BRCA1 haploinsufficiency promotes chromosomal amplification under Fenton reaction-based carcinogenesis through ferroptosis-resistance

Abstract

Germline-mutation in BRCA1 tumor suppressor gene is an established risk for carcinogenesis not only in females but also in males. Deficiency in the repair of DNA double-strand breaks is hypothesized as a responsible mechanism for carcinogenesis. However, supporting data is insufficient both in the mutation spectra of cancers in the patients with BRCA1 germline-mutation and in murine knockout/knock-in models of Brca1 haploinsufficiency. Furthermore, information on the driving force toward carcinogenesis in BRCA1 mutation carriers is lacking. Here we applied Fenton reaction-based renal carcinogenesis to a rat heterozygously knockout model of BRCA1 haploinsufficiency (mutant [MUT] model; L63X/+). Rat MUT model revealed significant promotion of renal cell carcinoma (RCC) induced by ferric nitrilotriacetate (Fe-NTA). Array-based comparative genome hybridization of the RCCs identified significant increase in chromosomal amplification, syntenic to those in breast cancers of BRCA1 mutation carriers, including c-Myc, in comparison to those in the wild-type. Subacute-phase analysis of the kidney after repeated Fe-NTA treatment in the MUT model revealed dysregulated iron metabolism with mitochondrial malfunction assessed by expression microarray and electron microscopy, leading to renal tubular proliferation with iron overload. In conclusion, we for the first time demonstrate that biallelic wild-type BRCA1 provides more robust protection for mitochondrial metabolism under iron-catalyzed oxidative stress, preventing the emergence of neoplastic cells with chromosomal amplification. Our results suggest that oxidative stress via excess iron is a major driving force for carcinogenesis in BRCA1 haploinsufficiency, which can be a target for cancer prevention and therapeutics.

Keywords: BRCA1; Chromosomal amplification; Iron; Mitochondria; Renal cell carcinoma.

Graphical abstract

Fig. 1

Brca1 heterozygous knockout (L63X/+) in rat reveals promotional effect under ferric nitrilotriacetate (Fe-NTA)-induced renal carcinogenesis. (A) Probability of survival under Fe-NTA-induced renal carcinogenesis protocol in the wild-type (WT; n = 20) and Brca1 heterozygous knockout^L63X/+ (Mutant [MUT]; n = 19) groups (MUT vs WT; P < 0.05). Untreated groups of WT (n = 20) and MUT (n = 21) showed no renal cell carcinoma (RCC) during the experimental period (left panel; refer to Table 1). Probability of RCC-dependent death in the WT and MUT groups under Fe-NTA-induced renal carcinogenesis protocol (right panel; *P < 0.05). (B) RCC confirmed by computed tomography (CT) prior to euthanization and autopsy (top panel, transverse section CT; middle panel, coronal section CT; bottom panel, macroscopic appearance of the corresponding RCC). N, normal kidney; Ca, renal cell carcinoma. (C) A case of Fe-NTA-induced RCC in BRCA1 MUT rat (top panel, macroscopic appearance; middle panel, histology of the primary RCC; bottom panel, histology of pulmonary metastasis of RCC (bar = 200 μm; 50 μm in the inset). Li, liver; Lu, lung.

Fig. 2

Array-based comparative genome hybridization (aCGH) analysis discloses a preference to chromosomal amplification, including c-Myc amplification, in the Fe-NTA-induced RCCs in Brca1 MUT(L63X/+) rats in comparison to those in the WT rats. (A) Summary of aCGH analysis, which is divided into 4 groups of WT with no pulmonary metastasis (n = 4), WT with pulmonary metastasis (n = 4), MUT with no pulmonary metastasis (n = 4) and MUT with pulmonary metastasis (n = 4). Individual data on all the 16 RCCs are included in Fig. S2 and Table S2. Blue arrowhead, gene-desert area of chromosome 15 with contrasting difference in genomic alteration between WT and MUT RCCs. (B) Representative fluorescent in situ hybridization (FISH) analysis of c-Myc. c-Myc signal/chromosome 7 centromere signal = 7/2 (left 4 panels); extrachromosomal area (micronucleus) includes c-Myc amplification (dotted white circle; #C10K3LRCC; right 4 panels). Representative pictures are shown. Refer to text for details.

Fig. 3

Expression microarray analysis at the subacute phase of Fe-NTA-induced renal carcinogenesis sorts out mitochondrial and iron metabolisms as candidate responsible pathways in Brca1 MUT(L63X/+) rat. (A) Results of hierarchical clustering and GO term analysis for the genes differentially expressed between WT and Brca1 MUT at 3 weeks in the Fe-NTA-induced renal carcinogenesis protocol (refer to Table S4). (B) Renal Brca1 expression during the subacute phase of Fe-NTA-induced renal carcinogenesis protocol with qPCR (left panel; means ± SD) and immunoblot analysis (right panels). (C) Examples of altered metabolisms and mitochondrial dysfunction with expression microarray analysis specifically in the kidney of Brca1 MUT rats during the subacute phase of Fe-NTA-induced renal carcinogenesis (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 vs wild-type Fe-NTA renal carcinogenesis protocol at 3 weeks). Refer to text for details.

Fig. 4

Mitochondrial dysregulation and malfunction in the kidney of in Brca1 MUT(L63X/+) rat. (A, B) Electron microscopic analysis reveals increased lysosome*/autophagosome** in the renal tubular cells, and small-sized deformed mitochondria with immature cristae formation in the MUT rat in the untreated control (yellow arrowheads) and at 3 weeks in the Fe-NTA-induced renal carcinogenesis protocol (blue arrowheads; bar = 2.0 μm in the left panels; 500 nm in the right panels). (C) Round rate of mitochondria defined as the ratio of the minor axis to the major axis of mitochondria. CTRL, no treatment control; NW, no treatment wild-type; NM, no treatment MUT; FW, Fe-NTA-treated wild-type at 3 weeks; FM, Fe-NTA-treated MUT at 3 weeks (n = 4–30; ns, not significant, *P < 0.05, ***P < 0.001 vs wild-type). (D) Decreased expression of mitochondrial regeneration/fission-associated genes in the kidney of Brca1 MUT rats during the subacute phase of Fe-NTA-induced renal carcinogenesis (n = 3; *P < 0.05 vs wild-type). Also refer to Fig. S4 for additional data. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Excess iron accumulation in the kidney of Brca1 MUT(L63X/+) rat during Fe-NTA-induced renal carcinogenesis. (A) Iron deposition by Perl's iron staining and its quantitation. (B) Immunohistochemical/immunoblot detection of transferrin (Tf). (C) Immunoblot analysis of transferrin receptor 1 (Tfr1) and ferritin heavy chain (Fth1; n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 vs wild-type Fe-NTA at 3 weeks; bar = 100 μm; 50 μm in the inset in A; 200 μm; 50 μm in the inset in B).

Fig. 6

BRCA1 haploinsufficiency provides both nuclear mutagenic and cytoplasmic ferroptosis-resistant environments. (A) Immunostaining shows higher amounts of 8-OHdG [41] both in the untreated control kidney and in the kidney at 3 weeks of the Fe-NTA-induced renal carcinogenesis protocol of Brca1 MUT(L63X/+) rat in comparison to WT rat. (B) Ki-67 immunostaining as proliferation index was higher in the Brca1 MUT kidney than WT at 3 weeks in Fe-NTA-induced renal carcinogenesis protocol. (C) Immunostaining shows higher amounts of γ-H2AX (marker of DNA double-strand breaks [42]) in the kidney at 3 weeks of the Fe-NTA-induced renal carcinogenesis protocol of both WT and Brca1 MUT rats. However, MUT group reveals significantly lower immunostaining than WT group due to BRCA1 haploinsufficiency, suggesting impaired recognition/repair of DNA double-strand breaks induced by Fe-NTA. Refer to text for details. (D) Immunohistochemistry by HNEJ-1 monoclonal antibody [29] reveals significantly lower positivity in the kidney at 3 weeks of the Fe-NTA-induced renal carcinogenesis protocol in the Brca1 MUT(L63X/+) rats than WT, indicating that BRCA1 haploinsufficiency more efficiently generates ferroptosis-resistance in response to iron catalyzed oxidative stress (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 vs wild-type Fe-NTA at 3 weeks; bar = 200 μm; 50 μm in the inset in A, C and D; 100 μm; 50 μm in the inset in B).

Fig. 7

Schematic presentation of the present work.