3D collagen migration patterns reveal a SMAD3-dependent and TGF-β1-independent mechanism of recruitment for tumour-associated fibroblasts in lung adenocarcinoma

Abstract

Background The TGF-β1 transcription factor SMAD3 is epigenetically repressed in tumour-associated fibroblasts (TAFs) from lung squamous cell carcinoma (SCC) but not adenocarcinoma (ADC) patients, which elicits a compensatory increase in SMAD2 that renders SCC-TAFs less fibrotic. Here we examined the effects of altered SMAD2/3 in fibroblast migration and its impact on the desmoplastic stroma formation in lung cancer. Methods We used a microfluidic device to examine descriptors of early protrusions and subsequent migration in 3D collagen gels upon knocking down SMAD2 or SMAD3 by shRNA in control fibroblasts and TAFs. Results High SMAD3 conditions as in shSMAD2 fibroblasts and ADC-TAFs exhibited a migratory advantage in terms of protrusions (fewer and longer) and migration (faster and more directional) selectively without TGF-β1 along with Erk1/2 hyperactivation. This enhanced migration was abrogated by TGF-β1 as well as low glucose medium and the MEK inhibitor Trametinib. In contrast, high SMAD2 fibroblasts were poorly responsive to TGF-β1, high glucose and Trametinib, exhibiting impaired migration in all conditions. Conclusions The basal migration advantage of high SMAD3 fibroblasts provides a straightforward mechanism underlying the larger accumulation of TAFs previously reported in ADC compared to SCC. Moreover, our results encourage using MEK inhibitors in ADC-TAFs but not SCC-TAFs.

Aberrant TIMP‐1 production in tumor‐associated fibroblasts drives the selective benefits of nintedanib in lung adenocarcinoma

“…Likewise, our observed involvement of SMAD3 in promoting TIMP‐1 secretion more strikingly than transcription could be part of complex regulatory mechanisms affecting TIMP‐1 expression and secretion in ADC‐TAFs that remains to be fully elucidated. Interestingly, the higher levels of TIMP1 mRNA and secreted protein levels in ADC‐TAFs compared to SCC‐TAFs were attributable to the more extensive epigenetic repression of the SMAD3 promoter in SCC‐TAFs 15,40 . The heightened SMAD3 production and activation in ADC‐TAFs enhances the secretion of bioactive TGF‐β1, creating a positive feedback loop that amplifies the profibrotic activity of the TGF‐β1/SMAD3 pathway 15 .…”

“…Interestingly, the higher levels of TIMP1 mRNA and secreted protein levels in ADC‐TAFs compared to SCC‐TAFs were attributable to the more extensive epigenetic repression of the SMAD3 promoter in SCC‐TAFs. 15 , 40 The heightened SMAD3 production and activation in ADC‐TAFs enhances the secretion of bioactive TGF‐β1, creating a positive feedback loop that amplifies the profibrotic activity of the TGF‐β1/SMAD3 pathway. 15 Because we showed that nintedanib attenuates the TGF‐β1/SMAD3 pathway through the partial inhibition of the TGF‐β1 type I receptor ALK5, 15 our current findings strongly support that the hyperactive TGF‐β1/SMAD3 pathway in ADC‐TAFs is responsible for their excessive production of TIMP‐1 and subsequent selective positive response to nintedanib; conversely, the low SMAD3 expression and limited TIMP‐1 production in SCC‐TAFs may confer intrinsic resistance to this drug.…”