Non-canonical functions of SNAIL drive context-specific cancer progression

SNAIL-driven cancer progression is highly context-specific

To investigate the function of SNAIL in different cancer types in vivo, we created a latent Snail allele silenced by a lox-stop-lox (LSL) cassette as a knock-in (KI) at the mouse Rosa26 locus (LSL-R26Snail/+ mouse line, termed SnailKI/+; Supplementary Fig. S1a–c). SNAIL expression was then activated in several genetically engineered murine autochthonous cancer models: i) a PDAC model that depends on Cre-induced expression of oncogenic KRASG12D in the Ptf1a lineage of the pancreas (Ptf1aCre/+;LSL-KrasG12D/+, termed PKrasG12D/+); ii) a classical WNT-driven intestinal cancer model, induced by loss of the tumour suppressor adenomatosis polyposis coli (Apc) due to Cre-mediated deletion of a floxed Apc allele in intestinal epithelial cells (Villin-Cre;Apclox/+, termed VApcΔInt); and iii) two different models of serrated intestinal cancer driven by either oncogenic KRASG12D or BRAFV637E, based on Villin-Cre-induced activation of latent oncogenic KrasG12D (Villin-Cre;LSL-KrasG12D/+ termed VKrasG12D/+) or BrafV637E (Villin-Cre;LSL-BrafV637E/+, termed VBrafV637E/+). In this way we mimicked the acquisition of SNAIL expression in different tumour types and subtypes driven by distinct oncogenes and signalling pathways.

Concomitant transgenic expression of SNAIL and activation of oncogenic KRASG12D in the pancreas of Ptf1aCre/+;LSL-KrasG12D/+;LSL-R26Snail/+ mice (termed PKrasG12D/+;SnailKI/+) accelerated the formation of acinar to ductal metaplasia (ADM) and PDAC precursor lesions (pancreatic intraepithelial neoplasia (PanIN)), and substantially increased cancer development (Fig. 1a–h and Supplementary Fig. S1d–f). Consistent with this, PDAC gene set enrichment was already apparent in one-month old mice with aberrant SNAIL expression (Fig. 1f). All animals in the tumour watch cohort developed PDAC with a median survival of 190 days, compared to 465 days in PKrasG12D/+ animals. Biallelic Snail expression in Ptf1aCre/+;LSL-KrasG12D/+,LSL-R26Snail/Snail mice (termed PKrasG12D/+;SnailKI/KI) drastically reduced survival further to a median of only 64 days (Fig. 1g, h).

To assess the impact of SNAIL on tumour development, progression and survival of intestinal cancer, where SNAIL is aberrantly expressed in 78% of cases18, we used three different genetically engineered models that mirror major histopathological and molecular disease subtypes19,20,21 (Fig. 2 and Supplementary Fig. S2). Surprisingly, in the classical Apc loss-of-function model (VApcΔInt) that is driven by activation of canonical WNT signalling20, there was a trend towards prolonged survival (median survival of 419 vs. 355 days; p = 0.12) and reduced number of adenomas and carcinomas per animal upon aberrant SNAIL expression (Fig. 2a–e; p = 0.08 and 0.14, respectively). SNAIL had no major effect on tumour morphology and histopathology (Fig. 2f). This stands in sharp contrast to the dramatic pro-tumorigenic effects observed in the KRAS-driven PDAC model and suggests that SNAIL has context-specific functions in different tumour entities and/or oncogenic backgrounds. To test the hypothesis that SNAIL cooperates specifically with KRAS, but not WNT pathway activation, to induce cancer progression across tumour entities, we activated SNAIL in vivo in a KRASG12D-driven model of serrated intestinal cancer (Fig. 2g). This specific CRC subtype is characterized by a serrated histopathological morphology and progresses through a hyperplasia - serrated adenoma - serrated carcinoma sequence distinct from the classical WNT-driven CRC progression model described by Vogelstein and colleagues22, which is characterized by an adenoma-carcinoma sequence without hyperplasia and serrated morphology23,24. Unexpectedly, aberrant SNAIL expression failed to accelerate oncogenesis. Instead, we observed a weak trend towards prolonged survival in the KRAS-driven intestinal cancer model (median survival of 502 days (VKrasG12D/+;SnailKI/+) vs 354 days (VKrasG12D/+); p = 0.29; Fig. 2h, i). Accordingly, we detected no obvious change in the number of adenomas and carcinomas per animal, as well as in the grading of the tumours (Fig. 2j–n). These data indicate that the tissue of origin and not the oncogenic driver dictates the functional role of SNAIL in tumour progression in vivo.

In the BrafV637E-driven model of serrated intestinal cancer, we observed more invasive carcinomas in animals with aberrant SNAIL expression (Villin-Cre;LSL-BrafV637E/+;SnailKI/+; termed VBrafV637E/+;SnailKI/+) than in VBrafV637E/+ animals, and also more adenomas per animal (Supplementary Fig. S2a–f). Median survival was reduced to 392 days in the VBrafV637E/+;SnailKI/+ cohort vs. 481 days in VBrafV637E/+; mice (Supplementary Fig. S2b; p = 0.0321). However, there was no overt consistent change in the grade of carcinomas observed towards more undifferentiated tumours in any of the three intestinal cancer models (Fig. 2f, m, n and Supplementary Fig. S2f).