Abstract 5849: SMAD3 in tumor associated fibroblasts drives enhanced fibroblast accumulation in lung adenocarcinoma through increased migration

Lung cancer is characterized by a stiff fibrotic microenvironment rich in activated/pro-fibrotic tumor-associated fibroblasts (TAFs). We previously reported a larger accumulation of TAFs in lung adenocarcinoma (ADC) compared to lung squamous cell carcinoma (SCC), the two most frequent lung cancer subtypes. However, the underlying mechanisms remain elusive. TAF accumulation is largely contributed by the proliferation and/or migration of resident fibroblasts. Moreover, we recently showed that SMAD3 is epigenetically repressed in SCC-TAFs compared to ADC-TAFs owing to an excessive exposure to cigarette smoke particles, which elicited a compensatory increase in its closely related homolog SMAD2 in SCC-TAFs. However, it remains unknown whether the differential SMAD2/3 expression between ADC- and SCC-TAFs contributes to the larger accumulation of TAFs in ADC. To address this question, we knocked-down SMAD2 or SMAD3 in control pulmonary fibroblasts by shRNA and used them as ADC-like or SCC-like models. To assess proliferation, we examined cell number density and found that shSMAD2 (ADC-like) fibroblasts exhibited a significantly lower number density in basal conditions compared to shSMAD3 (SCC-like) fibroblasts, which was confirmed in TAFs. In the presence of TGF-β1, number density increased and attained similar values in shSMAD2 and shSMAD3 fibroblasts as well as in ADC- and SCC-TAFs. To assess fibroblast migration, we used a microfluidic device to quantify biophysical descriptors of protrusions and subsequent migration within a 3D collagen culture. Notably, both protrusions and migration descriptors were increased in basal conditions selectively in shSMAD2 (ADC-like) fibroblasts concomitantly with Erk1/2 hyperactivation This enhanced migration was abrogated by TGF-β1 as well as by the MEK inhibitor Trametinib. In contrast, high SMAD2 fibroblasts were poorly responsive to TGF-β1 and Trametinib, exhibiting impaired migration in all conditions. Consistent results were observed in ADC-TAFs upon knocking-down SMAD3 as well as using the Transwell migration Boyden assay, thereby revealing that the enhanced migration in high SMAD3 conditions as in shSMAD2 and ADC-TAFs is caused by an intrinsic migratory priming. These results reveal that altered SMAD2/3 expression provide growth and migration advantages only in the absence of TGF-β1, although in opposing directions, since growth advantage was observed in high SMAD2 conditions (as in SCC), whereas migration advantage was found in high SMAD3 conditions (as in ADC). These findings strongly support that the larger TAF accumulation in ADC occurs at early stages (under low TGF-β1) and is driven by the enhanced migration of ADC-TAFs due to their high SMAD3 and Erk1/2 activation. Moreover, our results encourage the therapeutic use of MEK inhibitors in ADC-TAFs but not SCC-TAFs. Citation Format: Natalia Isabel Diaz Valdivia, Yago Juste Lanas, Alejandro Llorente, Rafael Ikemori, Alejandro Bernardo, Noemi Reguart, Jose M. Garcia Aznar, Jordi Alcaraz. SMAD3 in tumor associated fibroblasts drives enhanced fibroblast accumulation in lung adenocarcinoma through increased migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5849.