Inactivation of Brca2 promotes Trp53-associated but inhibits KrasG12D-dependent pancreatic cancer development in mice

Abstract

Background & aims: Inherited mutations in the BRCA2 tumor suppressor have been associated with an increased risk of pancreatic cancer. To establish the contribution of Brca2 to pancreatic cancer we developed a mouse model of pancreas-specific Brca2 inactivation. Because BRCA2-inactivating mutations cause defects in repair of DNA double-strand breaks that result in chromosomal instability, we evaluated whether Brca2 inactivation induced instability in pancreatic tissue from these mice and whether associated pancreatic tumors were hypersensitive to DNA damaging agents.

Methods: We developed mouse models that combined pancreas-specific Kras activation and Trp53 deletion with Brca2 inactivation. Development of pancreatic cancer was assessed; tumors and nonmalignant tissues were analyzed for chromosomal instability and apoptosis. Cancer cell lines were evaluated for sensitivity to DNA damaging agents.

Results: In the presence of disrupted Trp53, Brca2 inactivation promoted the development of premalignant lesions and pancreatic tumors that reflected the histology of human disease. Cancer cell lines derived from these tumors were hypersensitive to specific DNA damaging agents. In contrast, in the presence of KrasG12D, Brca2 inactivation promoted chromosomal instability and apoptosis and unexpectedly inhibited growth of premalignant lesions and tumors.

Conclusions: Trp53 signaling must be modified before inactivation of the Brca2 wild-type allele, irrespective of Kras status, for Brca2-deficient cells to form tumors.

Figure 1. Brca2 inactivation promotes pancreatic cancer when combined with Trp53 inactivation

(A) Schematic representation of the Brca2^F11 allele and the Trp53^F2-10 allele before and after pdx-1-cre dependent recombination. (B) PCR analysis demonstrating rearrangement of the Brca2 and Trp53 alleles in response to pdx-1-cre expression using DNA from mouse tail and pancreas. (C) Kaplan-Meier plots showing pancreatic cancer free survival of aged CPB2^Δ11/Δ11 (n=47), CPB2^wt/Δ11 (n=41), and CPB2^wt/wt (n=34) mice. p- values were determined by a log rank test. (D) Frequency of histological subtypes of tumors detected in CPB2^Δ11/Δ11, CPB2^wt/Δ11 and CPB2^wt/wt mice. (E) Representative images from tumors derived from CPB2^Δ11/Δ11 mice stained with H&E (i,iv,vii,x), cytokeratin 19 (ii,v,viii,xi) or amylase (iii,vi,ix,xii). (F) PanIN lesions (i,ii) and multinucleated cells (iii) from a CPB2^Δ11/Δ11 pancreas.

Figure 2. Inactivation of Brca2 in pancreatic cancer cell lines induces sensitivity to DNA damage and promotes chromosomal instability

(A–C) Apoptosis in cell lines in response to ABT-888, gemcitabine, and cisplatin. (D) Quantification of radial structures in tumor cell lines treated with 100nM MMC. (E) Multinucleation and centrosome amplification in murine pancreatic tumor cell lines. (F) Aneuploidy and polyploidy in tumor cell lines with and without BRCA2.

Figure 3. Inactivation of Brca2 in pancreatic cancer cell lines disrupts localization of midbody proteins during cytokinesis

(A) Immunofluorescence images of intercellular bridges and midbody structures in tumor cell lines stained with antibodies against α-tubulin, BRCA2, CEP55, and CHMP1B. (B) Expression of BRCA2 in a reconstituted CPB2^Δ11/Δ11 cell line. (C) Immunofluorescence images showing the presence of endobrevin (red) and BRCA2 (green) at the midbody of a CPB2^Δ11/Δ11 cell line (top) reconstituted with GFP-BRCA2 (bottom). (D) Multinucleation and centrosome amplification measured by immunofluorescence in a CPB2^Δ11/Δ11 cell line reconstituted with empty vector or wild type BRCA2. Error bars represent SEM.

Figure 4. Brca2 mutant alleles prevent development of premalignant lesions and pancreatic tumors in LSL-Kras^G12D mice

(A) Schematic representation of the LSL-Kras^G12D allele before and after pdx-1-cre dependent rearrangement. (B) PCR analysis of the Brca2^F11 and Kras^G12D alleles in response to pdx-1-cre expression in the mouse tail and pancreas. (C) (i)H&E, (ii)cytokeratin 19, (iii)amylase and (iv)insulin staining of a normal pancreas from a CKB2^Δ11/Δ11 mouse. (v)H&E, (vi)cytokeratin 19 and (vii)amylase staining of a metaplastic lesion from a CKB2^wt/wt mouse. (viii, xii)H&E staining of PanIN lesions from an 8-month old Kras^G12D mouse. (ix)H&E (x)cytokeratin 19 and (xi)amylase staining of a ductal tumor from a CKB2^Δ11/Δ11 mouse. (D,E) Quantitation of PanINs (D) and metaplastic lesions (E) from the pancreata of CKB2^wt/wt, CKB2^wt/Δ11 and CKB2^Δ11/Δ11 mice. Error bars represent SEM. (F) Tumor incidence in CKB2^Δ11/Δ11 mice (n=28), CKB2^wt/Δ11 mice (n=35) and CKB2^wt/wt mice (n=30). p- values were determined by a log rank test.

Figure 5. Tumors from CKB2^wt/wt, CPB2^wt/wt and CPB2^Δ11/Δ11 mice display differences in biomarker expression

Representative images of pancreatic tumors from CPB2^Δ11/Δ11, CPB2^wt/wt, and CKB2^wt/wt stained by IHC for (i–iii)Hes1, (iv–vi)Shh, (vii–ix)Ki-67, (x–xii)phospho-Erk1/2 and (xiii–xv)alcian blue dye.

Figure 6. Brca2 mutant alleles promote chromosomal instability and apoptosis in mouse pancreatic tissue and MEFs

(A) FISH analysis of pancreatic tissue from mice showing the percentage of cells with aneuploidy. (B) Metaphase spreads of MEFs scored for the presence of aneuploidy. (C) Percentage of MEFs with multinucleation measured by immunofluorescence microscopy after staining with α-tubulin antibody. (D,E). Quantitation of cleaved caspase 3 expression measured by IHC in the pancreas of 4 month old CPB2 and CB2 mice (D) and CKB2 (E) mice. Error bars represent SEM.

Figure 7. A model of BRCA2 deficient tumorigenesis in the pancreas

The model reflects germline inheritance of a BRCA2 mutation. Inactivation of Tp53 signaling precedes inactivation or loss of the 2^nd BRCA2 allele and facilitates cancer development. Early loss of the 2^nd BRCA2 allele prior to disruption of Tp53 is inconsistent with cell growth/cell survival and tumor formation. Activation of Kras or other oncogenes prior to disruption of the 2^nd BRCA2 allele is insufficient to maintain tumor formation if wildtype Tp53 signaling remains intact. Activation of oncogenes after inactivation of the 2^nd BRCA2 allele and Tp53 signaling may promote tumor development.