhibitor (TKI) treatment. Owing to the difficulty of obtaining carcinomatous meningeal tissue for analysis, cerebrospinal fluid (CSF) might be an alternative. OBJECTIVE: To investigate the EGFR mutation detection in the CSF of lung adenocarcinoma patients with LM. METHODS: Twenty-five lung adenocarcinoma patients with LM diagnosed by CSF cytology were retrospectively evaluated. The results of EGFR mutation detection in CSF, the treatment plan, and clinical outcome information were recorded. RESULTS: Nineteen patients had a known EGFR status in their primary tumors. Twenty patients received EGFR mutation analysis in CSF after LM diagnosis and 14 of them with a known EGFR mutation status of both primary tumors and CSF. Ten (71.4%) had the same EGFR gene status. In primary tumors, no T790M mutations were detected, whereas in CSF, 2 L858R cases and 1 19del case had T790M mutations at the same time. The detection rate of T790M mutations in CSF was 18.1% (2 of 11) in all cases with EGFR-sensitive mutations in the primary lesion. CONCLUSIONS: EGFR mutation detection in CSF of lung adenocarcinoma patients with LM might be an alternative when leptomeningeal biopsy cannot be applied and may help to guide TKI treatments.

Characterization of genetic alterations in brain metastases from non-small cell lung cancer.

Brain metastasis (BM) is the primary contributor to mortality in non-small cell lung cancer (NSCLC) patients. Although the findings of NSCLC genetic sequencing studies suggest the potential for personalizing therapeutic approaches, the genetic profiles and underlying mechanisms of BM progression remain poorly understood. Here, we investigated the genetic profiles of brain metastases from NSCLC in six patients with primary tumors and corresponding BM samples via whole exome sequencing and targeted panel sequencing. We have demonstrated considerable genetic heterogeneity between primary lung cancer and corresponding brain metastases specimens. High-frequency mutations were found in NOTCH2,NOTCH2NL,FANCD2,EGFR, and TP53. Additionally, EGFR and TP53 consistently exhibited high frequencies of mutation between primary tumors and corresponding brain metastases. The implication is that most of the genetic alterations may be acquired or lost during malignant progression, and the stable EGFR and TP53 mutational status between paired primary tumors and metastatic sites confirms that most mutations detected on analysis of the primary tumor or metastases are sufficient for clinical decision-making, and suggest there is no need to re-biopsy recurrent tumors or metastases for most NSCLC patients.

High probability and frequency of EGFR mutations in non-small cell lung cancer with brain metastases.

Lung cancer is the leading cause of cancer death in men and women worldwide. Brain metastasis (BMs) of non-small cell lung cancer (NSCLC) is the most important cause of death. This study aimed to explore the association of epidermal growth factor receptor (EGFR) mutations and BMs in NSCLC. We analyzed 50 NSCLC patients with BMs and 50 match-paired NSCLC patients with no brain metastases (NBMs). The EGFR mutation status of primary lesions was detected using the amplification refractory mutation system polymerase chain reaction. The BMs patients had a higher frequency of EGFR mutations than the NBMs patients (52.0 vs. 22.0% respectively, P < 0.001), in both adenocarcinoma (60.5 vs. 30.6%, P = 0.003) and squamous carcinoma (37.5 vs. 0%, P = 0.04). The incidence of BMs in patients with EGFR mutations was higher than in patients with wild-type EGFR (70.3 vs. 38.1%, P = 002). NSCLC patients with BMs had a higher incidence of EGFR mutations and those with mutant EGFR had a higher frequency of BMs. EGFR mutations may promote brain metastasis growth of NSCLC.

FRAS1 knockdown reduces A549 cells migration and invasion through downregulation of FAK signaling.

Distal metastasis is the major cause of death for the vast majority of lung cancer patients. Many extracellular matrix (ECM)-related molecules are proposed to be associated with the migration and invasion of cancer cells. FRAS1 encodes an ECM protein, however, little is known about its function on tumorigenesis and metastasis of lung cancer. In this work, FRAS1 was silenced by shRNA in non-small cell lung cancer (NSCLC) A549 cell line. The capacities of A549 cells to migrate and invade were decreased markedly after FRAS1 knockdown. The shRNA knockdown of FRAS1 was found to be specific and had no effect on A549 cells proliferation. Western blot experiments demonstrated that FRAS1 knockdown inhibited FAK signaling but not Src signaling. Overall, we found that FRAS1 knockdown reduces A549 cells migration and invasion ability through downregulation of FAK signaling.