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. 2024 Sep 28;43(1):265.
doi: 10.1186/s13046-024-03189-3.

Inactivation of HIPK2 attenuates KRASG12D activity and prevents pancreatic tumorigenesis

Silvia Sozzi #  1   2 , Isabella Manni #  3 , Cristiana Ercolani  4 , Maria Grazia Diodoro  4 , Armando Bartolazzi  5 , Francesco Spallotta  6 , Giulia Piaggio  3 , Laura Monteonofrio  1 , Silvia Soddu #  1 , Cinzia Rinaldo #  7   8 , Davide Valente #  9   10
Affiliations

Inactivation of HIPK2 attenuates KRASG12D activity and prevents pancreatic tumorigenesis

Silvia Sozzi et al. J Exp Clin Cancer Res. .

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) features KRAS mutations in approximately 90% of human cases and excessive stromal response, termed desmoplastic reaction. Oncogenic KRAS drives pancreatic carcinogenesis by acting on both epithelial cells and tumor microenvironment (TME). We have previously shown that Homeodomain-Interacting Protein Kinase 2 (HIPK2) cooperates with KRAS in sustaining ERK1/2 phosphorylation in human colorectal cancers. Here, we investigated whether HIPK2 contributes to oncogenic KRAS-driven tumorigenesis in vivo, in the onset of pancreatic cancer.

Methods: We employed an extensively characterized model of KRASG12D-dependent preinvasive PDAC, the Pdx1-Cre;LSL-KRasG12D/+ (KC) mice. In these mice, HIPK2 was inhibited by genetic knockout in the pancreatic epithelial cells (KCH-/-) or by pharmacologic inactivation with the small molecule 5-IodoTubercidin (5-ITu). The development of preneoplastic acinar-to-ductal metaplasia (ADM), intraepithelial neoplasia (PanIN), and their associated desmoplastic reaction were analyzed.

Results: In Hipk2-KO mice (KCH-/-), ERK phosphorylation was lowered, the appearance of ADM was slowed down, and both the number and pathologic grade of PanIN were reduced compared to Hipk2-WT KC mice. The pancreatic lesion phenotype in KCH-/- mice was characterized by abundant collagen fibers and reduced number of αSMA+ and pSTAT3+ desmoplastic cells. These features were reminiscent of the recently described human "deserted" sub-TME, poor in cells, rich in matrix, and associated with tumor differentiation. In contrast, the desmoplastic reaction of KC mice resembled the "reactive" sub-TME, rich in stromal cells and associated with tumor progression. These observations were confirmed by the pharmacologic inhibition of HIPK2 in KC mice.

Conclusion: This study demonstrates that HIPK2 inhibition weakens oncogenic KRAS activity and pancreatic tumorigenesis providing a rationale for testing HIPK2 inhibitors to mitigate the incidence of PDAC development in high-risk individuals.

Keywords: HIPK2; KRAS; Pancreatic tumorigenesis.

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Conflict of interest statement

The authors declare that they have no competing interest.

Figures

Fig. 1
Fig. 1
HIPK2 is expressed in human PDACs. A WB analysis of HIPK2 in whole cell lysates of the indicated human pancreatic and PDAC-derived cell lines. HSP70 was used as loading control. The histogram indicates the mean ± standard error of HIPK2 fold change expression relative to HPDE in three independent experiments. B Representative images of IHC for HIPK2 in three different human PDAC samples. C Representative images for HIPK2 immunostaining in normal, PanIN and PDAC ducts are reported on the left. Scale bars are 200 µm. Quantification of the percentage of HIPK2+ cells in the indicated tissues is reported as dot plot on the right. ANOVA test with Tukey’s multiple comparison test, * P < 0.05, n.s.: not significant. D TCGA analysis of the HIPK2 gene alteration frequency as reported in cBioPortal (https://www.cbioportal.org/). S.V.: Structural variant; Mut.: Mutation. CNA: Copy Number Alteration. Each histogram shows the frequency of HIPK2 gene alteration in the indicated dataset
Fig. 2
Fig. 2
The levels of pERK are reduced in KCH−/− pancreata. A Schematic representation of LSL-KRasG12D/+ and Hipk2flox/flox recombinant alleles. B Representative genotyping analysis for Hipk2flox and LSL-KRasG12D alleles of tail (T) and pancreas (P) from Pdx1-Cre;LSL-KRasG12D/+ (KC) and Pdx1-Cre;LSL-KRasG12D/+;Hipk2flox/flox (KCH−/−) mice. Floxed (2lox) and recombined (1lox) alleles are indicated by arrows. C WB analysis of pERK levels in KC and KCH−/− pancreata. HSP70 was used as loading control. D Normalized levels of pERK represented as ratio of pERK and total ERK densitometry values. Mean ± Standard Deviation (SD) is reported. Student’s t-test, * P < 0.05. E Representative IHC images for each pERK score. Scale bars are 100 µm. F Representative IHC images of pERK in KC and KCH−/− pancreata. Scale bars are 200 µm. G Scatter plot bar of pERK score in all KC and KCH−/− mice. Mean ± SD is reported at the bottom. Mann–Whitney’s test, * P < 0.05
Fig. 3
Fig. 3
Depletion of HIPK2 reduces pancreatic tumorigenesis in KC mice. A Representative images of pancreas slices from KC and KCH−/− stained with H&E. PanIN and ADM lesions are visible. Scale bars are 300 µm. B Representative images of ADM and PanIN in KC and KCH−/− pancreata. Bar is 50 µm. C ADM in each visual field were counted in 12 KC and 14 KCH−/− pancreatic samples stained by H&E. Corresponding quantification for each mouse is reported in the scatter plot bar. Mean ± SD, Mann–Whitney’s test, n.s. not significant. D-E Data in B were divided into two subgroups based on the mouse age at the euthanization to obtain ADM histograms in mice younger or older than 7 months ( 7mo and > 7mo), respectively. Mean ± SD, Mann–Whitney’s test, n.s. not significant. F The percentage of PanINs relative to unaffected ducts was evaluated on the same samples described in B and shown as scatter plot bar. Mean ± SD, Mann–Whitney’s test, *** P < 0.001. G-H Data in E were divided in two subgroups (≤ 7mo and > 7mo) as described above. Mean ± SD, Mann–Whitney’s test, * P < 0.05. I, PanINs present in the H&E samples were counted based on their grade. The percentage of each PanIN grade is shown and the number of counted PanINs is indicated inside the histograms. Fisher’s exact test, **** P < 0.0001
Fig. 4
Fig. 4
Loss of HIPK2 determines different oncogenic KRAS sub-TMEs. Pancreatic lesions of all KC and KCH−/− mice were analyzed with the indicated staining by serially cut slices. Scale bars are 200 µm A, B, Representative images of IHC for aSMA are shown in A; the percentages of the different score staining intensity (from 0 to 3) are reported in B and the number of lesions counted for each score is indicated inside the histograms; Fisher’s exact test, **** P < 0.0001. C, D Representative images of PR staining are shown in C; the percentages of the different score staining intensity (from 0 to 4) are reported in D and the number of lesions counted for each score is indicated inside the histograms; Fisher’s exact test, **** P < 0.0001. E, F Representative images of IHC for pERK are shown in E; the percentages of lesions positive for pERK (pERK+) are reported as histograms in F, Fisher’s exact test, ** P < 0.01. G, H Representative images of IHC for pSTAT3 are shown in G; the percentages of lesions positive for pSTAT3 (pSTAT3.+) are reported as histograms in H, Fisher’s exact test, *** P < 0.001. I Representative images of αSMA (upper images) and Picrosirius Red (lower images) showing reactive and deserted sub-TMEs in the indicated mice. L The percentages of lesions with reactive and deserted sub-TME are reported and the number of lesions counted for each sub-TME is indicated inside the histograms. Fisher’s exact test, **** P < 0.0001
Fig. 5
Fig. 5
Inhibition of HIPK2 by 5-ITu prevents the onset of ADM and PanIN. A Schematic representation of the experimental design. B ADM in each visual field were counted in all DMSO and 5-ITu treated FVB-KC mice. The corresponding number of ADM per field for each mouse is reported in the scatter plot bar. Mean ± SD, Mann–Whitney’s test *P < 0.05. C Quantification of PanINs in the same DMSO and 5-ITu treated FVB-KC mice analyzed in B. The corresponding percentages are reported and the total number of normal ducts and PanINs counted is indicated inside the histograms. Fisher’s exact test, ** P < 0.01 D Representative images of pERK staining in DMSO and 5-ITu treated FVB-KC mice. For each image a 2x-magnified detail is shown in the upper-right corner. E The percentages of lesions with reactive and deserted sub-TMEs in DMSO and 5-ITu treated samples are reported. The total number of reactive and deserted subTMEs counted is indicated inside the histograms. Fisher’s Exact test, * P < 0.05. F Representative images of reactive and deserted sub-TMEs stained with αSMA (upper images) and Picrosirius Red (lower images). Scale bars are 200 µm
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