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(1) Background: Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) is an intractable problem for many clinical oncologists. The mechanisms of resistance to EGFR-TKIs are complex. Long non-coding RNAs (lncRNAs) may play an important role in cancer development and metastasis. However, the biological process between lncRNAs and drug resistance to EGFR-mutated lung cancer remains largely unknown. (2) Methods: Osimertinib- and afatinib-resistant EGFR-mutated lung cancer cells were established using a stepwise method. A microarray analysis of non-coding and coding RNAs was performed using parental and resistant EGFR-mutant non-small cell lung cancer (NSCLC) cells and evaluated by bioinformatics analysis through medical-industrial collaboration. (3) Results: Colorectal neoplasia differentially expressed (CRNDE) and DiGeorge syndrome critical region gene 5 (DGCR5) lncRNAs were highly expressed in EGFR-TKI-resistant cells by microarray analysis. RNA-protein binding analysis revealed eukaryotic translation initiation factor 4A3 (eIF4A3) bound in an overlapping manner to CRNDE and DGCR5. The CRNDE downregulates the expression of eIF4A3, mucin 1 (MUC1), and phospho-EGFR. Inhibition of CRNDE activated the eIF4A3/MUC1/EGFR signaling pathway and apoptotic activity, and restored sensitivity to EGFR-TKIs. (4) Conclusions: The results showed that CRNDE is associated with the development of resistance to EGFR-TKIs. CRNDE may be a novel therapeutic target to conquer EGFR-mutant NSCLC.

DOI： 10.3390/ijms22084005

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Language：Japanese   Publisher：(一社)日本呼吸器学会  

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Language：Japanese   Publisher：(一社)日本呼吸器学会  

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Language：Japanese   Publisher：(一社)日本呼吸器学会  

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Language：English   Publishing type：Research paper (scientific journal)  

Overcoming acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is critical in combating EGFR-mutant non-small cell lung cancer (NSCLC). We tried to construct a novel therapeutic strategy to conquer the resistance to second-and third-generation EGFR-TKIs in EGFR-positive NSCLC patients. We established afatinib- and osimertinib-resistant lung adenocarcinoma cell lines. Exome sequencing, cDNA array and miRNA microarray were performed using the established cell lines to discover novel therapeutic targets associated with the resistance to second-and third-generation EGFR-TKIs. We found that ANKRD1 which is associated with the epithelial-mesenchymal transition (EMT) phenomenon and anti-apoptosis, was overexpressed in the second-and third-generation EGFR-TKIs-resistant cells at the mRNA and protein expression levels. When ANKRD1 was silenced in the EGFR-TKIs-resistant cell lines, afatinib and osimertinib could induce apoptosis of the cell lines. Imatinib could inhibit ANKRD1 expression, resulting in restoration of the sensitivity to afatinib and osimertinib of EGFR-TKI-resistant cells. In EGFR-mutant NSCLC patients, ANKRD1 was overexpressed in the tumor after the failure of EGFR-TKI therapy, especially after long-duration EGFR-TKI treatments. ANKRD1 overexpression which was associated with EMT features and anti-apoptosis, was commonly involved in resistance to second-and third-generation EGFR-TKIs. ANKRD1 inhibition could be a promising therapeutic strategy in EGFR-mutant NSCLC patients.

DOI： 10.1038/s41598-018-33190-8

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Language：Japanese   Publisher：(NPO)日本肺癌学会  

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Language：Japanese   Publisher：(NPO)日本肺癌学会  

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Language：Japanese   Publisher：(NPO)日本肺癌学会  

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Language：Japanese   Publisher：(NPO)日本肺癌学会  

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Language：English   Publishing type：Research paper (scientific journal)  

Anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKIs) induce a dramatic response in non-small cell lung cancer (NSCLC) patients with the ALK fusion gene. However, acquired resistance to ALK-TKIs remains an inevitable problem. In this study, we aimed to discover novel therapeutic targets to conquer ALK-positive lung cancer. We established three types of ALK-TKI (crizotinib, alectinib and ceritinib)-resistant H2228 NSCLC cell lines by high exposure and stepwise methods. We found these cells showed a loss of ALK signaling, overexpressed AXL with epithelial-mesenchymal transition (EMT), and had cancer stem cell-like (CSC) properties, suggesting drug-tolerant cancer cell subpopulations. Similarly, we demonstrated that TGF-β1 treated H2228 cells also showed AXL overexpression with EMT features and ALK-TKI resistance. The AXL inhibitor, R428, or HSP90 inhibitor, ganetespib, were effective in reversing ALK-TKI resistance and EMT changes in both ALK-TKI-resistant and TGF-β1-exposed H2228 cells. Tumor volumes of xenograft mice implanted with established H2228-ceritinib-resistant (H2228-CER) cells were significantly reduced after treatment with ganetespib, or ganetespib in combination with ceritinib. Some ALK-positive NSCLC patients with AXL overexpression showed a poorer response to crizotinib therapy than patients with a low expression of AXL. ALK signaling-independent AXL overexpressed in drug-tolerant cancer cell subpopulations with EMT and CSC features may be commonly involved commonly involved in intrinsic and acquired resistance to ALK-TKIs. This suggests AXL and HSP90 inhibitors may be promising therapeutic drugs to overcome drug-tolerant cancer cell subpopulations in ALK-positive NSCLC patients for the reason that ALK-positive NSCLC cells do not live through ALK-TKI therapy.

DOI： 10.18632/oncotarget.25531

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BACKGROUND/AIM: We evaluated the usefulness of reverse transcription-polymerase chain reaction (RT-PCR) for detecting anaplastic lymphoma kinase (ALK) translocations using cytology samples from lung cancer patients. MATERIALS AND METHODS: We analyzed ALK translocations by RT-PCR in cytology samples from lung cancer patients diagnosed at the Nippon Medical School Hospital between 2013 and 2015. Immunochemistry (IHC) and break-apart fluorescence in situ hybridization (FISH) were also performed on available tissue samples. RESULTS: A total of 155 cytology samples were analyzed in our study. We obtained 115 (68%) samples from bronchial lavage. We were able to determine 153 (99%) results by RT-PCR with 4 (3%) positive samples. The four samples positive by RT-PCR were also positive by IHC and FISH performed on the tissue samples collected simultaneously. CONCLUSION: RT-PCR is a suitable method for detecting ALK translocations using cytology samples from patients with primary lung cancer, especially when tissue samples are not available.

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Language：English   Publishing type：Research paper (scientific journal)  

PURPOSE: This study aimed to evaluate the clinicopathological significance of cancer stem-like cell (CSLC) markers in high-grade neuroendocrine carcinoma (HGNEC) of the lung, including small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC). METHODS: We retrospectively studied patients who underwent surgical resection of SCLC (n = 60) and LCNEC (n = 45) to analyze their clinicopathological profiles and the immunohistochemical expression of putative CSLC markers (Caveolin, Notch, CD44, CD166, SOX2, ALDH1, and Musashi1). Staining scores for these markers in tumor cells were calculated by multiplying the percentage of positive tumor cells per lesion by the staining intensity level (0, 1, and 2); a score of ≥ 10 represented positive expression. RESULTS: There was a difference between SCLC and LCNEC with respect to both SOX2 (55 vs. 27 %, p = 0.003) and CD166 (27 vs. 47 %, p = 0.034) expression. ALDH1 expression was equally observed in SCLC and LCNEC (67 vs. 73 %, p = 0.46), and patients with ALDH1-positive HGNEC had significantly worse recurrence-free survival (RFS) and overall survival (OS) rates than those with ALDH1-negative HGNEC (5-year RFS: 39 vs. 67 %, p = 0.009; 5-year OS: 50 vs. 79 %, p = 0.021). A multivariate analysis revealed that positive ALDH1 expression was an independent unfavorable prognostic factor with respect to both RFS and OS. CONCLUSIONS: The differences in the expression profiles of CSLC markers might reflect morphological differences between SCLC and LCNEC. Positive ALDH1 expression in lung HGNEC was associated with an unfavorable patient prognosis, which suggested that ALDH1-positive tumor cells might be future therapeutic targets for the treatment of lung HGNEC.

DOI： 10.1007/s00432-015-1985-3

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Cancer-associated fibroblasts (CAFs) expressing podoplanin (PDPN) are a favorable prognosticator in surgically resected small cell lung cancer (SCLC). Here we explore whether CAFs expressing PDPN influence proliferation of SCLC cells. Compared with control group (SCLC cells co-cultured with CAFs-Ctrl), numbers of SCLC cells co-cultured with CAFs overexpressing PDPN were decreased. Suppression of PDPN expression by shRNA in CAFs resulted in increased numbers of SCLC cells. In surgically resected human SCLC specimens, the frequency of Geminin-positive cancer cells was significantly higher in the cases with PDPN-positive CAFs than in the cases with PDPN-negative CAFs. Thus CAFs expressing PDPN inhibit growth of SCLC cells, suggesting that CAFs expressing PDPN represent a tumor inhibitory stromal cell component in SCLC.

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INTRODUCTION: The information regarding therapeutically relevant genomic alterations in small cell lung cancer (SCLC) is not well developed. We analyzed the SCLC genome using an integrative approach to stratify the targetable alterations. METHODS: We performed whole exon sequencing (n = 51) and copy number analysis (n =47) on surgically resected tumors and matched normal tissue samples from treatment-naive Japanese SCLC patients. RESULTS: The demographics of the 51 patients included in this study were as follows: median age, 67 years (range, 42-86 years); female, 9 (18%); history of smoking, 50 (98%); and pathological stage I/II/III/IV, 28/13/9/1, respectively. The average number of nonsynonymous mutations was 209 (range, 41-639; standard deviation, 130). We repeatedly confirmed the high prevalence of inactivating mutations in TP53 and RB1, and the amplification of MYC family members. In addition, genetic alterations in the PI3K/AKT/mTOR pathway were detected in 36% of the tumors: PIK3CA, 6%; PTEN, 4%; AKT2, 9%; AKT3, 4%; RICTOR, 9%; and mTOR, 4%. Furthermore, the individual changes in this pathway were mutually exclusive. Importantly, the SCLC cells harboring active PIK3CA mutations were potentially targetable with currently available PI3K inhibitors. CONCLUSIONS: The PI3K/AKT/mTOR pathway is distinguishable in SCLC genomic alterations. Therefore, a sequencing-based comprehensive analysis could stratify SCLC patients by potential therapeutic targets.

DOI： 10.1097/JTO.0000000000000250

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PURPOSE: The immunophenotypes of cancer stromal cells have been recognized as prognostic factors of cancer. The purpose of this study was to analyze the prognostic markers of high-grade neuroendocrine carcinomas of the lung (HGNEC; both small cell carcinoma and large cell neuroendocrine carcinoma) by examining the immunophenotypes of cancer stromal cells. MATERIALS AND METHODS: One hundred and fifteen patients who underwent a complete resection of HGNEC were included in this study. We examined the presence of CD204-positive tumor-associated macrophages (TAMs), Foxp3-positive regulatory T cells (Tregs), and podoplanin-positive cancer-associated fibroblasts (CAFs) to evaluate the prognostic values of these markers. RESULTS: The number of CD204-positive TAMs and Foxp3-positive Tregs did not influence the overall survival (OS) or the relapse-free survival (RFS) of the patients. However, patients with podoplanin-positive CAFs had a significantly better prognosis than those with podoplanin-negative CAFs [OS: p = 0.002, RFS: p = 0.002, 5-year overall survival (5YR): 74 vs. 45 %]. According to subgroup analyses, patients with podoplanin-positive CAFs displayed a better prognosis for both small cell carcinoma (OS: p = 0.046, 5YR: 74 vs. 46 %) and large cell neuroendocrine carcinoma (OS: p = 0.020, 5YR: 74 vs. 45 %). Moreover, in multivariate analyses, the podoplanin status of the CAFs was shown to be a statistically significant independent predictor of recurrence. CONCLUSION: The presence of podoplanin-positive CAFs had a favorable prognostic value, suggesting that the evaluation of podoplanin expression by CAFs would lead to a novel risk classification of patients.

DOI： 10.1007/s00432-013-1502-5

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Histological vascular invasion (VI) by tumors is reportedly a risk factor influencing recurrence or survival after surgical treatment; however, few studies have evaluated which VI features affect recurrence or survival. The objective of this study was to evaluate how VI features affect recurrence in lung adenocarcinoma patients. We selected 106 patients with pathological stage I lung adenocarcinoma who showed VI and examined the properties of intravascular tumors associated with recurrence. First we investigated the relationship between the frequency of VI in a histological cross-section and the incidence of recurrence; however, a significant impact was not observed. Microscopic examination revealed the intravascular tumors were composed of not only cancer cells but also non-cancerous cells. To examine whether the characteristics of intravascular cancer cells and/or non-cancerous cells have prognostic value, we examined the expression levels of epithelial-mesenchymal transition-related markers in cancer cells and the numbers of infiltrating non-cancerous cells, including macrophages, endothelial cells, and fibroblasts. High levels of E-cadherin expression in the intravascular cancer cells were significant predictors of recurrence (P = 0.004), whereas the expressions of CD44, CD44 variant 6, and vimentin were not. Large numbers of intravascular CD204(+) macrophages (P = 0.016), CD34(+) microvessels (P = 0.007), and α-smooth muscle actin (+) fibroblasts (P = 0.033) were also significant predictors of recurrence. Our results indicated VI with abundant stromal cell infiltrates might be a predictor of recurrence and suggested the tumor microenvironment created by cancer cells and stromal cells within the blood vessel may play an important role during the metastatic process.

DOI： 10.1111/cas.12219

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