Triple SILAC identified progestin-independent and dependent PRA and PRB interacting partners in breast cancer

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          Progesterone, an important ovarian steroid hormone regulating sexual differentiation and reproductive development,  was isolated by Willard M. Allen and George W. Corner more than 90 years ago (1). Its biological functions are mediated through its cognate receptor - progesterone receptor (PR).  In breast cancer, PR is used as a predictive marker in conjunction with Estrogen receptor (ER) for breast cancer classification and prognosis.  Expression of PR is a good prognosis factor in ER-positive breast cancer (2).  PR primarily expresses as two isoforms, PRA and PRB.  These two isoforms have similar structures, except the PRA is shorter than PRB by 164 amino acids at the N-terminus (3). Significantly, these two isoforms are differentially modulated breast cancer progression.    Breast cancer patients with a high PRA/PRB ratios are associated with poor prognosis and resistance to treatments (4, 5).  Several studies uncovered the different biological functions of PRA and PRB in breast cancer.  However, the exact mechanisms and how they altered breast cancer biologies remain unclear.  

            Our research laboratory has long been interested in uncovering novel functions of the progesterone receptor. One of our laboratory projects aims to elucidate how PRA and PRB differentially modulate breast cancer biologies.      We first identified proteins that are differentially expressed in breast cancer cells expressing either PRA or PRB, both in the presence or absence of progestins (6). However, to better understand how PRA and PRB mediate different biological functions, we seek to identify PRA- and PRB- interacting partners in the presence and absence of progestin in breast cancer cells.  We constructed a tetracycline-inducible PRA or PRB breast cancer cell model.  Both PRA and PRB were tagged with an HA-tag at the C-terminus.  Cells expressing inducible PRA or PRB exhibited normal PR transcriptional and biological functions.  Co-immunoprecipitation (Co-IP) technique was used to isolate individual PRA or PRB complexes containing PR interacting partners.  However, the challenge for this project is how we specifically identify PRA or PRB interacting partners, as traditional CO-IP often pulls down the non-specific binding in IgG control.  

            Thanks to our research team - Dr. Gyorgy Hutvagner, Dr. Eileen M. McGowan, Dr. Matthew P. Padula, and Dr. Sarah Bajan at the University of Technology Sydney, Australia, for their tremendous assistance with the advanced labeling mass-spectrometer technology to help us specifically identify PRA and PRB interacting proteins. 

            In this study, we applied affinity purification coupled with stable isotope labeling of amino acids in cell culture (SILAC) mass spectrometry technique to identify PRA and PRB interacting partners comprehensively. We performed both forward and reversed SILAC strategies to eliminate false interacting partners to achieve high confidence PRA and PRA interacting protein lists.  The dataset obtained from this study will be helpful in mapping PRA- and PRB-specific molecular networks and uncover novel differential PRA and PRB networks.          A better understanding of how PRA and PRB differentially mediated different biological functions of breast cancer may help develop a novel therapeutic strategy for breast cancer in the future.

Our full paper is available here: https://doi.org/10.1038/s41597-021-00884-0

 

 

References

  1. Ali S, Balachandran K, O'Malley B. 90 Years of progesterone: Ninety years of progesterone: the 'other' ovarian hormone. J Mol Endocrinol. 2020;65(1):E1-E4.
  2. Bardou VJ, Arpino G, Elledge RM, Osborne CK, Clark GM. Progesterone receptor status significantly improves outcome prediction over estrogen receptor status alone for adjuvant endocrine therapy in two large breast cancer databases. J Clin Oncol. 2003;21(10):1973-9.
  3. Graham JD, Yeates C, Balleine RL, Harvey SS, Milliken JS, Bilous AM, et al. Characterization of progesterone receptor A and B expression in human breast cancer. Cancer Res. 1995;55(21):5063-8.
  4. Mote PA, Bartow S, Tran N, Clarke CL. Loss of co-ordinate expression of progesterone receptors A and B is an early event in breast carcinogenesis. Breast Cancer Res Treat. 2002;72(2):163-72.
  5. Hopp TA, Weiss HL, Hilsenbeck SG, Cui Y, Allred DC, Horwitz KB, et al. Breast cancer patients with progesterone receptor PR-A-rich tumors have poorer disease-free survival rates. Clin Cancer Res. 2004;10(8):2751-60.
  6. Pateetin P, Pisitkun T, McGowan E, Boonyaratanakornkit V. Differential quantitative proteomics reveals key proteins related to phenotypic changes of breast cancer cells expressing progesterone receptor A. The Journal of Steroid Biochemistry and Molecular Biology. 2020;198:105560.

Prangwan Pateetin

Postdoctoral fellow, Chulalongkorn University