The phospholipid transporter PITPNC1 links KRAS to MYC to prevent autophagy in lung and pancreatic cancer

PITPNC1 is regulated by KRAS oncogene and predicts poor survival in LUAD and PDAC

To identify novel effectors with relevance to KRAS oncogenesis, we used a gene signature derived from experimental models expressing mut KRAS (n = 41 genes) reported by our group [35]. We queried this gene signature against TCGA data set by comparing gene expression profiles of wt and mut KRAS LUAD patients. Among the 15 differentially expressed genes, SPRY4, DUSP6, CCND1, PHLDA1, DUSP4, and PITPNC1 were the most robustly upregulated in KRAS-mutated patients (p < 0.0001) (Fig. 1A). We focused our attention on the phosphatidylinositol cytoplasmic transfer protein 1 (PITPNC1) which, unlike the other genes, had not been previously linked to KRAS oncogene biology. PITPNC1 was also upregulated when compared to normal lung tissue, indirectly suggesting a link to the tumour phenotype (Fig. 1B). PITPNC1 mRNA increase was dependent on KRAS oncogene expression, since the presence of other dominant oncogenic drivers (e.g. BRAF or EGFR) did not affect PITPNC1 transcript levels (Suppl. Figure 1A and B). PITPNC1 upregulation was not due to differential PITPNC1 amplification in mut vs wt KRAS LUAD patients either (p = 0.815) (Fig. 1C). Thus, the differential transcriptional regulation of PITPNC1 may be a consequence of aberrant KRAS activation. We further tested PITPNC1’s clinical role in human cancer by performing survival analysis in LUAD patients. High PITPNC1 expression was associated with poor overall survival in mut KRAS patients but not in wt (Fig. 1D). Since PITPNC1 was part of a mut KRAS signature that included genes with a role in LUAD and PDAC, we studied human PDAC specimens. Notably, high PITPNC1 was also a worse prognosis marker in PDAC (Fig. 1E).

Fig. 1figure 1

PITPNC1 is upregulated in KRAS-mutated LUAD and PDAC and predicts poor survival. A Heatmap of upregulated genes in The Cancer Genome Atlas (TCGA) LUAD data set comparing expression profiles of wt and mut KRAS LUAD patients. PITPNC1 mRNA expression levels in normal lung (N), wild type (wt) and mutant (mut) KRAS LUAD. Mut vs wt KRAS (p < 0.0001) or vs N (p < 0, 0001). C PITPNC1 gene amplification percentage (GISTIC2 analysis) in mut and wt KRAS LUAD samples, or both (p = 0.815). D Kaplan–Meier survival analysis of LUAD patients, stratified based on KRAS status and PITPNC1 expression. Data from TCGA database: wt KRAS (Log-rank test p = 0.96) and mut KRAS (Log-rank test p = 0.04). E Kaplan–Meier survival analysis of PDAC patients stratified by PITPNC1 expression. Data from ICGC database (Log-rank test p = 0.027). F Western blot of PITPNC1 and KRAS expression in H2126 and H6C7 cells, expressing a control (LacZ) or overexpressing KRAS (wt KRAS4B or mut KRASG12D, G12C or G12V). Twenty μg of protein were loaded per sample. HSP90 and β-TUBULIN were used as loading markers. G Western blot of PITPNC1 and KRAS expression in A549, H2009, PATU8902 and HPAFII cells, expressing a control (GFPsh) or an inducible KRAS shRNA (KRASsh) (activated by 1 µg/ml doxycycline). Twenty μg of protein were loaded per sample. HSP90 were used as loading markers. H Western blot of PITPNC1 expression in A549, H2009 and HPAFII cells treated for 24 h with pharmacologic inhibitors: trametinib (MEKi, 0.5 μmol/L), BIX02189 (MEK5i, 10 μmol/L), SP600125 (JNKi, 10 μmol/L) or GSK2126458 (PI3Ki, 0.1 μmol/L). Twenty μg of protein were loaded per sample. β-TUBULIN was used as loading marker. I Western blot of PITPNC1 and KRAS expression in Kraslox/lox MEFs transduced with different human HA-tagged KRAS mutants (G12C, G12D, G12V, G12R, G12S, G13D and Q61H). 4OHT: 600 nM. PITPNC1 mRNA expression levels in no loss of heterozygosity (no LOH) and loss of heterozygosity (LOH) TCGA LUAD patients. no LOH vs LOH (p = 0.047)

The clinical data led us to test the connection between KRAS and PITPNC1 via genetic gain- and loss-of-function experiments in lung and pancreas cellular models. Overexpression of mut KRAS (G12D, G12C and G12V) in wt KRAS LUAD cells (H2126 and H1568) and in immortalized normal human pancreatic duct epithelial cells (H6C7) increased PITPNC1 protein and mRNA levels (Fig. 1F). Such PITPNC1 upregulation was also observed in human LUAD patients with different KRAS mutations (Suppl. Figure 1C). Conversely, KRAS inhibition in LUAD (A549, H2009, H1792) and PDAC (PATU8902, HPAFII) cells using a specific shRNA decreased PITPNC1 protein (Fig. 1G, and Suppl. Figure 1D and E). PITPNC1 was consistently downregulated upon inactivation of MEK1/2 and JNK1/2 in both LUAD (A549, H2009) and PDAC (HPAFII) cells (Fig. 1H and Suppl. Figure 1F), indicating a regulation by KRAS through different effector pathways. Notably, PITPNC1 was the unique member of the PITP family controlled by KRAS, as the expression of PITPNA, PITPNB, PITPNM1, PITPMN2 and PITPNM3 did not change upon KRAS genetic modulation (Suppl. Figure 1G-I).

In addition to KRAS activating mutations [36], an imbalance between wt and mut KRAS alleles can influence cancer cells’ fitness, expression profile and therapy response in LUAD and PDAC [37,38,39,40]. Thus, we investigated PITPNC1 levels in relationship to KRAS dosage. First, we used Kraslox/lox MEFs expressing different KRAS mutations to study PITPNC1 in the context of loss-of-heterozygosity (LOH) [39]. Similar to human cell lines, exogenous expression of the various KRAS mutations increased PITPNC1 expression (Suppl. Figure 1J). Notably, Cre-excision of the wt allele in MEFs via 4-OHT treatment reduced PITPNC1 levels in all mutants but G12V (Fig. 1I). Such decrease was also observed in mut KRAS human samples of the LUAD TCGA data set (p = 0.047) (Fig. 1J). Second, we assessed the impact of mut KRAS amplification on PITPNC1 expression in the LUAD data set. However, no significant differences were found (Suppl. Figure 1K). These data may indicate that PITPNC1 represents a functional node downstream of KRAS integrating signals from receptor tyrosine kinases which become activated upon mut KRAS expression and require wt KRAS for downstream signalling.

Given the relevance of concurrent mutations in mut KRAS LUAD prognosis and response to therapy [41], we explored the association of PITPNC1 expression with prevalently mutated tumour suppressor genes (TSGs). LKB1 mutations were mostly found in mut KRAS with high PITPNC1 expression (p = 0.005) while ARID1A mutations appeared mostly in low PITPNC1-expressing tumours (p = 0.0208) (Suppl. Figure 2A). These results led us to test the impact of LKB1 mutations on PITPNC1 expression. CRISPR/Cas9-based LKB1 knockout in KRAS-mutated LUAD cells (H2009) enhanced PITPNC1 expression (Suppl. Figure 2B). This finding was further recapitulated in mouse LUAD cell lines driven by mut Kras (KLA and LKR10) upon LKB1 abrogation with specific sgRNAs (Suppl. Figure 2C). Thus, PITPNC1 is regulated by KRAS through MEK1/2 and JNK1/2 signalling pathways, and its expression may be exacerbated by LKB1 loss.

PITPNC1 inhibition reduces cell proliferation in vitro and impairs tumour growth in vivo in LUAD and PDAC

To characterise the functional role of PITPNC1, genetic depletion using two independent shRNAs, one of which had been previously validated via rescue experiments [17], was carried out in a panel of LUAD (n = 6) and PDAC (n = 4) cell lines (Fig. 2A). PITPNC1 inhibition consistently reduced cell proliferation of all cell lines (Fig. 2B). Likewise, a decreased colony-forming capacity was also observed in both tumour types (Fig. 2C). However, we did not find a consistent effect on apoptosis in PITPNC1-depleted cells (Suppl. Figure 3A and B).

Fig. 2figure 2

PITPNC1 inhibition in LUAD and PDAC cells reduce cell proliferation and impair tumour growth in vivo. A Western blot of PITPNC1 expression in A549, H358, H2009, H1792 LUAD cell lines and PATU8902, Panc1, MiaPaca2 PDAC cell lines transfected with a control (GFPsh) or a specific shRNA against PITPNC1 (PITPNC1 sh6 and sh7). Twenty μg of protein were loaded per sample. β-TUBULIN was used as loading marker. B Relative proliferation of A549 H23, H358, H2009, H1792, H2347 LUAD cell lines and PATU8902, Panc1, MiaPaca2 and HPAFII PDAC cell lines. Cells were transfected with a control (GFPsh) or a specific shRNA against PITPNC1 (PITPNC1 sh6 and sh7) (Dunnett´s multiple comparation test). C Representative images and quantification of clonogenic ability (mean ± std. error). D Tumour volume (mm3) of A549-derived xenografts (n = 6) (Dunnett’s multiple comparison test). E Representative images of tumours of D. F Tumour weight (g) of A549-derived xenografts (n = 6) of D at end point. G Tumour volume (mm3) of PATU8902-derived xenografts (n = 8) (Dunnett’s multiple comparison test). H Representative images of tumours of G. I Tumour weight (g) of PATU8902-derived xenografts (n = 8) of G at end point. J pH3 and CC3 quantification of A549-derived xenografts of D at end point. (Mann Whitney test). K pH3 and CC3 quantification of PATU8902-derived xenografts of G at end point (Mann Whitney test). L Representative images of lung photon flux ratio of A549 GFP/luciferase PITPNC1-overexpressing cells OE compared with the control (GFP/luciferase) (n = 8) at the indicated days. M Lung photon flux ratio of L (Bonferroni´s multiple comparison test). N Lung tumour nodules quantification on the lungs extracted from L (Mann Whitney test). O Liver foci quantification in the liver extracted from L (Mann Whitney test). P Representative images of lung tumour nodules quantification from N. Q Representative images of liver foci quantification from O

Next, we investigated if PITPNC1 is necessary for KRAS-driven tumourigenesis in vivo. First, LUAD cell lines infected with PITPNC1 shRNAs were subcutaneously injected in immunocompromised mice. PITPNC1 knocked-down cells generated tumours of a smaller volume and weight than controls (Fig. 2D-F). PITPNC1 abrogation in PDAC cells also impaired tumour growth, yielding lighter tumours (Fig. 2G-I). The effect of PITPNC1 loss in vivo was related to decreased tumour proliferation and enhanced cytotoxic activity in these models (Fig. 2J and K, and Suppl. Figure 3C).

Complementary to PITPNC1 inhibition experiments, the effect of its overexpression was also assessed in mut KRAS LUAD cell lines (A549 and H358) (Suppl. Figure 3D). No effect on colony formation was observed (Suppl. Figure 3E). Moreover, exogenous PITPNC1 did not confer a growth advantage in vivo when cells were injected subcutaneously in immunodeficient mice (Suppl. Figure 3F-K).

Mut KRAS LUAD harbouring inactivating LKB1 mutations display poor prognosis, in part due to an enhanced metastatic potential [41, 42]. Since increased PITPNC1 expression was observed upon LKB1 loss, we explored PITPNC1 overexpression in the metastatic setting. A549 cells were first constructed to express luciferase and transduced with a PITPNC1-expressing or a control vector. We used a mouse model of lung colonization in vivo where cancer cells initially seed in the lungs after intravenous injection (~ 10–15 min post-injection) (Fig. 2L). Subsequent bioluminescence monitoring revealed that, on week 1, PITPNC1-expressing cells colonized the lung more efficiently. Interestingly, while the bioluminescence signal of the two groups become closer by week 2, it increased in cancer cells over-expressing PITPNC1 at week 3 and 4 (Fig. 2M). Macroscopic and microscopic analysis of tissues at endpoint revealed a higher number of metastatic liver foci in the group of mice injected with PITPNC1-overexpressing cells while the lung tumor burden was similar (Fig. 2N-Q), suggesting that secondary metastasis to the liver contribute to the distinct bioluminescence signal. No differences in the migratory capacity in vitro or the metastatic tropism in vivo of control and PITPNC1-overexpressing cells were detected that could explain these findings (Suppl. Figure 3L-N), suggesting the involvement of heterotypic interactions as described in breast cancer [17]. Thus, PITPNC1 upregulation contributes to the metastatic phenotype of mut KRAS LUAD.

A PITPNC1 gene signature features KRAS-regulated genes and predicts poor survival in LUAD and PDAC

To get a better understanding of PITPNC1 as a KRAS effector, we interrogated the transcriptome of KRAS-mutated LUAD cells (A549) after PITPNC1 inhibition with two shRNAs. A total of 429 genes were found differentially expressed (logFC ± 1, B > 0) with regard to control cells (Fig. 3A). The downregulated PITPNC1 gene signature (dPITPNC1 GS; n = 233 genes), which a priori would contain transcriptional targets whose overexpression fosters the oncogenic phenotype, was used. This signature was queried against two independent data sets where genetic or pharmacological blockade of KRAS, via a tet-inducible KRAS shRNA or the KRASG12C ARS160 inhibitor respectively, was carried out. A consistent enrichment of the dPITPNC1 GS in genes repressed upon KRAS inhibition was found (Fig. 3B). To expand these findings to the pancreas setting, we took advantage of gene expression data from cancer cell lines (iKrasC) and xenograft tumours (iKrasT) derived from an inducible genetically engineered mouse (GEM) model of Kras-driven PDAC in which doxycycline administration activates expression of a mut Kras allele [43] (Fig. 3C). In both data sets, a large overlap of the dPITPNC1 GS with genes decreased after oncogenic KRAS inactivation was found, suggesting that multiple PITPNC1-regulated genes are part of the KRAS signalling pathway.

Fig. 3figure 3

A PITPNC1 gene signature features KRAS-regulated genes and predicts poor LUAD and PDAC patients’ outcome. A Heat map of downregulated and upregulated genes in A549 cells after PITPNC1 inhibition with two specific shRNAs (sh6 and sh7) or control (GFPsh). B Gene set enrichment analysis (GSEA) of the dPITPNC1 gene signature in the comparison of both genetically and pharmacologically KRAS inhibition (tet-shKRAS, activated by 1 µg/ml doxycycline, or KRASiARS1620 respectively) vs control (GFP or DMSO respectively). C GSEA of the dPITPNC1 gene signature in the comparison of gene expression data from cancer cell lines (iKrasC) and xenograft tumours (iKrasT) derived from an inducible genetically engineered mouse (GEM) model of Kras-driven PDAC in which doxycycline administration activates expression of a mutant Kras allele. D GSEA of the dPITPNC1 gene signature in the comparison of mut vs wt KRAS LUAD in four data sets. E GSEA of the dPITPNC1 gene signature in the comparison of PDAC vs normal tissue in two data sets. F Survival analysis of LUAD patients (TCGA data set) stratified by the dPITPNC1 gene signature (Log-rank test p = 0.0059). G Survival analysis of LUAD patients (Shedden et al. data set) stratified by the dPITPNC1 gene signature (Log-rank test p = 0.01772). H Survival analysis of PDAC patients (ICGC data set) stratified by the dPITPNC1 gene signature (Log-rank test p = 0.0081). I Survival analysis of PDAC patients (TCGA data set) stratified by the dPITPNC1 gene signature (Log-rank test p = 0.0137)

To test the PITPNC1-regulated genes in a more clinically relevant setting, we performed GSEA using human LUAD data sets (n = 4) with information on the KRAS mutational status. A general enrichment of the dPITPNC1 GS was found in LUAD tumours harbouring KRAS mutations compared to those with native alleles (Fig. 3D). Likewise, we found a strong enrichment of the dPITPNC1 GS in human PDAC samples with regard to normal pancreas in two data sets (Fig. 3E). Additional analysis of genes whose expression was diminished in response to PITPNC1 were recurrently present in the leading edges of the previously investigated data sets was done by qPCR. A dramatic reduction in mRNA expression was detected for all genes (Suppl. Figure 4A), validating the RNAseq data.

We next explored the clinical relevance of the dPITPNC1 GS. We observed that high dPITPNC1 GS levels were associated with the LUAD and PDAC patient subgroup with the worst prognosis (Fig. 3F-I). Analysis of the signature in the context of tumour stage revealed no differences in either tumour type (Suppl. Figure 4B and C). Likewise, no significant changes in two of the main patient subgroups of LUAD (mut KRAS/P53-mutated and mut KRAS/LKB1-mutated) and PDAC (classical and basal), which display differential outcome, response to therapy and gene expression profiles [41, 44, 45], were found (Suppl. Figure 4D and E). Collectively, these results indicate that PITPNC1 controls the expression of a gene signature with clinical implications for KRAS-mutated tumours.

PITPNC1 loss induces a G1 phase arrest and MYC downregulation

To expand our understanding of PITPNC1’s functional role in KRAS-driven oncogenesis, we performed Gene Ontology analysis to infer the biological pathways (BP) related to PITPNC1-regulated genes. First, the dPITPNC1 GS was used as input. The top BP included general cell cycle, sodium ion transmembrane transport, regulation of hormone levels or establishment/maintenance of cell polarity (Fig. 4A). These findings prompted us to inquiry about the impact of PITPNC1 loss on the cell cycle. We found a consistent G1 arrest and S phase decrease across all LUAD cell lines (Fig. 4B and Suppl. Figure 5A). These observations were extended to the PDAC setting (Fig. 4C), suggesting the regulation of common cellular mechanisms across mut KRAS tumours.

Fig. 4figure 4

PITPNC1 loss induces a G1 phase arrest linked to MYC downregulation. A Gene Ontology analysis of the downregulated PITPNC1 gene set (dPITPNC1 GS). B and C Cell cycle analysis by EdU labelling in the human LUAD A549 and H2009 (B), and PDAC HPAFII and Panc1 (C) cell lines after PITPNC1 knockdown with a specific shRNA (sh6 or sh7) compared to control (GFPsh). (Bonferroni´s multiple comparison test). MYC mRNA expression in A549, H2009 and H1792 LUAD and PDAC PATU8902, Panc1 and HPAFII cell lines expressing a specific shRNA (sh6 or sh7) compared to control (GFPsh) (Dunnet´s multiple comparison test). E MYC protein expression in the A549, H2009 and H1792 LUAD cell lines after PITPNC1 knockdown with a specific shRNA (sh6 or sh7) compared to control (GFPsh). Twenty μg of protein were loaded per sample. β-TUBULIN was used as loading marker. F MYC protein expression in Panc1, HPAFII and MiaPaca2 PDAC cell lines after PITPNC1 knockdown with a specific shRNA (sh6 or sh7) compared to control (GFPsh). Twenty μg of protein were loaded per sample. β-TUBULIN was used as loading marker. G E2F1 and p27 protein expression in the A549, H2009 and H1792 LUAD cell lines after PITPNC1 knockdown with a specific shRNA (sh6 or sh7) compared to control (GFPsh). Twenty μg of protein were loaded per sample. β-TUBULIN was used as loading marker. H E2F1 and p27 protein expression in the PATU8902, HPAFII and MiaPaca2 PDAC cell lines after PITPNC1 knockdown with a specific shRNA (sh6 or sh7) compared to control (GFPsh). Twenty μg of protein were loaded per sample. HSP90 was used as loading marker. I MYC and PITPNC1 protein expression in H2009 and PATU8902 MYC-overexpressing cells after PITPNC1 knockdown with a specific shRNA (sh6 or sh7) compared to control (GFPsh) and treated with DMSO or MG132 (10 μM, 6 h). Twenty μg of protein were loaded per sample. HSP90 was used as loading marker. J AURKA and PLK1 protein expression in A549, H2009 and H1792 LUAD cell lines after PITPNC1 knockdown with a specific shRNA (sh6 or sh7) compared to control (GFPsh). Twenty μg of protein were loaded per sample. HSP90 was used as loading marker. K AURKA and PLK1 protein expression in Panc1, HPAFII and MiaPaca2 PDAC cell lines after PITPNC1 knockdown with a specific shRNA (sh6 or sh7) compared to control (GFPsh). Twenty μg of protein were loaded per sample. HSP90 was used as loading marker. L MYC protein levels in A549 and H1792 LUAD and Panc1 and HPAFII PDAC cell lines treated with DMSO, PLK1i (BI2536, 50–100 nM, 48 h), or both PLK1i plus proteasome inhibitor (MG132, 10 μM 6 h). Twenty μg of protein were loaded per sample. HSP90 was used as loading marker

BP analysis also featured a MYC active pathway, which led us to test MYC expression in mut KRAS LUAD and PDAC cells with depleted PITPNC1. We found an overt MYC downregulation across all cell lines studied, which mainly occurred at the protein level (Fig. 4D-F), positioning MYC downstream of PITPNC1 and providing a direct link to the KRAS pathway. Such MYC downregulation was recapitulated upon PITPNC1 inhibition in vivo (Suppl. Figure 5B). MYC cooperates with oncogenic RAS to regulate G1 to S phase transition of cell cycle [46], a phenotype observed in PITPNC1-depleted cells. Indeed, MYC inhibition using two specific shRNAs revealed a G1 arrest similar to that found in cells with PITPNC1 loss (Suppl. Figure 5C). This mechanism involves repression of various cyclin kinase inhibitors, such as CDKN1B (p27) and CDKN1C (p57), and activation of E2F transcription factors [47], a link sustained in our experimental models (Suppl. Figure 5D). These observations led us to investigate the molecular consequences of PITPNC1 loss on the cell cycle. Detailed analysis of transcriptomics data showed upregulation of p27 and p57, and downregulation of E2F1 (Suppl. Figure 5E). These results were validated using qPCR and Western blot analyses in independent samples (Fig. 4G and H, and Suppl. Figure 5F and G).

To investigate if PITPNC1 regulates cell cycle through MYC, exogenous MYC was overexpressed in PITPNC1-depleted cells. No rescue of the proliferative phenotype was found, most likely because MYC levels were still low even in the overexpressing cells (Suppl. Figure 5H and I). Notably, blocking the proteasome activity with the specific inhibitor MG132 rescued MYC expression, suggesting post-translational regulatory mechanisms (Fig. 4I). This prompted us to scan the PITPNC1-knockdown RNAseq data for potential kinases involved in MYC protein regulation, and found downregulation of AURKA and PLK1 (Suppl. Figure 5J), two kinases previously reported to stabilize MYC protein via direct phosphorylation [48, 49]. Notably, only PLK1 was consistently decreased across the various LUAD and PDAC cell lines upon PITPNC1 loss, with an expression pattern mimicking that of MYC protein (Fig. 4J and K). We next tested the possibility that PLK1 regulates MYC protein. Using the PLK1 inhibitor BI-2536, we found reduced MYC protein expression that is rescued by proteasome inhibition (Fig. 4L). Thus, PITPNC1 may be regulating MYC protein expression in part by PLK1. Taken together, these results suggest that PITPNC1 represents a functional link that connects oncogenic KRAS to MYC.

PITPNC1 controls mTOR localization via MYC to prevent autophagy

To complement the previous findings, we explored those genes upregulated upon PITPNC1 abrogation (i.e., uPITPNC1 GS). The top 5 BPs of the GO analysis involved P53 transcriptional gene network, regulation of mTORC1 signalling, antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, natural killer cell-mediated toxicity, and genotoxicity pathway (Fig. 5A). We focused on mTOR as it is an effector of the PI3K pathway that can function within the KRAS signalling network. Enriched genes in the regulation of mTORC1 signalling BP feature included CASTOR1, RRAGD, SESN1, SESN3, and GPR137C. Upregulation of SESN1, SESN2 and SESN3 was validated at the mRNA level using qPCR and the results confirmed in additional PITPNC1-depleted cells (H2009 and HPAFII) (Fig. 5B and C, and Suppl. Figure 6A). These results suggested that activation of the mTOR pathway is altered upon PITPNC1 inhibition.

Fig. 5figure 5

PITPNC1 controls mTOR localization to prevent autophagy. A Gene Ontology analysis of the upregulated PITPNC1 gene set (uPITPNC1 GS). B and SESN1, SESN2 and SESN3 expression levels in A549 (B) and HPAFII (C) cell lines were measured by qPCR. Cells were virally infected to express a control (GFPsh) or a PITPCN1 shRNA (sh6 and sh7) (Dunnet’s multiple comparison test). GAPDH was used as housekeeping gene. D and E mTOR/LAMP1 colocalization analysis by immunofluorescence in A549 (D) and HPAFII (E) PITPNC1-depleted cells. F and G Quantification of mTOR/LAMP1 Mander’s overlap coefficient (MOC) in A549 (F) and (G) of D and E (Dunnett’s multiple comparison test). H Lysosomes per cell and average lysosomes size in A549 of D (Dunn’s multiple comparison test). I Lysosomes per cell and average lysosomes size in HPAFII of E (Dunn’s multiple comparison test). J Western blots of LC3-I and LC3-II protein levels in a LUAD (n = 3) and PDAC (n = 3) cell lines expressing a shRNA control (C) or two PITPNC1 shRNAs (sh6 and sh7). Twenty μg of protein were loaded per sample and HSP90 was used as loading control. K Western blots of protein levels of LC3-I and LC3-II in a LUAD (n = 3) and PDAC (n = 1) cell lines expressing a shRNA control (C) or two MYC shRNAs (sh42 snd sh89). Twenty μg of protein were loaded per sample and HSP90 was used as loading control. L Proposed model for the role of PITPNC1 in KRAS-driven LUAD and PDAC

SESTRINS (SESN1-3) inactivate GATOR2 to inhibit mTOR activity, constraining the localization of mTOR to the lysosome where it gets activated [50, 51]. Thus, we investigated mTOR localization in response to PITPNC1 abrogation by immunofluorescence. A549, H2009 and HPAFII control cells showed mTOR activation, as inferred from the overlapping signal with the lysosome marker LAMP1. However, this colocalization was impaired when PITPNC1 was inhibited (Fig. 5D-G, and Suppl. Figure 6B and C). This modification occurred without changes in mTOR protein abundance (Suppl. Figure 6D), suggesting that PITPNC1 controls mTOR lysosomal recruitment.

A close visualization of the lysosomes in PITPNC1-depleted cells revealed increased number and size compared to PITPNC1-proficient ones (Fig. 5H and I, and Suppl. Figure 6E). Expansion of the lysosomal compartment or lysosomal biogenesis has been related to enhanced autophagy [52]. mTOR functions as a counter-regulator of autophagy [53, 54]. Thus, we analysed the level of the autophagy marker LC3-II, which tightly correlates with the number of autophagosomes/autophagolysomes [55]. Increased LC3-II was observed in cell lines lacking PITPNC1 (Fig. 5J). In keeping with autophagy induction, downregulation of gene signature featuring autophagy and lysosome biogenesis [56] was also found in PITPNC1-inhibited cells (Suppl. Figure 6F).

Enhanced LC3-II expression could indicate either upregulation of autophagic flux (i.e., autophagosome formation) or blockade of autophagic degradation [57]. To confirm the underlying mechanism, we compared changes in LC3-II under the presence of the lysosomal protease inhibitor hydrochloroquine, which accumulates within lysosomes leading to lysosome neutralization and the inhibition of autophagic flux/autophagosome formation [55]. Hydroxichloroquine treatment elicited a further accumulation of LC3-II (Suppl. Figure 6G and H), indicating that PITPNC1 inhibition enhances autophagic flux. This mechanism occurred without activation changes in S6K and 4EBP1 (Suppl. Figure 6I).

MYC suppresses autophagy in B cell lymphomas by antagonizing the function of TFEB transcription factors [58], raising the possibility that PITPNC1 could control autophagy through MYC in LUAD and PDAC. To address this possibility, we first tested if MYC regulated autophagy in our experimental systems. MYC inhibition by specific shRNAs induced LC3II/I ratio in LUAD and PDAC cell lines (Fig. 5K). Autophagy induction was associated with reduced mTOR localization to lysosomes (Suppl. Figure 7A-D). This was associated with an increase in number and size of lysosomes (Suppl. Figure 7E and F). To define how MYC regulates autophagy, we tested if MYC could be transcriptionally controlling the negative regulators of mTOR localization, SESTRIN1-3, which are downregulated after PITPNC1 inhibition. qPCR analysis of MYC-depleted cells showed that MYC inhibition significantly enhanced their expression (Suppl. Figure 7G and H), positioning SESTRINS downstream of MYC. Taken together, these observations suggest that PITPNC1 controls mTOR activity via MYC to prevent autophagy. A proposed model for the role of PITPNC1 in LUAD AND PDAC KRAS-driven tumours is depicted in Fig. 5L.

JAK2 inhibitors reverse the expression of a PITPNC1-regulated transcriptome and synergize with Sotorasib

Given the lack of pharmacological tools to inhibit PITPNC1 and aiming to increase the translational value of our findings, we followed a drug repurposing strategy that predicts compounds capable of reversing the expression profile of the PITPNC1-regulated transcriptome. The top 200 up and down differentially expressed genes obtained after PITPNC1 knockdown (logFC ± 1, B > 0) were used as input and a repurposing score > 90 was used as cut-off. The top 5 drug families predicted to reverse the PITPNC1 transcriptome were JAK, HDAC, DNA synthesis, bromodomain and DNA dependent protein kinase inhibitors (i). Additional drug families scoring in this analysis were PI3Ki, mTORi or MEKi, known downstream effectors of KRAS oncogene (Fig. 6A). The same drug repurposing approach applied to a KRAS-dependent transcriptome uncovered common drug families (Suppl. Figure 8A), consistent with the overlap of PITPNC1- and KRAS-regulated genes. JAK inhibitors, particularly those against JAK2, scored highest in both repurposing studies and were selected for downstream analyses.

留言 (0)

沒有登入
gif