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

Abstract

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.

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

“…2 Several research groups, including us, have reported that Src, a non-receptor type tyrosine kinase, has a role in the anoikis resistance in lung adenocarcinomas. [3][4][5] We have demonstrated that 1) intralymphatic floating lung adenocarcinoma cells observed in primary tumor tissues form compact nests expressing E-cadherin and phosphorylated Src; 2) these two proteins are also expressed in tumor spheroids of four lung adenocarcinoma cell lines (LC-KJ, HCC827, H1650, and H1975) cultured in suspension, the shape of which is similar to intralymphatic tumor cell nests observed in tissues. 5 We thus consider tumor spheroids of the four cell lines in suspension culture system to be a suitable in vitro model of circulating tumor cells.…”

“…[3][4][5] We have demonstrated that 1) intralymphatic floating lung adenocarcinoma cells observed in primary tumor tissues form compact nests expressing E-cadherin and phosphorylated Src; 2) these two proteins are also expressed in tumor spheroids of four lung adenocarcinoma cell lines (LC-KJ, HCC827, H1650, and H1975) cultured in suspension, the shape of which is similar to intralymphatic tumor cell nests observed in tissues. 5 We thus consider tumor spheroids of the four cell lines in suspension culture system to be a suitable in vitro model of circulating tumor cells. In addition, we have shown that ABT-263, a potent Bcl-2 inhibitor, 6,7 significantly enhances the susceptibility for apoptosis in suspended lung adenocarcinoma cells treated with Src tyrosine kinase inhibitors (TKIs), 8 as ABT-737, the predecessor of ABT-263, can increase the levels of epidermal growth factor receptor (EGFR) TKI-induced apoptosis in EGFR-mutant lung adenocarcinomas.…”

“…7,9,10 However, the effect of the combination therapy of ABT-263 and a Src TKI on apoptosis in lung adenocarcinoma cells upon detachment was obtained from short-term (24-48 h) in vitro experiments. 5,8 We have not yet confirmed whether long-term treatment with a Src TKI and ABT-263 can ultimately eradicate lung adenocarcinoma cells in suspension. Clinical and experimental observations reveal that even highly effective therapies, such as EGFR TKIs for EGFR-mutant lung adenocarcinomas cannot eradicate tumor cells.…”

“…H1975 cells, which considerably resist Src or EGFR TKI therapy, contain two point mutations of T790M in exon 20 and L858R in exon 21 in the EGFR gene. 5,11,14,15 The EGFR T790M mutation may have a key role in the decreased susceptibility to Src TKIs in H1975 cells, because mutant EGFR and Src interact with each other. 16 In this study, we focused on the two EGFR-mutant lung adenocarcinoma cell lines to elucidate to what extent acute and chronic combination therapy with a Src TKI, a Bcl-2 inhibitor, and an HDAC inhibitor, or an EGFR TKI, a Bcl-2 inhibitor, and an HDAC inhibitor, can induce apoptosis in the two cell lines in suspension.…”

“…Two EGFR-mutant lung adenocarcinoma cell lines (HCC827 and H1975) cultured in either monolayer 14,15 or suspension 5 show different sensitivity to Src TKI treatment. The difference may be because of the fact that only H1975 cells have a point mutation of T790M in exon 20, which confers resistance to first-generation EGFR TKIs such as gefitinib and erlotinib; HCC827 cells, which are sensitive to Src or EGFR TKIs, harbor a deletion mutation in exon 19 (del E746-A750).…”