BRAF V600E promotes anchorage-independent growth but inhibits anchorage-dependent growth in hTERT / Cdk4-Immortalized normal human bronchial epithelial cells

Certain oncogenes, including mutant RAS and BRAF , induce a type of senescence known as oncogene-induced senescence (OIS) in normal cells in a cell-type-specific manner. OIS serves as a barrier to transformation by activated oncogenes. Our previous studies showed that mutant KRAS V12 did not efficiently induce OIS in an hTERT/Cdk4 -immortalized normal human bronchial epithelial cell line (HBEC3), but it did enhance both anchorage-dependent and anchorage-independent growth. In this study, we investigated whether mutant BRAF , a well-known inducer of OIS, could trigger OIS in HBEC3 cells. We also assessed the impact of mutant BRAF on the growth of HBEC3 cells, as no previous studies have examined this using a normal bronchial epithelial cell line model. We established an HBEC3 cell line, designated as HBEC3-BIN, that expresses mutant BRAF V600E in a doxycycline-regulated manner. Unlike our previous finding that KRAS V12 upregulated both pERK and pAKT, mutant BRAF V600E upregulated pERK but not pAKT in HBEC3-BIN cells. Similar to KRAS V12 , BRAF V600E did not efficiently induce OIS. Interestingly, while BRAF V600E inhibited colony formation in anchorage-dependent conditions, it dramatically enhanced colony formation in anchorage-independent conditions in HBEC3-BIN. In HBEC3 cells without BRAF V600E or KRAS V12 expression, p21 was only detected in the cytoplasm, and its localization was not altered by the expression of BRAF V600E or KRAS V12 . Next-generation sequencing analysis revealed an enrichment of gene sets known to be involved in carcinogenesis, including IL3/JAK/STAT3, IL2, STAT5, and the EMT pathway. Our results indicate that, unlike KRAS V12 , which promoted both, BRAF V600E enhances anchorage-independent growth but inhibits anchorage-dependent growth of HBEC3. This contrast may result from differences in activation signaling in the downstream pathways. Furthermore, HBEC3 cells appear to be inherently resistant to OIS, which may be partly due to the fact that p21 remains localized in the cytoplasm upon expression of BRAF V600E or KRAS V12 .