Non-canonical functions of SNAIL drive context-specific cancer progression
Issuing time:2023-03-15 14:23
Abstract
SNAIL is a key transcriptional regulator in embryonic development and cancer. Its effects in physiology and disease are believed to be linked to its role as a master regulator of epithelial-to-mesenchymal transition (EMT). Here, we report EMT-independent oncogenic SNAIL functions in cancer. Using genetic models, we systematically interrogated SNAIL effects in various oncogenic backgrounds and tissue types. SNAIL-related phenotypes displayed remarkable tissue- and genetic context-dependencies, ranging from protective effects as observed in KRAS- or WNT-driven intestinal cancers, to dramatic acceleration of tumorigenesis, as shown in KRAS-induced pancreatic cancer. Unexpectedly, SNAIL-driven oncogenesis was not associated with E-cadherin downregulation or induction of an overt EMT program. Instead, we show that SNAIL induces bypass of senescence and cell cycle progression through p16INK4A-independent inactivation of the Retinoblastoma (RB)-restriction checkpoint. Collectively, our work identifies non-canonical EMT-independent functions of SNAIL and unravel its complex context-dependent role in cancer.
Introduction
SNAIL is overexpressed in > 70% of human pancreatic ductal adenocarcinomas (PDAC)1 and a wide number of other tumour entities, such as intestinal, breast, lung and liver cancer2,3,4,5,6. SNAIL expression is believed to be a key driver of tumour aggressiveness and metastasis formation via the induction of an epithelial-to-mesenchymal transition (EMT) program and the subsequent acquisition of stem cell-like features5,7,8,9,10,11,12. Accordingly, it is often correlated with poor prognosis and shortened survival of cancer patients. However, the specific in vivo functions of SNAIL and the role of EMT during tumour progression in different tumour types remain largely unexplored10,13,14,15,16. In an in vivo selection model of highly metastatic PDAC cells, we have demonstrated previously that SNAIL drives EMT and subsequently metastasis formation17. This runs counter to recent findings in an autochthonous mouse model of PDAC showing that Snail deletion does not influence the metastatic phenotype, but sensitizes tumours to chemotherapy13. This prompted us to re-investigate and mechanistically probe the function of SNAIL in vivo by systematic and comprehensive genetic gain- and loss-of-function approaches using a variety of disease-relevant genetically engineered autochthonous mouse models as well as human cancers.
Here, we show context-dependent oncogenic functions of the transcriptional regulator SNAIL in cancer, which are independent of its role as a regulator of the EMT process. SNAIL-induced phenotypes depend on both, the genetic context of the tumour and its tissue of origin, ranging from protective effects that delay tumour onset in intestinal cancer models to a dramatic acceleration of pancreatic cancer development and aggressiveness. Mechanistically, we demonstrate that SNAIL acts as transcriptional activator that bypasses oncogenic KRAS-induced senescence and drives the cell cycle by p16INK4A-independent inactivation of the Retinoblastoma (RB)-restriction checkpoint of senescence, thereby inducing context-dependent cancer progression. This knowledge provides opportunities to target SNAIL-driven cancers, which are a major clinical problem due to their high aggressiveness and lethality.
Results
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).
a Genetic strategy to activate SNAIL and KRASG12D expression in the pancreas. b Immunoblot analysis of SNAIL protein expression in pancreas of 1-month-old Ptf1aCre/+;LSL-KrasG12D/+ (PKrasG12D/+) and Ptf1aCre/+;LSL-KrasG12D/+;LSL-R26Snail/+ (PKrasG12D/+;SnailKI/+) compound mutant mice. c Representative hematoxylin and eosin (H&E), Alcian blue (AB), Muc5a, CK19 and BrdU stains of acinar to ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) in 1-month-old PKrasG12D/+ and PKrasG12D/+;SnailKI/+ mice. Note the almost complete remodelling of pancreatic tissue in PKrasG12D/+;SnailKI/+ animals. Scale bars, 50 μm. d Quantification of ADM and PanIN progression in % of total lesions at age of one-month (error bars, mean ± SEM; n = 4 per genotype; 3 representative slides per mouse; *p = 0.037, Mann-Whitney two-tailed test). e Quantification of ductal and acinar structures after in vitro culture of acinar explants for 5 days (n = 3 control/PKrasG12D/+ mice, n = 5 PKrasG12D/+;SnailKI/+ mice; mean ± SEM; *p = 0.036, Mann-Whitney two-tailed test). f Gene-set enrichment analysis (GSEA) using mRNA expression profiles of PKrasG12D/+;SnailKI/+ (red) and PKrasG12D/+ (blue) pancreata of 1-month-old mice (n = 2 per genotype) computed and corrected for multiple testing using the Benjamini–Hochberg procedure (for statistical details, please see methods section). Genes were ranked using Signal-to-Noise ratio statistics according to their correlation. Vertical black lines mark the position of each gene in the data set. Normalized Enrichment Score: 3.15; Nominal p-value < 0.0001; False Discovery Rate (FDR) q-value < 0.0001. g Kaplan-Meier survival curves of PKrasG12D/+ (n = 125; 465 days median survival), PKrasG12D/+;SnailKI/+ (n = 42; 190 days median survival) and PKrasG12D/+;SnailKI/KI (n = 28; 64 days median survival) mice (***p < 0.0001, log-rank test, Bonferroni correction). h Macroscopic view and representative H&E and BrdU staining of pancreata with PDAC of three endpoint mice per genotype (tu tumour; st stomach; sp spleen). Scale bars, 50 µm. Note: The PKrasG12D/+ cohort in (g) is the same shown in Figs. 4b, 6e, f and the PKrasG12D/+;SnailKI/+ cohort is the same shown in Figs. 6e, l. Source data of Fig. 1 are provided in the Source Data file.
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.
a Strategy to activate Snail expression in intestinal epithelium in the Villin-Cre;Apclox/+ (termed VApcΔInt) model of intestinal cancer. b Kaplan-Meier survival curves of Villin-Cre;Apclox/+;LSL-R26Snail/+ (termed VApcΔInt;SnailKI/+; n = 10, median survival 419 days) and VApcΔInt mice (n = 8, median survival 355 days); p = 0.12, log-rank test. c qRT-PCR of Snail mRNA expression normalized to Cyclophilin A in intestinal tumours of VApcΔInt (n = 3) and VApcΔInt;SnailKI/+ (n = 4) endpoint mice (Mean ± SEM; **p = 0.0035, unpaired two-tailed t-test with We’ch’s correction). d Number of adenomas in VApcΔInt (n = 8) and VApcΔInt;SnailKI/+ (n = 9) endpoint mice. Mean ± SEM, p = 0.08, Mann-Whitney two-tailed test. e Percentage of carcinoma-bearing mice (left) and carcinoma number (right) in VApcΔInt (n = 8) and VApcΔInt;SnailKI/+ (n = 9) endpoint mice. Left, two-tailed Fisher’s exact test, p > 0.9999; right, Mann-Whitney two-tailed test, mean ± SEM, p = 0.14. f Representative H&E and Ki67 staining of invasive intestinal carcinoma of three VApcΔInt and VApcΔInt;SnailKI/+ mice. g Strategy to express Snail in intestinal epithelium in the Villin-Cre;LSL-KrasG12D/+ model (termed VKrasG12D/+) of intestinal cancer. h Kaplan-Meier survival curves of VKrasG12D/+ (n = 5, median survival 354 days) and Villin-Cre;KrasG12D/+;LSL-R26Snail/+ (termed VKrasG12D/+;SnailKI/+) mice (n = 5, median survival 502 days); p = 0.29, log-rank test. i qRT-PCR of Snail mRNA expression in the intestine of VKrasG12D/+ (n = 5) and VKrasG12D/+;SnailKI/+ endpoint mice (n = 3). Mean ± SEM, **p = 0.005, unpaired two-tailed t-test with Welch’s correction. j Number of adenomas in VKrasG12D/+ (n = 5) and VKrasG12D/+;SnailKI/+ (n = 5) endpoint mice. Mean ± SEM, Mann-Whitney two-tailed test; p = 0.795 (ns, not significant). k Representative H&E and Ki67 staining of intestinal adenoma in three VKrasG12D/+ and VkrasG12D/+;SnailKI/+ mice. l Percentage of carcinoma-bearing mice (left) and carcinoma number (right) in VKrasG12D/+ (n = 5) and VKrasG12D/+;SnailKI/+ (n = 5) endpoint mice. Left, two-tailed Fisher’s exact test (p > 0.9999); right, two-tailed Student’s t-test (p = 0.72), mean ± SEM. m Representative H&E staining of intestinal carcinoma in two VKrasG12D/+ and three VKrasG12D/+;SnailKI/+ mice. n Pathological grading of intestinal carcinomas from VApcΔInt (n = 44), VApcΔInt;SnailKI/+ (n = 21), VKrasG12D/+ (n = 2), VKrasG12D/+;SnailKI/+ (n = 4), VBrafV637E/+ (n = 8) and VBrafV637E/+;SnailKI/+ (n = 6) mice; two-tailed Fisher’s exact test. FC, fold change; ns, not significant; scale bars, 50μm for all images. Source data are provided in the Source Data file.
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).
These observations support the notion that SNAIL acts as a classical cancer-promoting oncogene particularly in KRAS-driven pancreatic and to a far lesser extent in BRAF-driven intestinal cancer, demonstrating its context-specific functions in cancer.
Snail activation fails to repress E-cadherin and does not induce an EMT program in PDAC
SNAIL is a master regulator of EMT that is associated with cancer aggressiveness, metastasis and decreased patient survival in various cancer types, such as PDAC5,7,14,25,26. We therefore analysed the effect of Snail activation on morphological and transcriptional EMT readouts in our autochthonous cancer models in vivo.
H&E, E-cadherin (Cdh1) and CK19 stainings revealed differentiated and undifferentiated cancers regardless of genotype in our three different PDAC models (Fig. 3a, b and Supplementary Fig. 3a–c). However, histopathological grading indicated a trend towards less undifferentiated/sarcomatoid cancers (Grade 4) in the PKrasG12D/+;SnailKI model in a Snail gene dose-dependent fashion (Fig. 3a and Supplementary Fig. S3a–c). Morphologically, PKrasG12D/+;SnailKI/+ and PKrasG12D/+;SnailKI/KI mice displayed a phenotype characterized by budding of epithelial tumour cells with the formation of small solid tumour cell nests (Supplementary Fig. S3b, c). These areas retained epithelial differentiation, visible as CK19 and E-cadherin positivity, adjacent to tubular ductal structures (Supplementary Fig. S3b). Aberrant SNAIL expression therefore did not drive PDAC development to a more undifferentiated or sarcomatoid phenotype.
a Grading of PKrasG12D/+ (n = 32), PKrasG12D/+;SnailKI/+ (n = 19) and PKrasG12D/+;SnailKI/KI PDAC mice (n = 17). Grade 4=undifferentiated/sarcomatoid86. b Representative staining of SNAIL and E-cadherin in PDAC sections of endpoint mice (n = 3 per genotype). c E-cadherin western blot of pancreas of 1-month-old mice (n = 2 per genotype). d qRT-PCR of Cdh1 mRNA expression normalized to Cyclophilin A (CypA) in pancreas of 1-month-old mice (Control, n = 3; PKrasG12D/+n = 5; PKrasG12D/+;SnailKI/+n = 6; PKrasG12D/+;SnailKI/KIn = 4). Mean ± SEM, unpaired two-tailed t-test with Welch’s and Bonferroni correction. e qRT-PCR of Snail (left) and Cdh1 (right) mRNA expression of PDAC cells with or without transgenic Snail expression (epi, epithelial (n = 27); mes, mesenchymal (n = 11)). Each dot represents one PDAC cell line. Mean ± SEM. ***p = 0.0005, unpaired two-tailed t-test with Welch’s correction. f Percentage of PKrasG12D/+ (n = 20), PKrasG12D/+;SnailKI/+ (n = 12) and PKrasG12D/+;SnailKI/KI (n = 8) PDAC cell lines with indicated morphology. g E-cadherin immunocytochemistry (green) of PDAC cells with or without transgenic Snail expression (n = 3 independent experiments). DAPI counterstain (blue). h SNAIL and E-cadherin western blot of PKrasG12D/+;SnailKI/+ (n = 2), PKrasG12D/+;SnailKI/KI (n = 1) (left) and Snail-transduced (RCAS-TVA system) PDAC cells (right) (n = 1). i PDAC cells from PKrasG12D/+, PKrasG12D/+;SnailKI/+ and floxed PKrasG12D/+;SnailKO/KO knock-out (KO) mice (n = 2 per genotype) treated with TGFβ for 72 h. j Total liver (left) and lung (right) metastasis rate of endpoint PKrasG12D/+ (n = 16), PKrasG12D/+;SnailKI/+ (n = 17) and PKrasG12D/+;SnailKI/KI (n = 17) PDAC mice. **p = 0.01 (liver) and p = 0.005 (lung), two-tailed Fisher’s exact test with Bonferroni correction. Nd. Not detected. k Representative H&E, CK19 and Ki67 staining of liver and lung metastases of three PKrasG12D/+;SnailKI/+ mice. l Representative E-cadherin staining of intestinal tumours of VApcΔInt and VApcΔInt;SnailKI/+ endpoint mice (n = 3 per genotype). m qRT-PCR of Cdh1 mRNA expression in intestinal tumours of VApcΔInt (n = 3) and VApcΔInt;SnailKI/+ (n = 4) endpoint mice. Mean ± SEM, unpaired two-tailed t-test with Welch’s correction. n–o qRT-PCR of Cdh1 mRNA expression in colon samples of (n) VKrasG12D/+ (n = 5) and VKrasG12D/+;SnailKI/+ (n = 3), and (o) VBrafV637E/+ (n = 5) and VBrafV637E/+;SnailKI/+ (n = 7) mice. Mean ±SEM, unpaired two-tailed t-test with Welch’s correction. FC Fold change, ns not significant; scale bars 50 μm. Source data are provided in the Source Data file.
SNAIL is known to repress the transmembrane glycoprotein CDH1 (E-cadherin) in several contexts, thereby inducing EMT, migration, invasion and metastasis7,27. Surprisingly, expression of SNAIL had no effect on CDH1 expression in vivo, nor in cultured primary PDAC cell isolates in vitro (Fig. 3b–e). E-cadherin protein and mRNA expression levels were comparable in cells with and without transgenic Snail expression (Fig. 3b–e). There was a marked decrease in the proportion of mesenchymal, and an increase in the epithelial phenotype in tumour cell lines from the PKrasG12D/+;SnailKI/+ model compared to PKrasG12D/+ controls (Fig. 3f and Supplementary Fig. S3d). Thus, there was no shift to a mesenchymal phenotype in the Snail-transgenic cell lines, not even with biallelic Snail expression, although we observed a > 5-fold higher expression of Snail in mesenchymal vs. epithelial PDAC cells without transgene (Fig. 3e, f and Supplementary Fig. S3d). Furthermore, global mRNA expression profiles revealed no EMT-related signatures and epithelial cancer cell isolates from PKrasG12D/+;SnailKI transgenic mice clustered exclusively with epithelial PDAC cells from PKrasG12D/+-driven models, irrespectively of the Trp53 mutational status (Supplementary Fig. S3e). To activate SNAIL expression in established epithelial PDAC cells, we transduced them with a retroviral Snail expression cassette. E-cadherin localization, expression levels, and cell morphology remained unchanged (Fig. 3g, h, Supplementary Fig. S3f), indicating that SNAIL expression alone is not sufficient to induce an overt EMT program in PDAC cells. To investigate whether SNAIL expression influences the ability of PDAC cells to undergo EMT, epithelial PDAC cells isolated from PKrasG12D/+, PKrasG12D/+;SnailKI/+ and from conditional pancreas-specific Snail knock-out (KO) mice (PKrasG12D/+;SnailKO/KO; see also Fig. 4a–d) were treated with the strong EMT-inducer TGFβ. All TGFβ-treated cells, regardless of genotype, underwent rapid EMT and displayed mesenchymal morphology (Fig. 3i), providing genetic evidence that SNAIL is dispensable for EMT-induction.
a Upper panel: Genetic strategy to conditionally delete a floxed Snail allele (Snaillox) in the pancreas of KrasG12D expressing mice. Lower panel: Genotyping PCR to test Snail-deletion (Snail-KO) using DNA from PDAC cells (cells) and tumour tissue with non-recombined stroma (tu) of PKrasG12D/+ and PKrasG12D/+;SnailKO/KO mice (n = 2 per genotype). Lower left panel: Floxed Snail allele (fl): 480 bp, Snail WT allele (WT): 395 bp, deleted Snail allele: no band. Lower right panel: deleted Snail allele (del): 492 bp; floxed Snail and WT allele: no band. b Kaplan-Meier survival curves of indicated genotypes of PKrasG12D/+;SnailKO/KO mice (n = 8; median survival 380 days), compared to PKrasG12D/+ (n = 125; median survival 465 days). ns, not significant, log-rank test. c Representative H&E-stained PDAC tissue sections of PKrasG12D/+;SnailKO/KO mice with undifferentiated (upper panel) and differentiated (lower panel) morphology (n = 6). d Pathological grading of PdACs of PKrasG12D/+ (n = 32) and PKrasG12D/+;SnailKO/KO mice (n = 6). e Genetic strategy to conditionally delete Cdh1 and express Snail in the pancreas of PKrasG12D/+ mice. f Kaplan-Meier survival curves of indicated genotypes of Pdx1-Cre;KrasG12D/+;SnailKI/+;Cdh1KO/+ (n = 8; median survival 78 days), compared to Pdx1-Cre;KrasG12D/+;SnailKI/+ mice (n = 8; median survival 166 days). ***p = 0.0008, log rank test. g qPCR analysis of Cdh1 mRNA expression in PDACs of Pdx1-Cre;KrasG12D/+;SnailKI/+;Cdh1KO/+ (n = 5) and Pdx1-Cre;KrasG12D/+;SnailKI/+ (n = 3) endpoint mice. Cdh1 mRNA levels were normalized to Cyclophilin A. Mean ± SEM, *p = 0.036, Mann-Whitney two-tailed test. FC, fold change. h Representative H&E-stained PDAC tissue sections of indicated Pdx1-Cre;KrasG12D/+;SnailKI/+ and Pdx1-Cre;KrasG12D/+;SnailKI/+;Cdh1KO/+ mice with differentiated and undifferentiated morphology (n = 8 per genotype). i Pathological grading of PDACs in Pdx1-Cre;KrasG12D/+;SnailKI/+ (n = 8) and Pdx1-Cre;KrasG12D/+;SnailKI/+;Cdh1KO/+ mice (n = 8). Note: The PKrasG12D/+ cohort in (b) is the same shown in Figs. 1g, 6e, f. Source data of Fig. 4 are provided in the Source Data file.
Beside the unchanged tumour differentiation status, what is also surprising is that aberrant SNAIL expression did not increase metastasis into liver and lung. Indeed, heterozygous transgenic PKras;SnailKI/+ mice had a trend towards reduced metastases, and biallellic PKras;SnailKI/KI mice significantly fewer (Fig. 3j, k). The capacity of autochthonous tumours to metastasize to liver and lung is thus independent of SNAIL expression, as demonstrated earlier13.
Results from the three different models of intestinal cancer were notably similar. Adenomas and carcinomas were mainly well differentiated and showed no histopathological signs or features of EMT induction regardless of SNAIL expression. Consistent with this, E-cadherin expression was retained in SNAIL-expressing adenomas and carcinomas (Fig. 3l–o), indicating that aberrant SNAIL expression is insufficient to drive a full EMT program in the intestinal epithelium.
To validate our findings in genetic loss-of-function models in vivo, we deleted Snail in KRAS-driven PDAC using the Cre/loxP system (Fig. 4a). As previously demonstrated13, this did not significantly alter PDAC development. Snail knock-out animals displayed similar tumour burden and overall survival to control mice (Fig. 4b–d). Importantly, and in line with our previous findings, loss of Snail did not drive PDAC to a well-differentiated epithelial phenotype nor block development of undifferentiated cancers that had already undergone an EMT program (Fig. 4c, d). Deletion of Snail does not therefore block EMT in vitro or in vivo.
Using a genetic approach to assess E-cadherin/Cdh1 function in the aberrant Snail expression model, we knocked out one floxed allele of the Cdh1 tumour suppressor in Pdx1-Cre;KrasG12D/+;SnailKI/+ mice (Fig. 4e). E-cadherin mRNA levels were reduced in Pdx1-Cre;KrasG12D/+;SnailKI/+;Cdh1lox/+ animals, which showed dramatically shortened median survival (78 days) compared to Pdx1-Cre; KrasG12D/+;SnailKI/+ (166 days) mice (Fig. 4f, g) and a clear shift of the tumours towards an undifferentiated mesenchymal phenotype (Fig. 4h, i). These data reveal that E-cadherin expression and function is independent of SNAIL in PDAC, and suppresses tumour progression and mesenchymal transition in vivo.
SNAIL bypasses senescence during pancreatic carcinogenesis
To discern how aberrant SNAIL expression promotes rapid tumour progression in the PKrasG12D/+;SnailKI/+ PDAC model, independent of overt EMT induction, we investigated early tumour barriers and events in tumour formation. Oncogene-induced senescence (OIS), a feature of KRAS-driven premalignant PanIN lesions of the pancreas28,29, is a cellular stress response, which blocks proliferation so protecting cells from neoplastic transformation30. As reported previously, nearly all PanINs of the PKrasG12D/+ model displayed positive senescence-associated β-galactosidase (SA-β-gal) staining, the most reliable marker of OIS in the pancreas31. In contrast, we observed a dramatically reduced rate of OIS in PKrasG12D/+;SnailKI/+ mice (Fig. 5a, b), which displayed an almost complete loss of OIS in premalignant PanIN lesions (Fig. 5b). In addition, we observed loss of senescence-associated gene sets controlled by the retinoblastoma (RB) tumour suppressor gene in expression profiles of PanIN bearing PKrasG12D/+;SnailKI/+ pancreata (Fig. 5c). In contrast, deletion of Snail in the PKrasG12D/+ model (PKrasG12D/+;SnailKO/KO) induced a strong senescence phenotype as evidenced by SA-β-gal staining of PanIN bearing pancreatic tissue sections (Fig. 5d), indicating that SNAIL is indeed capable of impacting OIS.
a Representative images of senescence-associated β-galactosidase (SA-β-gal) staining of pancreata with PanIN lesions of PKrasG12D/+ (n = 13) and PKrasG12D/+;SnailKI/+ (n = 12) mice. Scale bars, 50 μm. LG, low grade; HG, high grade. b Quantification of SA-β-gal-stained PanIN lesions from PKrasG12D/+ (n = 13) and PKrasG12D/+;SnailKI/+ (n = 12) mice. Mean ± SEM, *p < 0.0001, Mann-Whitney two-tailed test. LG, low grade; HG, high grade. c Gene set enrichment analysis (GSEA) of mRNA expression profiling of 1-month-old mice (n = 2 per genotype) computed and corrected for multiple testing using the Benjamini–Hochberg procedure (for statistical details, please see methods section) shows significant enrichment of Rb1 targets senescent genes (CHICAS_RB1_TARGETS_SENESCENT) in PKrasG12D/+;SnailKI/+ (red) vs. PKrasG12D/+ (blue) pancreata. Normalized Enrichment Score: 2.68; Nominal p-value < 0.001; False Discovery Rate (FDR) q-value < 0.001. d Representative images of SA-β-gal staining of pancreata with PanIN lesions of PKrasG12D/+;SnailKO/KO mice (n = 2). Scale bars, 50 μm. LG Low grade, HG High grade. e Viability of Human Pancreatic Duct Epithelial (HPDE) cells after activation of KRASG12D alone or in combination with SNAIL. HPDE cells transduced with lentiviral constructs for doxycycline-inducible expression of EGFP, KRASG12D ( + mock vector) or KRASG12D + SNAIL were treated with 100 ng ml-1 doxycycline. Viability was assessed by CellTiter-Glo assay after 72 h and is displayed as % of the respective untreated controls. Mean ± SEM. n = 3 independent experiments; *p = 0.033, unpaired two-tailed t-test with Welch’s correction. f Representative images of SA-β-Gal staining of HPDE cells treated for 3 days with doxycycline (100 ng ml-1) to induce activation of EGFP, KRASG12D + mock or KRASG12D + SNAIL. n = 3 independent experiments. The percentage of SA-β-gal+ cells is indicated in the upper right corner. Scale bar, 10 µm. Source data of Fig. 5 are provided in the Source Data file.
To probe SNAIL function in a human model and validate the relevance of our findings for human PDAC development, we employed immortalized human pancreatic duct epithelial (HPDE) lineage cells and engineered them with doxycycline-inducible human KRASG12D and SNAIL expression vectors (Fig. 5e, f and Supplementary Fig. S4a). Activation of oncogenic KRASG12D in HPDE cells induced strong morphological changes as well as OIS, as demonstrated by SA-β-gal staining, accompanied by a decrease in cell viability compared to EGFP transduced controls. In contrast, co-expression of KRASG12D and SNAIL reverted this phenotype almost completely, resulting in a loss of OIS and increased HPDE proliferation (Fig. 5e, f, Supplementary Fig. S4a). PDAC cells isolated from full blown tumours displayed no SA-β-gal staining and senescence phenotype, indicating that OIS is bypassed during early steps of pancreatic carcinogenesis as described previously28 (Supplementary Fig. S4b). Thus, we demonstrate in genetic mouse and engineered human pancreatic epithelial lineage cells that SNAIL expression bypasses OIS, thereby driving pancreatic cancer initiation.
SNAIL overcomes senescence without inactivating the TRP53/p21CIP1 axis
Oncogene-induced senescence is associated with induction of the tumour suppressor TRP53 and/or p16INK4A depending on cellular context and their loss facilitates tumor formation (Supplementary Fig. S4c)30,32. As shown by immunohistochemistry, TRP53 and its functional downstream target p21CIP1 are expressed in premalignant PanIN lesions and full-blown PDAC cells of PKrasG12D/+;SnailKI/+ mice in vivo (Fig. 6a, b). Because mutant gain- or loss-of-function TRP53 is unable to transcriptionally activate p21CIP128, these data support the notion that i) expression and function of the TRP53/p21CIP1 axis is intact in our model, and ii) the observed bypass of senescence in PKrasG12D/+;SnailKI/+ pancreata is independent of the TRP53 pathway. To further substantiate these findings, we show upregulation of TRP53 and p21CIP1 protein abundance in full-blown PKrasG12D/+;SnailKI/+ PDAC cells upon treatment with the topoisomerase inhibitor etoposide in vitro (Fig. 6c) and enrichment of TRP53 downstream targets in pancreatic gene expression profiles of PanIN-bearing PKrasG12D/+;SnailKI/+ mice (Fig. 6d).
a, b Representative Trp53 and p21CIP1 stainings of PanINs (a) and PDAC (b) of PKrasG12D/+;SnailKI/+ animals (n = 3 each). c Western blot of TRP53 and p21CIP1 expression in PKrasG12D/+;SnailKi/+ (n = 3) and PKrasG12D/+ (n = 1) PDAC cell lines after 6 h 20 μM etoposide (Eto) or vehicle (DMSO) treatment. d GSEA of mRNA expression of 1-month-old mice (n = 2 per genotype) computed and corrected for multiple testing using the Benjamini–Hochberg procedure (for statistical details, see methods section) shows significant enrichment of KEGG p53 signalling pathway genes in PKrasG12D/+;SnailKI/+ (red) vs. PKrasG12D/+ (blue). Normalized Enrichment Score: 1.98; Nominal p-value < 0.001; False Discovery Rate (FDR) q-value < 0.001. e, f Kaplan-Meier survival curves of PKrasG12D/+ (n = 125; 465 days), PKrasG12D/+;SnailKI/+ (n = 42; 190 days), PKrasG12D/+;Trp53R172H/+ (n = 28; 117 days) and PKrasG12D/+;SnailKI/+;Trp53R172H/+ (n = 22; 90 days) animals. ***p < 0.0001, log-rank test with Bonferroni correction. g Representative p16INK4A staining of PanINs (left) and PDAC (right) of PKrasG12D/+;SnailKI/+ mice (n = 3 each). h qRT-PCR analysis of p16Ink4a (left) and p19Arf (right) mRNA expression in PDAC of endpoint mice (PKrasG12D/+n = 5; PKrasG12D/+;SnailKI/+n = 12; PKrasG12D/+;SnailKI/KIn = 12). Mean ± SEM, **p = 0.0094, *p = 0.0365, Mann-Whitney two-tailed test. i Scheme of Cdkn2a gene locus and p16Ink4a genotyping strategy. The non-related proteins p16INK4A and p19ARF are encoded both by the Cdkn2a locus. Red arrows, primer positions. UTR, untranslated region. Scheme according to35. j PCR of p16Ink4a genomic sequence integrity in PDAC cell lines of PKrasG12D/+ (n = 30), PKrasG12D/+;SnailKI/+ (n = 13); PKrasG12D/+;SnailKI/KI (n = 9) endpoint mice. Gabra, internal positive control. k Quantification of PCR analysis of p16Ink4a genomic sequence integrity of data in panel (j). **p = 0.0016, two-tailed Fisher’s exact test. l Kaplan-Meier survival curves of PKrasG12D/+;SnailKI/+ (n = 42; 190 days), PKrasG12D/+;SnailKI/+;p16Ink4a*/+ (n = 22; 156 days) and PKrasG12D/+;SnailKI/+;Cdkn2alox/+ with loss of p16INK4A and p19ARF (n = 17; 108 days). ***p < 0.0001, log-rank test with Bonferroni correction. Note: PKrasG12D/+ cohort in panel (e, f) is the same shown in Figs. 1g, 4b, and PKrasG12D/+;SnailKI/+ cohort of panel e and l is the same shown in Fig. 1g. Source data of Fig. 6 are provided in the Source Data file. Scale bars, 50 μm. ns, not significant.
To test the functional relevance of the TRP53 pathway in an in vivo model, we used a genetically engineered p53 mutant allele, which lacks canonical TRP53 function and p21CIP1 induction33. These animals harbour the murine orthologue of the Li-Fraumeni hotspot mutation R175H at the endogenous murine Trp53 locus (Trp53R172H). In this p53 mutant background, PKrasG12D/+;SnailKI/+;Trp53R172H/+ mice displayed a dramatically accelerated PDAC formation and showed in vivo evidence for synergy between aberrant SNAIL expression and functional p53 inactivation. All animals in the tumour watch cohort developed invasive PDAC. Median survival of PKrasG12D/+;SnailKI/+;Trp53R172H/+ mice (90 days) was significantly less than PKrasG12D/+;SnailKI/+ (190 days) and PKrasG12D/+;Trp53R172H/+ (117 days) mice (Fig. 6e, f). These data demonstrate that TRP53 and SNAIL function, at least in part, via non-overlapping and non-redundant pathways and tumour barriers.
The cell cycle regulator p16INK4A, which blocks G1 to S phase progression of the cell cycle, is implicated in OIS and tumour suppression and is frequently lost during KRAS-driven pancreatic carcinogenesis (Fig. 6g–k and Supplementary Fig. S4c)32. It is encoded by the Cdkn2a locus together with p19ARF, which is involved in TRP53 activation by inhibiting Mdm234. Immunohistochemistry revealed strong expression of p16INK4A in PanIN lesions and full-blown PDAC of PKrasG12D/+;SnailKI/+ mice (Fig. 6g), indicating intact p16INK4A function. Furthermore, p16Ink4a/p19Arf mRNA expression in PDAC tissue was greater in PKrasG12D/+;SnailKI/+ than PKrasG12D/+ animals, and increased further in PKrasG12D/+;SnailKI/KI animals (Fig. 6h). We therefore tested the integrity of the genomic sequence of the p16Ink4a locus in cell lines isolated from the three different models (Fig. 6i, j). We observed loss of p16Ink4a in 60% of PKrasG12D/+ PDAC cell lines tested. In contrast, only 38,5% of PKrasG12D/+;SnailKI/+ and none of the PKrasG12D/+;SnailKI/KI cell lines were deficient for p16Ink4a (Fig. 6j, k). These findings suggest that Snail might inactivate the p16-RB controlled cell cycle/OIS restriction checkpoint downstream of p16Ink4a, to block OIS and drive PDAC progression.
To test this hypothesis, we again used genetic in vivo models of pancreatic tumour evolution. We crossed PKrasG12D/+ and PKrasG12D/+;SnailKI/+ animals with either p16Ink4a* mutant loss of function mice35, or Cdkn2alox mice with conditional knock-out of both p16Ink4a and p19Arf gene products36. All animals of the tumour watch cohort developed invasive PDAC. However and as hypothesized, PKrasG12D/+;SnailKI/+;p16Ink4a*/+ mice showed no statistically significant difference in median survival compared to PKrasG12D/+;SnailKI/+ mice (Fig. 6l). These data demonstrate at the level of genetics that p16Ink4a and SNAIL function via overlapping pathways/shared tumour barriers. In line, PKrasG12D/+;SnailKI/+;Cdkn2alox/+ mice with loss of both, the p16Ink4a and the p19ARF tumour suppressor barriers, had dramatically reduced median survival (Fig. 6l), confirming our data obtained with the Trp53R172H mutant in vivo model (Fig. 6e, f).
Taken together, our mechanistic dissection of three important tumor suppressor genes of KRAS-driven carcinogenesis provides strong in vivo genetic evidence that SNAIL bypasses senescence and drives tumour development independent of TRP53 inactivation and downstream of the p16Ink4a cell cycle restriction check-point, e.g., via blocking the RB-controlled senescence-pathway.
Consistent with this hypothesis, we observed increased proliferation of premalignant PanIN lesions, as evidenced by BrdU labelling and Ki67 staining (Fig. 7a, b and Supplementary Fig. S4d). In addition, we demonstrate the enrichment of genes that promote cell cycle progression by gene expression profiling of PanIN bearing PKrasG12D/+;SnailKI/+ vs. PKrasG12D/+ pancreata of one-month-old mice (Fig. 7c), as well as increased DNA damage and apoptosis induction by aberrant SNAIL expression (Supplementary Fig. S4e–i). It has been previously shown that E2F activation in response to RB inactivation leads to p53-dependent apoptosis37,38,39,40,41. Thus, combining p53 inactivation with aberrant SNAIL expression accelerates tumorigenesis most likely due to the prevention of p53-dependent apoptosis.
a Representative BrdU stainings of ADMs and PanINs of PKrasG12D/+ (n = 11) and PKrasG12D/+;SnailKI/+ (n = 5) mice. b Percentage of BrdU positive cells in ADMs/PanINs of PKrasG12D/+;SnailKI/+ (n = 5) and PKrasG12D/+ (n = 11) mice. Mean ± SEM, **p = 0.006, unpaired two-tailed t-test with Welch’s correction. c GSEA 1-month-old mice (n = 2 per genotype) computed and corrected for multiple testing using Benjamini–Hochberg procedure (statistical details see methods) shows significant enrichment of KEGG cell-cycle genes in PKrasG12D/+;SnailKi/+ (red) vs. PKrasG12D/+ (blue) pancreata. Normalized Enrichment Score (NES): 2.38; Nominal p-value < 0.001; False Discovery Rate (FDR) q-value < 0.001. d, e Representative stainings (d) and quantification of pRb-S807/811 positive PanINs (e) of PKrasG12D/+;SnailKI/+ and PKrasG12D/+ animals (n = 4 per genotype). Mean ± SEM, *p = 0.029, Mann-Whitney two-tailed test. f GSEA corrected for multiple testing using Benjamini–Hochberg procedure shows significant enrichment of hallmark E2F target genes in PKrasG12D/+;SnailKI/+ (red) vs. PKrasG12D/+ (blue) in 1-month-old mice (n = 2 per genotype). NES: 2.44; Nominal p-value < 0.001; FDRq-value < 0.001. g Chromatin-immunoprecipitation (ChIP) of SNAIL binding to E-boxes of indicated promoters in PKrasG12D/+ (n = 3) and PKrasG12D/+;SnailKI/+ (n = 3) PDAC cell lines ± Trp53 mutation as indicated. %input calculation; IgG, negative control. Mean ± SEM. *p = 0.05, Mann-Whitney one-tailed test. hCcnb1 and E2f3 promoter activity in PKrasG12D/+ (n = 3) and PKrasG12D/+;SnailKI/KI (n = 3) PDAC cells (three independent experiments). Mean ± SEM, *p = 0.026, unpaired one-tailed Student’s t-test. i Volcano-plot representing enriched proteins in PKrasG12D/+;SnailKI/+ PDAC cells upon Snail or IgG ChIP, respectively, followed by mass-spectrometry based quantification of co-precipitated proteins (two independent experiments in triplicate for each condition). x-axis, log2-fold change; y-axis, adjusted p-value of the two-sample t-test (two-tailed, FDR < 0.05, s0 = 1). 141 of 1039 proteins were significant vs. IgG control. Pathway-enrichment analysis of significant proteins with MSigDB Hallmarks (upper right panel) and Reactome (lower panel) databases. j Scheme of genome-scale CRISPR/Cas9 negative-selection screen (PKrasG12D/+;SnailKO/KO, PKrasG12D/+;SnailKI/+, PKrasG12D/+;SnailKI/KI cells; n = 4). k Differences in β-scores (SnailKI overexpression (OE) - SnailKO knock-out (KO) cells) were used for Reactome database enrichment analysis (FDR ≤ 0.05; difference in β score < −1). Scale bars, 50 μm. ns not significant. LG low grade; HG high grade. Source data of Fig. 7 provided in Source Data file.
Phosphorylation and thereby inactivation of the tumour suppressor RB by cyclin dependent kinase (CDK)/cyclin complexes, which is negatively regulated by p16Ink4a, is an essential step to bypass the firm G1-phase arrest of senescent cells and initiate cell cycle activation and proliferation42. RB inactivation leads to dissociation of the E2F complex thereby activating the expression of E2F target genes, which can then drive cell cycle progression as well as apoptosis, as observed in our model40,41,43. In line, we detected phosphorylation of RB and thus inactivation of the RB-controlled cell cycle/senescence checkpoint (Fig. 7d, e) in PanIN lesions together with concomitant enrichment of E2F target genes (Fig. 7f) in the PKrasG12D/+;SnailKI/+ model. Although gene expression profiling of bulk tissues is confounded by the increased number of PanIN lesions in PKrasG12D/+;SnailKI/+ mice at an age of one month, and gene sets that promote cell cycle progression overlap substantially with E2F target genes, our various different in vivo models and datasets provide strong evidence for the hypothesis that SNAIL might bypass senescence downstream of p16Ink4a via interference with the RB-controlled cell cycle/senescence restriction check-point.
SNAIL is a transcriptional regulator of the cell cycle
Cyclins, such as cyclin A1 (CCNA1) or cyclin B1 (CCNB1) interact with cyclin-dependent kinases (CDK) to phosphorylate and thereby inactivate RB, which releases E2F transcription factors to enter the nucleus and activate transcription of target genes essential for the transition from G1 to S phase and progression of the cell cycle. Because our data provide genetic evidence that SNAIL bypasses senescence and drives the cell cycle in vivo, we validated the upregulation of important cell cycle regulators and downstream effectors. Transcriptomic profiling and qRT-PCR analysis revealed a marked increase in mRNA expression of several cell cycle-related genes, including cyclins and cyclin-dependent kinases, in pancreata with aberrant SNAIL expression compared with PKrasG12D/+ controls (Supplementary Fig. S5a, b), in line with the proliferation and E2F signature shown in Fig. 7c, f.
SNAIL, as a transcription factor, functions primarily via binding to promoter and enhancer regions of the target genes. To test whether SNAIL binds to promoter regions of genes that positively regulate the cell cycle, we analysed publicly available chromatin immunoprecipitation (ChIP)-seq cancer cell line datasets11 and compared them to significantly enriched genes in pancreas of one-month-old PKrasG12D/+;SnailKI/+ mice. Of 69 enriched genes implicated in proliferation and cell cycle progression, 62 were bound by SNAIL in their promoter region (Supplementary Fig. S5c). Calculation of the odds ratio for this enrichment (8.53; Supplementary Fig. S5c) strongly suggested that the presence of the vast majority (89.9%) of genes in the SNAIL-bound fraction was not due to chance. Thus, SNAIL has a clear preference for binding to genes that promote cell cycle progression. To validate these findings functionally in the PKrasG12D/+;SnailKI/+ PDAC model, we performed ChIP experiments using cell lines isolated from PKrasG12D/+ and PKrasG12D/+;SnailKI/+ mice with and without Trp53 mutation, and selected SNAIL targets from the ChIP-seq study. This revealed binding of SNAIL to E-boxes in promoter regions of multiple genes, such as Ccnb1, Ccnb2, Ccnd1, E2f2 and E2f3 (Fig. 7g and Supplementary Fig. S5d), all known to drive the cell cycle and bound by SNAIL in the published ChIP-seq data (Supplementary Fig. S5c). In line with the ChIP-seq data set, we did not observe binding of SNAIL to the Ccna1 promoter region, even though this cyclin mRNA is overexpressed in the PKrasG12D/+;SnailKI/+ model (Supplementary Fig. S5a). To test whether SNAIL is indeed capable of activating the expression of the identified cell cycle regulators, we performed promoter reporter assays using again our primary PDAC cell cultures. While aberrant SNAIL expression did not increase reporter gene activity of cyclin D1 (Ccnd1) and E2f2 promoter reporter constructs, cyclin B1 (Ccnb1; p = 0.026), cyclin B2 (Ccnb2; p = 0.0423) and E2f3 (p = 0.065) demonstrated evidence of gene activation (Fig. 7h and Supplementary Fig. S5e).
These findings support a context-specific function of SNAIL in vivo, which bypasses senescence by direct binding and activating important positive regulators of the cell cycle. To gain more insights into SNAIL-mediated gene activation, we studied potential co-regulators from chromatin cross-linked to SNAIL by proteomics and performed chromatin immunoprecipitation coupled to mass spectrometry (ChIP-MS). This allowed us to identify 141 significantly enriched putative chromatin-bound partners of SNAIL (Fig. 7i and Supplementary Data 1). Activating transcription factors, such as NFKB2 and SMAD2, nuclear receptors and coactivators, chromatin remodelers and histone modifiers, such as KDM1A were enriched together with SNAIL (Fig. 7i and Supplementary Data 1). Subsequent pathway analysis of significantly enriched genes revealed regulators of the cell cycle, such as MYC and E2F targets, and genes involved in progression through the G2M checkpoint (Fig. 7i). Further, several genes, e.g., RNA-binding proteins, involved in RNA pol II transcription and transcription termination, RNA metabolism, processing, splicing and transport were significantly enriched together with SNAIL, indicating a role of SNAIL in alternative splicing and RNA biology, which might contribute to its function in regulating cell cycle progression. Of note, NFKB/E2F interactions have previously been shown to control the timing of cell proliferation44 and SMAD2 silencing decreased PDAC cell division45. In addition, KDM1A has been recently linked to gene activation46 and PDAC cell cycle progression47. These data suggest that multiple interactions of SNAIL might contribute to its context-dependent role as a transcriptional activator and regulator of the cell cycle.
To identify functional relevant targets of SNAIL driving PDAC progression and maintenance, we performed pooled genome-wide CRISPR/Cas9 loss-of-function (viability) screens with cell lines isolated from PKrasG12D/+;SnailKI mice with aberrant SNAIL expression compared to PDAC cells from SNAIL-deficient PKrasG12D/+;SnailKO/KO animals (Fig. 7j, k, Supplementary Fig. S6 and Supplementary Data 2). We determined differential sensitivity scores48 by calculating the difference in β-score between SNAIL overexpressing and deficient cells and further analysed genes displaying a negative differential sensitivity score, pointing to enhanced depletion in PKrasG12D/+;SnailKI cells. This allowed us to identify 238 statistically significant genes, whose depletion led to the specific drop-out of cells with aberrant SNAIL expression.
Pathway analysis of these hits enabled us to uncover the specific genetic dependencies and vulnerabilities of cells with aberrant SNAIL expression, such as cell cycle and checkpoint regulation, E2F targets, NF-kB signaling, RNA pol II transcription and chromatin modifications (Fig. 7k and Supplementary Fig. S6b, c). Importantly, we observed that these pathways and processes correlated to a high degree with the ChIP-MS analysis of Fig. 7i, thereby cross-validating our findings by functional genetic screens. To probe the contribution of the differentially expressed cell cycle regulators for cell viability of SNAIL-driven PDAC, we correlated the β-scores of the PKrasG12D/+;SnailKI cells with their gene expression levels. As shown in Supplementary Fig. S6d, 32 out of a total of the 49 differentially expressed cell cycle-related genes of Supplementary Fig. S5 displayed a significant differential β-score indicating selective depletion in cells with aberrant SNAIL expression.
Aberrant SNAIL expression is prognostic in human PDAC
To assess a potential EMT-independent link of aberrant SNAIL expression with cell cycle progression in human PDAC, we analysed SNAIL and CDH1 abundance in differentiated and undifferentiated human PDAC specimens and cell lines, and analysed data of resected primary tumours49. We observed high levels of SNAIL expression in both differentiated and undifferentiated human PDAC specimens and cell lines in accordance with our findings in genetic mouse models corroborating EMT-independent functions of SNAIL also in human PDAC (Fig. 8a, b). In addition, we discerned undifferentiated specimens, which lack both, SNAIL and CDH1 expression (Fig. 8a). Gene expression profiling of primary resected differentiated human PDAC specimen with high CDH1 expression revealed no correlation with SNAIL abundance (p = 0.77; Fig. 8c). However, we observed a strong positive correlation between SNAIL and the expression of important cell cycle regulators, such as CDK4, CCNA1, CCND1, CCND2, CCNE1 and E2F3 (Fig. 8d). Strikingly, many of these genes have been identified as direct targets of SNAIL in murine PDAC (Fig. 7 and Supplementary Fig. S5) and functionally validated by genome wide CRISPR/Cas9-based negative selection screens (Fig. 8d right panel). In addition, tumours with high SNAIL expression were strongly associated with a poorer disease-free survival (DFS) and overall survival (OS) after surgical resection (Fig. 8e, f). Further, we observed a trend towards resistance against chemotherapy with gemcitabine in a small cohort with available clinical data of the resected PDAC patients (6 gemcitabine sensitive and 24 resistant PDAC cases; p = 0.065) (Fig. 8g). While intriguing and consistent with published experimental studies in mice, reporting that Snail knockout sensitizes PDAC tumours to gemcitabine treatment13, these human studies will require larger sample sets and prospective analyses in future.
a Left: Representative staining of SNAIL in human PDAC sections of differentiated (G1/2) and undifferentiated tumours (G3/4) of the Human Protein Atlas version 20.1 (http://www.proteinatlas.org)85. Images and clinical data are available from https://www.proteinatlas.org/ENSG00000124216-SNAI1/pathology/pancreatic+cancer#ihc. Right: Representative SNAIL and CDH1 staining in serial sections of an independent PDAC patient cohort with G1/2 and G3/4 tumours (n = 11). Scale bars, 50 µm. b qRT-PCR of SNAIL (SNAI1; left) and E-cadherin (CDH1; right) mRNA expression of human PDAC cell lines (epithelial, n = 9; mesenchymal, n = 16). Mean ± SEM. Left: ns, not significant (p = 0.835) unpaired two-tailed t-test; Right: p < 0.0001, Mann-Whitney two-tailed test. c SNAI1 (left) and CDH1 (middle) expression across SNAI1 quartile group of resected primary human PDAC samples (Q1 to Q4, n = 88). Mean ± SEM. p = 1.7e-26 (SNAI1) and 0.37 (CDH1), one-way ANOVA-test. Right: Pearson correlation of SNAIL and CDH1 expression across all PDAC samples (n = 88). Two-tailed Pearson correlation coefficient r = 0.031, r2 = 0.0009949, p = 0.7705 (not significant), 95% confidence interval −0.1791 to 0.2395. d Left: Heatmap of top 20 significant cell cycle related genes with the highest variance across SNAI1 quartile groups. Colour code, row-wise scaling of RNA expression. Row clustered using hierarchical clustering on Euclidean distance. Note: 11 out of the 20 human genes overlap with the cell cycle related genes of Supplementary Fig. S5b identified in the murine model (depicted in red). Right: Cross-species validation of cell cycle regulators. β-scores from genome-wide CRISPR/Cas9 negative-selection screen of PKrasG12D/+;SnailKI cell lines are indicated. Genes with FDR-q value > 0.05 are marked with an X on the bar. e, f Kaplan-Meier analysis of PDAC patients (n = 111). e Disease-Free Survival (DFS) p = 0.0178 log-rank test and (f) Overall Survival (p = 0.0094 log-rank test), in samples with aberrant high SNAI1 expression (Q4) compared to the rest (Q1-3). g Correlation of SNAIL expression with gemcitabine treatment resistance of human PDAC patients. Density distribution of SNAI1 mRNA expression across gemcitabine resistant (n = 6) or sensitive (n = 24) samples, p = 0.065, two-tailed Wilcoxon rank test. Source data of Fig. 8 are provided in the Source Data file.
Discussion
Understanding the specific in vivo functions of SNAIL, which is aberrantly expressed in a wide variety of epithelial cancers and often correlated with poor patient outcome, is crucial for improving patient stratification and clinical interventions5,14. SNAIL has been extensively and convincingly characterized as a master regulator of the embryonic EMT program, which triggers cancer cell plasticity, migration and metastatic spread in various tumour types5,8,11. In contrast, little is known about EMT-independent oncogenic functions of SNAIL in cancer initiation and progression. Specifically, non-redundant functions of this EMT transcription factor (TF) in autochthonous tumours remain elusive26. We employed complex genetic in vivo modelling to address this important question in a comprehensive and systematic manner across different cancer types, oncogenic drivers and pathways. This enabled us to discover a complex non-redundant context-specific EMT-independent framework of SNAIL function in epithelial PDAC that bypasses oncogenic KRAS-induced senescence and drives the cell cycle by p16INK4A-independent inactivation of the RB-restriction checkpoint of senescence and the cell cycle. Importantly, our data demonstrate that SNAIL acts in this context as a transcriptional activator, rather than via its canonical function as a transcriptional repressor50; it binds directly to the canonical E-boxes of a variety of important cell cycle regulators, such as cyclins, CDKs and E2F TFs to drive the cell cycle as evidenced by ChIP experiments and reporter gene assays. This allows sustained proliferation of epithelial PDAC cells and thus tumour progression independent of overt EMT induction and contrasts with WNT- and KRAS-driven intestinal cancer subtypes, which are refractory towards aberrantly expressed SNAIL. In line with our findings, SNAIL has recently been shown to be dispensable for the EMT process in PDAC, which is controlled by the EMT transcription factor Zeb151.
Collectively, our studies constitute a comprehensive analysis of SNAIL function in cancer. SNAIL has been identified and validated as an intrinsic cancer driver, and there are strong indications that both, the cell and tissue of origin as well as the genetic context dictates the function of SNAIL as a cancer driver. This improves our understanding of the diverse in vivo functions of SNAIL and will enable SNAIL downstream targets to be defined within the cell cycle machinery in epithelial PDAC. Our discovery has potentially important clinical implications, since it provides a framework for patient stratification and opens avenues for therapeutic interventions. Therapeutics targeting the cell cycle have been developed in recent years, which provide efficient opportunities to block cell cycle progression, e.g., via blockade of CDK4/6 activity52. Furthermore, considering the association of SNAIL with Gemcitabine resistance, it would seem worthwhile evaluating whether targeting SNAIL downstream effectors can improve the efficacy of current therapies for PDAC. Such treatment options are urgently needed. PDAC is a highly lethal and refractive disease with overall 5-year survival rates below 9%53.