Shoichi Matsukuma

Distinctive evaluation of nonmucinous and mucinous subtypes of bronchioloalveolar carcinomas in EGFR and K-ras gene-mutation analyses for Japanese lung adenocarcinomas: confirmation of the correlations with histologic subtypes and gene mutations.

Although adenocarcinomas of the lung are associated with epidermal growth factor receptor (EGFR) gene mutations and sensitivity to EGFR tyrosine kinase inhibitors, it remains unclear whether bronchioloalveolar carcinoma (BAC) components and/or subtypes affect these associations. We aimed to clarify correlations between EGFR gene mutations and BAC components and to establish the histologic features as reliable predictors for the mutations. We examined 141 non-small cell lung cancers (NSCLCs), including 118 adenocarcinomas, for mutations in exons 19 and 21 of the EGFR gene together with mutations in codon 12 of the K-ras gene using loop-hybrid mobility shift assays, a highly sensitive polymerase chain reaction-based method. Adenocarcinomas were subdivided into subtypes with a nonmucinous or mucinous BAC component and those without BAC components. In NSCLCs, EGFR mutations were detected in 75 cases (53.2%) and were significantly associated with adenocarcinoma, female sex, and never smoking. Among adenocarcinomas, nonmucinous and mucinous BAC components were significantly associated with EGFR and K-ras gene mutations, respectively. Because EGFR mutations were detected even in most pure nonmucinous BACs, ie, lung adenocarcinoma in situ, EGFR mutation is considered a critical event in the pathogenesis of nonmucinous BAC tumors.

The Associations Between RAS Mutations and Clinical Characteristics in Follicular Thyroid Tumors: New Insights from a Single Center and a Large Patient Cohort

BACKGROUND Many studies on thyroid follicular tumors have reported the presence of somatic mutations to three forms of RAS: HRAS, KRAS, and NRAS. However, the frequency and clinical significance of these RAS mutations remain unclear, in large part due to the different methodologies being used for mutation analysis and the limited number of cases featured in studies. To clarify the significance of RAS mutations, we examined a large number of follicular adenomas and carcinomas obtained from a single institute using established methods for the analysis of RAS. METHODS Tumor samples from 40 follicular adenoma and 58 follicular carcinoma patients treated at the Kanagawa Cancer Center Hospital were analyzed. The three RAS mutations at codons 12 and 61 were assessed with a polymerase chain reaction-based loop-hybrid mobility shift assay followed by confirmation with direct sequencing. The relationships between mutation status and clinicopathological features at the time of the initial operation and the prognosis of the patients were also analyzed. RESULTS Twelve out of 40 (30%) adenomas harbored RAS mutations. In contrast, 33 out of 58 (57%) follicular carcinomas harbored RAS mutations, and the mutation was predominantly found in the NRAS codon 61 (22/33, 67%, p<0.01). The rate of gene mutations was significantly higher in the carcinomas than in the adenomas (p<0.01). The NRAS codon 61 mutation in follicular carcinomas was positively associated with distant metastases through the entire clinical course of the patients (p<0.05), and RAS mutations were associated with poor overall patient survival (p<0.05). CONCLUSIONS We investigated the frequency of RAS mutations in follicular thyroid tumors from a large number of cases obtained from a single institute. The predominance of NRAS codon 61 mutations as a feature of carcinomas indicates that the diagnosis of adenoma alongside the presence of this mutation should be made cautiously. Our study raises the possibility that follicular adenomas with the RAS mutations have an inherent malignant potential; however, the clinical significance of this finding should be further investigated in more patients and over a longer follow-up period.

Novel heteroduplex method using small cytology specimens with a remarkably high success rate for analysing EGFR gene mutations with a significant correlation to gefitinib efficacy in non-small-cell lung cancer

We conducted a feasibility study to examine whether small numbers of cancer cells could be utilised for analysis of the EGFR gene status using the loop-hybrid mobility shift assay, which is a modified heteroduplex technique. Cytology specimens obtained by transbronchial abrasion were successfully used for analysis of the EGFR gene status in 50 of 52 (96.2%) patients diagnosed with class V non-small-cell carcinoma. Furthermore, the relationship between the EGFR gene status and clinical outcome was analysed in 25 patients treated with gefitinib. Overall, 10 of 11 patients with EGFR mutations in exon 19 or 21 showed tumour regression with gefitinib treatment, compared to only two of 14 patients with wild-type EGFR. The response rate was significantly higher in the EGFR mutation group than in the wild-type EGFR group (90.9 vs 14.3%, P=0.00014). Logistic regression analysis revealed that EGFR mutations in cytology specimens represented an independent predictor of the gefitinib response. The overall and progression-free survivals were significantly longer in the EGFR mutation group than in the wild-type EGFR group (P<0.05). In conclusion, cytology specimens could be useful for analysing the EGFR status in the majority of patients with non-small-cell lung cancer to determine whether they are likely to benefit from gefitinib treatment.

Epidermal growth factor receptor gene mutations in atypical adenomatous hyperplasias of the lung

Activating epidermal growth factor receptor (EGFR) gene mutations are frequently detected in lung adenocarcinomas, especially adenocarcinomas with a nonmucinous bronchioloalveolar carcinoma component. EGFR-mutated lung adenocarcinomas respond well to EGFR tyrosine kinase inhibitors. We previously found that most (88%) pure nonmucinous bronchioloalveolar carcinomas (adenocarcinoma in situ) already harbor EGFR mutations, indicating that the mutations are an early genetic event in the pathogenesis. We examined 54 atypical adenomatous hyperplasias, precursor lesions of lung adenocarcinomas, obtained from 28 Japanese patients for the hotspot mutations of EGFR exons 19 and 21 and K-ras codon 12. EGFR mutations were observed in 17 of the 54 (32%) atypical adenomatous hyperplasias examined: Ten and seven atypical adenomatous hyperplasias had deletion mutations at exon 19 or point mutations (L858R) at exon 21, respectively. We did not observe apparent histological differences between atypical adenomatous hyperplasias with and without EGFR mutations. K-ras mutation (G12S) was detected in only one atypical adenomatous hyperplasia. As EGFR mutational frequency of atypical adenomatous hyperplasias was much lower than that of nonmucinous bronchioloalveolar carcinomas, we surmise that EGFR-mutated atypical adenomatous hyperplasias, but not atypical adenomatous hyperplasias with wild-type EGFR, are likely to progress to nonmucinous bronchioloalveolar carcinomas.

Enhanced autophagy is required for survival in EGFR-independent EGFR-mutant lung adenocarcinoma cells.

Lung cancers harboring epidermal growth factor receptor (EGFR) mutations depend on constitutive activation of the kinase for survival. Although most EGFR-mutant lung cancers are sensitive to EGFR tyrosine kinase inhibitors (TKIs) and shrink in response to treatment, acquired resistance to TKI therapy is common. We demonstrate here that two EGFR-mutated lung adenocarcinoma cell lines, HCC827 and HCC4006, contain a subpopulation of cells that have undergone epithelial-to-mesenchymal transition and survive independent of activated EGFR. These EGFR-independent cancer cells, herein termed gefitinib-resistant (GR) cells, demonstrate higher levels of basal autophagy than their parental cells and thrive under hypoxic, reduced-serum conditions in vitro; this somewhat simulates the hypoxic environment common to cancerous tissues. We show that depletion of the essential autophagy gene, ATG5, by small interfering RNA (siRNA) or chloroquine, an autophagy inhibitor, markedly reduces GR cell viability under hypoxic conditions. Moreover, we show a significant elevation in caspase activity in GR cells following knockdown of ATG5. These results suggest that GR cells can evade apoptosis and survive in hostile, hypoxic environments with constant autophagic flux. We also show the presence of autophagosomes in some cancer cells from patient samples, even in untreated EGFR-mutant lung cancer tissue samples. Together, our results indicate that autophagy inhibitors alone or in combination with EGFR TKIs may be an effective approach for the treatment of EGFR-mutant lung cancers, where basal autophagy of some cancer cells is upregulated.

WZ4002, a third-generation EGFR inhibitor, can overcome anoikis resistance in EGFR-mutant lung adenocarcinomas more efficiently than Src inhibitors

Src has a role in the anoikis resistance in lung adenocarcinomas. We focused on two epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cell lines, HCC827 (E746-A750 deletion) and H1975 (L858R+T790M), in suspension to elucidate whether suspended lung adenocarcinoma cells are eradicated by long-term treatment with Src tyrosine kinase inhibitors (TKIs). We also examined metastasis-positive lymph nodes from 16 EGFR-mutant lung adenocarcinoma patients for immunohistochemical expression of mutant-specific EGFR. Almost all suspended HCC827 cells underwent apoptosis after 144 h of combination treatment with AZD0530, trichostatin A (TSA), and ABT-263, whereas many suspended H1975 cells survived the treatment. AZD0530 is a Src TKI, TSA is a histone deacetylase inhibitor, and ABT-263 is a Bcl-2 inhibitor. During the therapy, the phosphorylation of EGFR decreased in HCC827 cells and remained stable in H1975 cells. The phosphorylated EGFR of Src TKI-resistant H1975 cells, as well as HCC827 cells, was completely suppressed by the third generation EGFR TKI, WZ4002. Consequently, both the suspended cell lines were almost completely eradicated within 144 h, with the combined therapy of WZ4002, ABT-263, and TSA. Interestingly, treated suspended cells underwent apoptosis to a greater extent than did adherent cells. Intrasinus floating lung adenocarcinoma cells in the lymph nodes expressed a mutant-specific EGFR. These findings suggest that suspended EGFR-mutant lung adenocarcinoma cells depend significantly more on EGFR activation for survival than attached cells do. The tumor cells circulating in vessels, which express mutant-specific EGFR, would be highly susceptible to the combination therapy of WZ4002, ABT-263, and TSA.

Mea2/Golga3 gene is disrupted in a line of transgenic mice with a reciprocal translocation between Chromosomes 5 and 19 and is responsible for a defective spermatogenesis in homozygotes

Abstract. A line of transgenic mouse T604 transmitted a transgene to progeny together with a set of chromosomes with a reciprocal translocation. The transgene was integrated at a single site in the translocated chromosomes, as revealed by fluorescence in situ hybridization. The transgenic hemizygous males, also heterozygous for the translocation of chromosomes, showed apparently normal spermatogenesis, while the males homozygous for the transgene as well as for the translocated chromosomes showed a defect in spermatogenesis. Considering that the genetic rearrangement by either insertion of the transgene or the chromosome translocation in the T604 mouse line might have caused a recessive mutation in a gene indispensable for spermatogenesis, we have mapped the transgene integration site and the translocation breakpoints in mouse chromosomes. Linkage analysis with SSLP markers showed that the loci for the transgene and the translocation breakpoints were closely located to D5Mit24 on Chromosome (Chr) 5, and to a region between D19Mit19 and D19Jpk2 on Chr 19. Mea2 gene, mapped only 2 cM from D5Mit24 and known to show male-specific enhanced expression in the testis, was analyzed as a candidate for the gene disrupted in T604 transgenic mice. Southern blot analysis revealed that Mea2 gene was indeed disrupted in T604 mice, and Northern blot analysis of the testis RNA showed that the expression of Mea2 was annihilated in the testis of T604 transgenic homozygotes.

Lung adenocarcinoma cells floating in lymphatic vessels resist anoikis by expressing phosphorylated Src.

The ability to resist anoikis is critical for carcinoma cells to metastasize. Although several lung adenocarcinoma cell lines were shown to repress anoikis through the activation of Src, it remains unknown whether Src actually plays a crucial role in anoikis resistance in lung adenocarcinoma tissues. We examined 20 human lung adenocarcinoma tissues with lymphatic permeation and nine cell lines to investigate whether intralymphatic floating carcinoma cells in the tissues, used as an in vivo model of anoikis resistance, actually suppressed anoikis and whether cell lines in suspension culture, an in vitro model of anoikis resistance, survived through Src activation. We observed that the intralymphatic carcinoma cells aggregated tightly to form nests expressing E‐cadherin and phosphorylated Src (p‐Src). The apoptotic indices of these cells were comparable to those of extracellular matrix adhesive cells in all tissues, indicating that the intralymphatic cells actually evaded anoikis. Next, we found that the nine cell lines in suspension aggregated loosely (five cell lines) or tightly (four cell lines), and all cells resisted anoikis. Upon detachment, four cell lines (LC‐KJ, HCC827, H1650, and H1975) formed compact spheroids that expressed E‐cadherin and p‐Src. The spheroids were similar to intralymphatic tumour nests and were thus considered to be a suitable model of the nests. The spheroids of the four cell lines underwent apoptosis after treatment with the Src/Abl/Kit inhibitor PP1 or Src/Abl inhibitor bosutinib. On the other hand, the Abl/Kit inhibitor imatinib did not affect cell growth or apoptosis in the four types of spheroids. These results indicate that Src, but not Abl or Kit, plays an essential role in the development of anoikis resistance in lung adenocarcinomas. Copyright

ABT-263, a Bcl-2 inhibitor, enhances the susceptibility of lung adenocarcinoma cells treated with Src inhibitors to anoikis.

The tyrosine kinase Src plays an important role in the development of anoikis resistance in lung adenocarcinomas. Several suspension lung adenocarcinoma cell lines, which express phosphorylated Src, undergo apoptosis, or anoikis, in the presence of Src kinase inhibitors. However, lung adenocarcinoma cell lines vary in their sensitivity to Src inhibitors. We hypothesized that the addition of ABT-263, a potent Bcl-2 inhibitor, should significantly enhance the degree of anoikis in lung adenocarcinoma cells treated with Src inhibitors. In this study, we treated four suspension lung adenocarcinoma cell lines with ABT-263, an Src inhibitor (bosutinib or PP1), or a combination of both. In LC-KJ and HCC827 cells, combined treatment with ABT-263 and an Src inhibitor effectively induced anoikis, and the extent of anoikis was significantly greater than that induced by each agent alone; the synergy between the two drugs was apparent. Although we did not observe a marked increase in anoikis in LC-KJ and HCC827 cells treated with ABT-263 alone, H1650 and H1975 cells treated with ABT-263 (1 μM) upon detachment significantly underwent apoptosis, the levels of which were much greater than those observed upon attachment. However, the levels of anoikis induced by combination treatment were still greater than those by the individual agents in H1650 and H1975 cells. These findings provide a biological rationale to test combination therapy with ABT-263 and Src inhibitors in patients with lung adenocarcinoma.

NF-κB signaling is activated and confers resistance to apoptosis in three-dimensionally cultured EGFR-mutant lung adenocarcinoma cells.

Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells in suspension undergo apoptosis to a greater extent than adherent cells in a monolayer when EGFR autophosphorylation is inhibited by EGFR tyrosine kinase inhibitors (TKIs). This suggests that cell adhesion to a culture dish may activate an anti-apoptotic signaling pathway other than the EGFR pathway. Since the microenvironment of cells cultured in a monolayer are substantially different to that of cells existing in three-dimension (3D) in vivo, we assessed whether two EGFR-mutant lung adenocarcinoma cell lines, HCC827 and H1975, were more resistant to EGFR TKI-induced apoptosis when cultured in a 3D extracellular matrix (ECM) as compared with in suspension. The ECM-adherent EGFR-mutant cells in 3D were significantly less sensitive to treatment with WZ4002, an EGFR TKI, than the suspended cells. Further, a marked degradation of IκBα, the inhibitor of nuclear factor (NF)-κB, was observed only in the 3D-cultured cells, leading to an increase in the activation of NF-κB. Moreover, the inhibition of NF-κB with pharmacological inhibitors enhanced EGFR TKI-induced apoptosis in 3D-cultured EGFR-mutant cells. These results suggest that inhibition of NF-κB signaling would render ECM-adherent EGFR-mutant lung adenocarcinoma cells in vivo more susceptible to EGFR TKI-induced cell death.