MicroRNAs of the mir-17~92 cluster regulate multiple aspects of pancreatic tumor development and progression

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterized by resistance to currently employed chemotherapeutic approaches. Members of the mir-17~92 cluster of microRNAs (miRNAs) are upregulated in PDAC, but the precise roles of these miRNAs in PDAC are unknown. Using genetically engineered mouse models, we show that loss of mir-17~92 reduces ERK pathway activation downstream of mutant KRAS and promotes the regression of KRASG12D-driven precursor pancreatic intraepithelial neoplasias (PanINs) and their replacement by normal exocrine tissue. In a PDAC model driven by concomitant KRASG12D expression and Trp53 heterozygosity, mir-17~92 deficiency extended the survival of mice that lacked distant metastasis. Moreover, mir-17~92-deficient PDAC cell lines display reduced invasion activity in transwell assays, form fewer invadopodia rosettes than mir-17~92-competent cell lines and are less able to degrade extracellular matrix. Specific inhibition of miR-19 family miRNAs with antagomirs recapitulates these phenotypes, suggesting that miR-19 family miRNAs are important mediators of PDAC cell invasion. Together these data demonstrate an oncogenic role for mir-17~92 at multiple stages of pancreatic tumorigenesis and progression; specifically, they link this miRNA cluster to ERK pathway activation and precursor lesion maintenance in vivo and identify a novel role for miR-19 family miRNAs in promoting cancer cell invasion.

Keywords: PanIN; invasion; mir-17~92; pancreatic cancer; regression.

Figure 1. mir-17~92 null PanINs regress with age

Histological evaluation of PanIN lesions in pancreata from 4-month old (A–D) and 9-month old (E-H) KC and 17KC mice. Representative images from hematoxylin and eosin (A, B, E, F) and quadchrome (simultaneous Alcian Blue and Sirius Red staining; C, D, G, H) stains are shown. (I) Quantification of normal and neoplastic cell types as a percentage of total tissue area. The quadchrome stain marks collagen red, mucin blue, cytoplasm yellow-brown, and nucleic acids black. Arrowheads identify examples of PanIN lesions. Ac = Acinar tissue. Number of organs (n) analyzed for each group were 4 mo KC (4), 4 mo 17KC (7), 9 mo KC (3), 9 mo 17KC (2). Scale bar = 0.25 mm. p values by Student's t test: * < 0.05, ** < 0.01. Error bars represent standard deviation from the mean.

Figure 2. mir-17~92 null PanINs display reduced MEK/ERK pathway activation

Immunostaining of PanIN lesions identified in 4- and 9-month old KC and 17KC mice for phosphorylated ERK (A–D), phosphorylated MEK (E–H), AKT phosphorylated at Thr³⁰⁸ (I–L), and AKT phosphorylated at Ser⁴⁷³ (M–P). Scale bar = 0.1 mm.

Figure 3. ERK signaling is increased by mir-17~92 overexpression in PanIN cell lines

(A) Representative immunoblot for pERK and total ERK in the PanIN cell lines RP2294 and AH2375 stably infected with PIG-mir-17~92 or empty vector. (B) Average densitometry for three experiments performed in (A). Error bars represent standard deviation from the mean.

Figure 4. Loss of mir-17~92 prolongs survival in mice without metastases

(A) Kaplan-Meier survival plot for KPC and 17KPC mice. (B) Primary tumor burden identified in these mice upon euthanasia. (C) Kaplan-Meier survival plot comparing metastatic and localized KPC mice. (D) Kaplan-Meier survival plot comparing metastatic and localized 17KPC mice. (E) Kaplan-Meier survival plot comparing localized KPC and localized 17KPC mice. (F) Total primary tumor burden of localized mice. Number of animals (n) for each group were as follows: KPC (18), 17KPC (18), metastatic/localized KPC (5/13), metastatic/localized 17KPC (7/11), female/male KPC (7/11), female/male 17KPC (10/8). p values: * < 0.05. Error bars represent standard deviation from the mean.

Figure 5. mir-17~92 null PDAC cell lines have reduced invasive capacity in vitro

Characterization of in vitro phenotypes of PDAC cell lines derived from KPC and 17KPC tumors. (A) Invasion activity in transwell assays, displayed as invasion index [(number of invading cells/number of migrating cells) × 100]. (B) Migration activity in transwell assays. (C) Proliferation rate. (D) Colony formation in a soft agar. (E) Cell survival in serum-replete medium. (F) Cell survival in serum-free medium. Cell line nomenclature is noted as cage#animal# (e.g. 9025#2 is the cell line derived from the primary tumor of mouse #2 from cage #9025). All error bars represent SD. p value: * < 0.05.

Figure 6. mir-17~92 null PDAC cell lines form fewer invadopodia rosettes

Immunofluorescence staining for the invadopodia constituent proteins cortactin (A, E), actin (B, F) and paxillin (C, G) in representative KPC and 17KPC cell lines. Merged images are shown in (D) and (H). D′ and H′ are higher magnification views of panels D and H. Scale bar = 10 um. Quantification of invadopodia rosettes is shown in (I). n = 3 for each cell line. Areas of FITC-gelatin degradation, identified as dark regions, are shown for representative KPC and 17KPC cell lines (J, L). (K, M) Co-staining for actin and DNA (DAPI). (N) Quantification of gelatin degradation. n = 3 for each cell line. Error bars represent standard error of the mean. p values: * < 0.05, ** < 0.01, **** < 0.0001.

Figure 7. miR-19 family expression is absent in 17KPC cell lines

(A) Schematic representation of the mir-17~92 cluster and its paralogs mir-106b~25 and mir-106a~363. Constituent miRNAs are color-coded according to their seed families. (B–E) Quantitative RT-PCR measurement of mature miRNA expression across eighteen cell lines derived from primary KPC and 17KPC tumors. Some miRNAs share sufficient sequence similarity that standard oligonucleotides amplify both species equally (e.g. miR-19a and miR-19b), and therefore not all miRNAs are individually plotted. p values: **** < 0.0001. Error bars represent standard deviation from the mean.

Figure 8. PDAC cell line invasion and invadopodia formation is suppressed by miR-19 antagomirs

(A) Transwell invasion through Matrigel of KPC cell lines treated with control or miR-19-targeting antagomirs at the indicated concentrations. (B) Transwell migration activity of the cell lines shown in (A). (C) Quantitative RT-PCR measurement of baseline mature miR-19 levels in KPC cell lines. (D) Transwell invasion through Matrigel of the human PDAC cell lines MIA Paca-2 and PANC-1 treated with control or miR-19-targeting antagomirs at the indicated concentrations. (E) Transwell migration of activity of the cell lines shown in (D). (F) Quantitative RT-PCR measurement of baseline mature miR-19 levels in MIA Paca-2 and PANC-1 cells; miR-19 levels in the KPC cell line 9415#2 are shown for comparison. Invadopodia rosette formation (G) and FITC-gelatin degradation (H) in the KPC cell lines 9910#1 and 9248#1 treated with control or miR-19-targeting antagomirs. n = 3 for each cell line. p values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. (A–F) Error bars represent standard deviation from the mean. (G, H) Error bars represent standard error of the mean.