Unveiling the relationship between WWOX and BRCA1 in mammary tumorigenicity and in DNA repair pathway selection

Abstract

Breast cancer is the leading cause of cancer-related deaths in women worldwide, with the basal-like or triple-negative breast cancer (TNBC) subtype being particularly aggressive and challenging to treat. Understanding the molecular mechanisms driving the development and progression of TNBC is essential. We previously showed that WW domain-containing oxidoreductase (WWOX) is commonly inactivated in TNBC and is implicated in the DNA damage response (DDR) through ATM and ATR activation. In this study, we investigated the interplay between WWOX and BRCA1, both frequently inactivated in TNBC, on mammary tumor development and on DNA double-strand break (DSB) repair choice. We generated and characterized a transgenic mouse model (K14-Cre;Brca1^fl/fl;Wwox^fl/fl) and observed that mice lacking both WWOX and BRCA1 developed basal-like mammary tumors and exhibited a decrease in 53BP1 foci and an increase in RAD51 foci, suggesting impaired DSB repair. We examined human TNBC cell lines harboring wild-type and mutant BRCA1 and found that WWOX expression promoted NHEJ repair in cells with wild-type BRCA1. Our findings suggest that WWOX and BRCA1 play an important role in DSB repair pathway choice in mammary epithelial cells, underscoring their functional interaction and significance in breast carcinogenesis.

Fig. 1. Combined loss of Wwox and Brca1 results in basal- like mammary tumors in vivo.

A The K14-Cre;Brca1^fl/flWwox^fl/flTrp53^+/fl mice transgenic mouse model. K14-Cre conditionally expressed in the basal mammary epithelia, and basal epidermis. B Tumor frees survival curve of K14-Cre;Brca1^fl/fl (n = 6), K14-Cre;Brca1^fl/flWwox^fl/fl (n = 6) and K14-Cre;Brca1^fl/flWwox^fl/flTrp53^+/fl (n = 10) mice. Solid color- basal-like mammary tumor, yellow- cutaneous squamous cell carcinoma. Statistical significance calculated by Log-rank (Mantel-Cox) test. C H&E stain and IHC stain (WWOX, K14, ER) of normal mammary and mammary tumors from K14-Cre;Brca1^fl/fl, K14-Cre;Brca1^fl/flWwox^fl/fl and K14-Cre;Brca1^fl/flWwox^fl/fl mice. 40×. Scale- 200 µm.

Fig. 2. Combined loss of Wwox and Brca1 redirects the DSB repair to NHEJ in vivo.

A Left- Endogenous NHEJ DSB repair marked by 53BP1 foci in normal mammary or mammary tumors from: K14-Cre;WT (n = 3), K14-Cre;Brca1^fl/fl (n = 4), K14-Cre;Brca1^fl/flWwox^fl/fl (n = 3) and K14-Cre;Brca1^fl/flWwox^fl/flTrp53^+/fl (n = 8) mice. Arrow heads marking the foci. Merge- 160X scale- 10 µm, 750X scale- 20 µm. Right- quantification of 53BP1 foci per nuclei, three 40× fields were quantified from each mouse. Statistical analysis by t-test, P value < 0.05, error bars representing SEM. P value between normal and mammary tumor- not significant. B Left- Endogenous HDR DSB repair marked by RAD51 foci in normal mammary or mammary tumors from: K14-Cre;WT (n = 3), K14-Cre;Brca1^fl/fl (n = 4), K14-Cre;Brca1^fl/flWwox^fl/fl (n = 3) and K14-Cre;Brca1^fl/flWwox^fl/flTrp53^+/fl (n = 8). Arrow heads marking the foci. Merge- 160× scale- 10 µm, 750× scale- 20 µm. Right- quantification of RAD51 foci per nuclei, three 40X fields were quantified from each mouse. Statistical analysis by t-test, P value < 0.05, error bars representing SEM. P value between normal and mammary tumor- not significant.

Fig. 3. WWOX expression in BRCA1 WT TNBC cells elevates NHEJ as a response to direct DSB.

A In vitro experimental design. MDA-MB-231 or MDA-MB-436 TNBC cell line were electroporated with the See-Saw Repoter 2.0 system plasmid, 24 h later, a lenti virus expressing the systems endonuclease, IsceI, was added in addition to lenti- WWOX OE or lenti- empty vector (EV). 48 h later the cells were analyzed via flow cytometry. B WWOX immunoblot of MDA-MB 231 and MDA-MB 436 with empty vector (EV) and WWOX overexpression (OE). HSP90 as a house keeping gene. C Flow-cytometry scatter plot of the MDA-MB-231 or MDA-MB-436 with lenti EV or lenti WWOX OE and the See-Saw system. Living cells, expressing IsceI, presenting HDR marked by RFP or NHEJ repair marked by GFP. D Quantification graph of the NHEJ and HDR cell populations percentages from the flow-cytometry results. three experimental repeats, statistical significance calculated by t-test, P-value < 0.01, error bars representing SEM.

Fig. 4. Loss of WWOX significantly reduces HDR repair, especially when BRCA1 is present.

A HDR DSB repair marked by RAD51 foci in MDA-MB-231 TNBC cell line (sufficient BRCA1) with EV (left) or WWOX OE (right), with or without DNA damage agents (ATMi, cisplatin). B HDR DSB repair marked by RAD51 foci in MDA-MB-436 TNBC cell line (mutated BRCA1) with EV (left) or WWOX OE (right), with or without DNA damage agents (ATMi, Cisplatin). Scale: 120X-100uM, 600×-20uM. C Quantification of RAD51 foci per nuclei in MDA-MB-231 or 436 with EV or WWOX OE. At least 5 fields were quantified from each cell line and treatment. Statistical analysis by t-test, error bars representing SEM. D Fold change of RAD51 foci per nuclei treatment/ control of each of the cell lines. Statistical analysis by t-test, error bars representing SEM.

Fig. 5. Loss of WWOX doesn’t lead to more DNA damage but does increase NHEJ.

A DNA DSB (γH2AX) and NHEJ repair (53BP1) foci in MDA-MB-231 TNBC cell line with EV (left) or WWOX OE (right), with or without DNA damage agents (ATMi, cisplatin). B DNA DSB (γH2AX) and NHEJ repair (53BP1) foci in MDA-MB-436 TNBC cell line (mutated BRCA1) with EV (left) or WWOX OE (right), with or without DNA damage agents (ATMi, cisplatin). Scale: 120×-100μM, 600×-20μM. Quantification of γH2AX (C) and 53BP1 (E) foci per nuclei in MDA-MB-231 or 436 with EV or WWOX OE. At least 5 fields were quantified from each cell line and treatment, error bars representing SEM. Fold change of γH2AX (D) and 53BP1 (F) foci per nuclei treatment/ control of each of the cell lines. Statistical analysis by t-test, error bars representing SEM.

Fig. 6. Graphical abstract summarizing the role of WWOX in vivo and in vitro.

K14-Cre;Brca1^fl/flWwox^fl/fl mice harboring impaired HDR and insufficient NHEJ result in mammary tumor development, as compared to tumor-free K14-Cre; Brca1^fl/fl mice relying on NHEJ. MDA-MBA-231 cells with WT BRCA1, exhibit high levels of HDR, while 231-overexpressing WWOX shifts DSBs repair to NHEJ. MDA-MBA-436 with mutant BRCA1, exhibit low levels of HDR, while 436-overexpressing WWOX shifts DSBs repair to NHEJ mediated by unknown DDR player.