Inhibiting PLK1 activity in pathogenic BRCA1 variant cells reverses mutant phenotypes

Pathogenic mutations in the tumour suppressor gene BRCA1 hyperactivate PLK1, misorient cell divisions, and reduce luminal differentiation. These findings may explain the premalignant changes in the breast, and the type of tumors later produced in female BRCA1 mutation carriers.

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Young women who were born with a pathogenic Breast cancer 1 (BRCA1) mutation are at risk of developing breast cancer throughout their lifetime (1). Those who are diagnosed with cancer often develop a molecular subtype called “basal-like breast cancer” (2).  Despite its name, there is strong evidence to suggest that luminal progenitor cells, not basal cells, are the cell-of-origin for these tumours (3), generating interest in how these cells make the leap from normal and healthy, to cancerous. In addition, this type of breast cancer is often triple negative (2) - meaning that the tumour cells have little to no expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2- limiting treatment options for these patients. Understanding early changes that occur in pre-malignant BRCA1 tissue, or cells that have BRCA1 mutations, is critical to uncover new diagnostics and/or treatments for BRCA1 mutation carriers.

 In our new paper entitled “Pathogenic BRCA1 variants disrupt PLK1-regulation of mitotic spindle orientation” we make intriguing discoveries about how BRCA1 mutations impact cell division and differentiation in mammary cells isolated from young women and mice. We find that primary human cells that are heterozygous for BRCA1 mutations enter mitosis at an unusually fast rate, but fail to correctly orient their division axis. The orientation of a cell division in the mammary epithelium helps to regulate the fate of the two daughter cells by dictating their local environment (4). Consequently, if mammary epithelial cells fail to orient their division axes, the architecture of the mammary gland and the function of the daughter cells produced have the potential to change.  Using immortalized mammary epithelial cells that encode for a variety of different BRCA1 mutations, we show that cells with pathogenic mutations, but not those cells with benign mutations,  also acquire a defect in their division axis and fail to acquire mature luminal features. Lastly, we observe elevation in the activity of a mitotic kinase that regulates spindle orientation called Polo-like Kinase 1 (PLK1), in BRCA1 mutant cells. When we inhibit PLK1 activity through a small molecule inhibitor, we successfully reverse the mutant phenotypes caused by BRCA1 deficiency.

 We started by asking whether luminal progenitor cells from females with BRCA1 mutations display altered division axes compared to non-carrier controls. To answer this question, we grew cells on plates with L-shaped micropatterns coated with collagen. The L-shaped pattern provides adhesion cues to mitotic cells, so that normal, healthy cells divide along the hypotenuse of the pattern. We discovered that cells isolated from BRCA1 mutation carriers were unable to correctly orient their cell divisions along the hypotenuse like their wildtype counterparts could. Because the division axis plays a critical role in maintaining homeostasis in the mammary epithelium, we questioned if there is a connection between mutations that are clinically relevant, and that also misorient the division axis. Using MCF10A cells with different mutations that were either benign, pathogenic, or of uncertain clinical significance, we applied the same approach with L-shaped micropatterns to determine the impact that these mutations would have on the division axis of these cells. Excitingly, the cell lines with benign mutations were all able to orient their divisions, while cell lines with pathogenic mutations that expressed lower levels of the BRCA1 protein were unable to orient their division axis, suggesting a potential association between clinically relevant variants and the ability to orient cell divisions.

Cells dividing on L-shaped micropatterns. In the top row, a parental MCF10A cells is dividing along the hypotenuse.
In the bottom row, a pathogenic BRCA1 variant is dividing with a misoriented division axis.

After establishing that pathogenic BRCA1 variants misorient the cell division axis, we sought to gain insight into the mechanism by which loss of BRCA1 misorients divisions. Using MCF10A cells that express lower levels of BRCA1 through lentiviral transduced shBRCA1, we analyzed global protein expression compared to parental cells. We found that the mitotic kinase, PLK1, was overactive in the cells that have lower BRCA1 expression. This was an exciting discovery, as PLK1 activity on the spindle poles coordinates the position of the mitotic spindle apparatus (5). Accordingly, we asked whether inhibiting PLK1 with a low dose of a small molecule inhibitor could reposition the spindle and effectively reverse the impact of BRCA1 mutations on the cell division axis. After undertaking serial dilution experiments to ensure that exposing the cells to the drug would not affect viability, we discovered that indeed, inhibiting PLK1 in our BRCA1 mutant cells could re-orient cell divisions.

Finally, we set out to investigate the impact that misoriented cell divisions would have on luminal  differentiation, and if we could again rescue the phenotype by inhibiting PLK1 activity. We conducted a series of experiments to assess the impact that overexpressing PLK1, or under expressing BRCA1 would have on the ability of cultured cells to acquire luminal features. Aligning with our hypothesis, we observed that BRCA1 silenced or PLK1 overexpression colonies lacked typical features of luminal colonies, but inhibiting PLK1 in the BRCA1 silenced colonies promoted the colonies to gain luminal features. We then turned to using transgenic mice that lose expression of full length BRCA1 upon Cre recombination. After dissociating mammary tissue from these mice and transducing them with Cre, we cultured organoids from these mammary epithelial cells and assessed their abilities to acquire luminal features. Organoids grown from the cells without full length BRCA1 failed to acquire luminal features, but, after inhibiting PLK1, these organoids successfully developed luminal features, characterized by the expression of cytokeratin 8.

BRCA1 breast cancers are often characterized by lack of luminal features, leading to the belief that BRCA1 regulates differentiation in the mammary gland (2). BRCA1 cancer risk is also modified by genetic variation in HMMR (6) and DYNLL1 (7), genes that code for proteins that are involved in positioning the spindle with PLK1, implicating spindle positioning in BRCA1 tumourigenesis. The results from our paper reveal BRCA1 is involved in the intrinsic spindle positioning pathway, where, BRCA1 regulates PLK1 activity at spindle poles to orient cell divisions and modulate the acquisition of luminal features. We find that pathogenicity correlates with the inability to orient cell divisions, thus, the results depict a mechanism where pathogenic BRCA1 mutations lead to aberrant differentiation through misoriented divisions.

 

References:

1. Kuchenbaecker, K. B., Hopper, J. L., Barnes, D. R., Phillips, K. A., Mooij, T. M., Roos-Blom, M. J., ... & BRCA1 and BRCA2 Cohort Consortium. (2017). Risks of breast, ovarian, and contralateral breast cancer for BRCA1 and BRCA2 mutation carriers. Jama317(23), 2402-2416.

2. Foulkes, W. D., Smith, I. E., & Reis-Filho, J. S. (2010). Triple-negative breast cancer. New England journal of medicine363(20), 1938-1948.

3. Lim, E., Vaillant, F., Wu, D., Forrest, N. C., Pal, B., Hart, A. H., ... & Lindeman, G. J. (2009). Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nature medicine15(8), 907-913.

 4. Regan, J. L., Sourisseau, T., Soady, K., Kendrick, H., McCarthy, A., Tang, C., ... & Smalley, M. J. (2013). Aurora A kinase regulates mammary epithelial cell fate by determining mitotic spindle orientation in a Notch-dependent manner. Cell reports4(1), 110-123.

 5. Kiyomitsu, T., & Cheeseman, I. M. (2012). Chromosome-and spindle-pole-derived signals generate an intrinsic code for spindle position and orientation. Nature cell biology14(3), 311-317.

6. Maxwell, C. A., Benítez, J., Gómez-Baldó, L., Osorio, A., Bonifaci, N., Fernández-Ramires, R., ... & Pujana, M. A. (2011). Interplay between BRCA1 and RHAMM regulates epithelial apicobasal polarization and may influence risk of breast cancer. PLoS biology9(11), e1001199.

7. He, Y. J., Meghani, K., Caron, M. C., Yang, C., Ronato, D. A., Bian, J., ... & Chowdhury, D. (2018). DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells. Nature563(7732), 522-526.

Poster image Created with BioRender.com

Ryan Ghorayeb

Graduate Student, University of British Columbia