Limits...
Image based detection and targeting of therapy resistance in pancreatic adenocarcinoma

View Article: PubMed Central - PubMed

ABSTRACT

Pancreatic intraepithelial neoplasia (PanIN) is a premalignant lesion that can progress to pancreatic ductal adenocarcinoma, a highly lethal malignancy marked by its late stage at clinical presentation and profound drug resistance1. The genomic alterations that commonly occur in pancreatic cancer include activation of KRAS2 and inactivation of p53, and SMAD42-4. To date, however, it has been challenging to target these pathways therapeutically; thus the search for other key mediators of pancreatic cancer growth remains an important endeavor. Here we show that the stem cell determinant Musashi (Msi) is a critical element of pancreatic cancer progression in both genetic models and patient derived xenografts. Specifically, we developed Msi reporter mice that allowed image based tracking of stem cell signals within cancers, revealing that Msi expression rises as PanIN progresses to adenocarcinoma, and that Msi-expressing cells are key drivers of pancreatic cancer: they preferentially harbor the capacity to propagate adenocarcinoma, are enriched in circulating tumor cells, and are markedly drug resistant. This population could be effectively targeted by deletion of either Msi1 or Msi2, which led to a striking defect in PanIN progression to adenocarcinoma and an improvement in overall survival. Msi inhibition also blocked the growth of primary patient-derived tumors, suggesting that this signal is required for human disease. To define the translational potential of this work we developed antisense oligonucleotides against Msi; these showed reliable tumor penetration, uptake and target inhibition, and effectively blocked pancreatic cancer growth. Collectively, these studies highlight Msi reporters as a unique tool to identify therapy resistance, and define Msi signaling as a central regulator of pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus

Molecular targets of Msi signaling(a-b) Real-time PCR analysis of (a) Msi1 and (b) Msi2 expression in MIA PaCa-2 human pancreatic cancer cells relative to normal pancreas (n=3 independent experiments). (c-d) Analysis of shRNA knockdown efficiency in GFP+ sorted MIA PaCA-2 cells infected with GFP tagged lentiviral shRNA against scrambled control sequences, (c) MSI1 or (d) MSI2 (n=3 independent experiments). Analysis of direct Msi targets (e) Msi consensus binding sites in 3'UTR of Brd4, Hmga2 and c-Met transcripts. (f-g) Phospho-c-Met staining in WT-KPf/fC and (f) Msi1−/−-KPf/fC, (g) Msi2−/−-KPf/fC mice; Keratin (magenta), phospho-c-Met (green), DAPI (blue). See Figure 3b-c for quantified data. (h) Colony formation of MIA PaCa-2 cells infected with empty vector or c-MET overexpression vector (3 independent experiments) shows no impact of overexpressed c-Met on control MIA PaCa-2 (control for c-Met mediated rescue of MSI knockdown in Figure 3f). Data are represented as mean ± SEM. *** P < 0.001, **** P < 0.0001 by Student's t-test. Source Data for all panels are available online.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4998062&req=5

Figure 11: Molecular targets of Msi signaling(a-b) Real-time PCR analysis of (a) Msi1 and (b) Msi2 expression in MIA PaCa-2 human pancreatic cancer cells relative to normal pancreas (n=3 independent experiments). (c-d) Analysis of shRNA knockdown efficiency in GFP+ sorted MIA PaCA-2 cells infected with GFP tagged lentiviral shRNA against scrambled control sequences, (c) MSI1 or (d) MSI2 (n=3 independent experiments). Analysis of direct Msi targets (e) Msi consensus binding sites in 3'UTR of Brd4, Hmga2 and c-Met transcripts. (f-g) Phospho-c-Met staining in WT-KPf/fC and (f) Msi1−/−-KPf/fC, (g) Msi2−/−-KPf/fC mice; Keratin (magenta), phospho-c-Met (green), DAPI (blue). See Figure 3b-c for quantified data. (h) Colony formation of MIA PaCa-2 cells infected with empty vector or c-MET overexpression vector (3 independent experiments) shows no impact of overexpressed c-Met on control MIA PaCa-2 (control for c-Met mediated rescue of MSI knockdown in Figure 3f). Data are represented as mean ± SEM. *** P < 0.001, **** P < 0.0001 by Student's t-test. Source Data for all panels are available online.

Mentions: To understand the molecular basis of the effects of Msi loss, we genomically profiled Msi deficient tumor cells (Extended Data Fig. 6, 7a-d). Msi loss led to down-regulation of many key genes, including regulators of stem cell function (Wnt7a, Aldh, Lin28), proto-oncogenes (c-Met, Fos, Fyn) and Regenerating (Reg) family genes, linked to gastrointestinal cancers. Among these, analysis of 3'UTRs for Msi binding-sites and RIP-PCR identified BRD4, c-MET and HMGA2 as potential direct targets (Extended Data Fig. 7e, Fig. 3a). We focused on c-MET22, which was diminished in Msi pancreatic cancer and also bound MSI1 in CLIP-seq experiments (Fig. 3b-d, Extended Data 7f, g). c-Met could not only be activated molecularly by MSI but also effectively complemented MSI loss (Fig. 3e, f; Extended Data Fig. 7h). While these suggest that c-Met is a direct functional target of Msi, it is almost certainly one of many. In fact, Msi's powerful impact on cancer is probably because of its ability to control a broad range of programs (Extended Data Fig. 6). In this context, BRD4 and HMGA2 may be particularly attractive targets23,24, as they could act at an epigenetic level with c-Met to collectively mediate Msi function. Underscoring such a potential convergence of epigenetic and oncogenic pathways, inhibitors of both Brd4 and c-Met effectively targeted gemcitabine-resistant Msi2+ cells (Fig. 3g-h).


Image based detection and targeting of therapy resistance in pancreatic adenocarcinoma
Molecular targets of Msi signaling(a-b) Real-time PCR analysis of (a) Msi1 and (b) Msi2 expression in MIA PaCa-2 human pancreatic cancer cells relative to normal pancreas (n=3 independent experiments). (c-d) Analysis of shRNA knockdown efficiency in GFP+ sorted MIA PaCA-2 cells infected with GFP tagged lentiviral shRNA against scrambled control sequences, (c) MSI1 or (d) MSI2 (n=3 independent experiments). Analysis of direct Msi targets (e) Msi consensus binding sites in 3'UTR of Brd4, Hmga2 and c-Met transcripts. (f-g) Phospho-c-Met staining in WT-KPf/fC and (f) Msi1−/−-KPf/fC, (g) Msi2−/−-KPf/fC mice; Keratin (magenta), phospho-c-Met (green), DAPI (blue). See Figure 3b-c for quantified data. (h) Colony formation of MIA PaCa-2 cells infected with empty vector or c-MET overexpression vector (3 independent experiments) shows no impact of overexpressed c-Met on control MIA PaCa-2 (control for c-Met mediated rescue of MSI knockdown in Figure 3f). Data are represented as mean ± SEM. *** P < 0.001, **** P < 0.0001 by Student's t-test. Source Data for all panels are available online.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4998062&req=5

Figure 11: Molecular targets of Msi signaling(a-b) Real-time PCR analysis of (a) Msi1 and (b) Msi2 expression in MIA PaCa-2 human pancreatic cancer cells relative to normal pancreas (n=3 independent experiments). (c-d) Analysis of shRNA knockdown efficiency in GFP+ sorted MIA PaCA-2 cells infected with GFP tagged lentiviral shRNA against scrambled control sequences, (c) MSI1 or (d) MSI2 (n=3 independent experiments). Analysis of direct Msi targets (e) Msi consensus binding sites in 3'UTR of Brd4, Hmga2 and c-Met transcripts. (f-g) Phospho-c-Met staining in WT-KPf/fC and (f) Msi1−/−-KPf/fC, (g) Msi2−/−-KPf/fC mice; Keratin (magenta), phospho-c-Met (green), DAPI (blue). See Figure 3b-c for quantified data. (h) Colony formation of MIA PaCa-2 cells infected with empty vector or c-MET overexpression vector (3 independent experiments) shows no impact of overexpressed c-Met on control MIA PaCa-2 (control for c-Met mediated rescue of MSI knockdown in Figure 3f). Data are represented as mean ± SEM. *** P < 0.001, **** P < 0.0001 by Student's t-test. Source Data for all panels are available online.
Mentions: To understand the molecular basis of the effects of Msi loss, we genomically profiled Msi deficient tumor cells (Extended Data Fig. 6, 7a-d). Msi loss led to down-regulation of many key genes, including regulators of stem cell function (Wnt7a, Aldh, Lin28), proto-oncogenes (c-Met, Fos, Fyn) and Regenerating (Reg) family genes, linked to gastrointestinal cancers. Among these, analysis of 3'UTRs for Msi binding-sites and RIP-PCR identified BRD4, c-MET and HMGA2 as potential direct targets (Extended Data Fig. 7e, Fig. 3a). We focused on c-MET22, which was diminished in Msi pancreatic cancer and also bound MSI1 in CLIP-seq experiments (Fig. 3b-d, Extended Data 7f, g). c-Met could not only be activated molecularly by MSI but also effectively complemented MSI loss (Fig. 3e, f; Extended Data Fig. 7h). While these suggest that c-Met is a direct functional target of Msi, it is almost certainly one of many. In fact, Msi's powerful impact on cancer is probably because of its ability to control a broad range of programs (Extended Data Fig. 6). In this context, BRD4 and HMGA2 may be particularly attractive targets23,24, as they could act at an epigenetic level with c-Met to collectively mediate Msi function. Underscoring such a potential convergence of epigenetic and oncogenic pathways, inhibitors of both Brd4 and c-Met effectively targeted gemcitabine-resistant Msi2+ cells (Fig. 3g-h).

View Article: PubMed Central - PubMed

ABSTRACT

Pancreatic intraepithelial neoplasia (PanIN) is a premalignant lesion that can progress to pancreatic ductal adenocarcinoma, a highly lethal malignancy marked by its late stage at clinical presentation and profound drug resistance1. The genomic alterations that commonly occur in pancreatic cancer include activation of KRAS2 and inactivation of p53, and SMAD42-4. To date, however, it has been challenging to target these pathways therapeutically; thus the search for other key mediators of pancreatic cancer growth remains an important endeavor. Here we show that the stem cell determinant Musashi (Msi) is a critical element of pancreatic cancer progression in both genetic models and patient derived xenografts. Specifically, we developed Msi reporter mice that allowed image based tracking of stem cell signals within cancers, revealing that Msi expression rises as PanIN progresses to adenocarcinoma, and that Msi-expressing cells are key drivers of pancreatic cancer: they preferentially harbor the capacity to propagate adenocarcinoma, are enriched in circulating tumor cells, and are markedly drug resistant. This population could be effectively targeted by deletion of either Msi1 or Msi2, which led to a striking defect in PanIN progression to adenocarcinoma and an improvement in overall survival. Msi inhibition also blocked the growth of primary patient-derived tumors, suggesting that this signal is required for human disease. To define the translational potential of this work we developed antisense oligonucleotides against Msi; these showed reliable tumor penetration, uptake and target inhibition, and effectively blocked pancreatic cancer growth. Collectively, these studies highlight Msi reporters as a unique tool to identify therapy resistance, and define Msi signaling as a central regulator of pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus