Prolactin modulates TNBC aggressive phenotype limiting tumorigenesis

in Endocrine-Related Cancer
Correspondence should be addressed to S Ali: suhad.ali@mcgill.ca
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Triple-negative breast cancer (TNBC) accounts for ~20% of all breast cancer cases. The management of TNBC represents a challenge due to its aggressive phenotype, heterogeneity and lack of targeted therapy. Loss of cell differentiation and enrichment with breast cancer stem-like cells (BCSC) are features of TNBC contributing to its aggressive nature. Here, we found that treatment of TNBC cells with PRL significantly depletes the highly tumorigenic BCSC subpopulations CD44+/CD24 and ALDH+ and differentiates them to the least tumorigenic CD44/CD24 and ALDH phenotype with limited tumorsphere formation and self-renewal capacities. Importantly, we found PRL to induce a heterochromatin phenotype marked by histone H3 lysine 9 trimethylation (H3K9me3) and accompanied by ultra-structural cellular architecture associated with differentiation and senescence rendering the cells refractory to growth signals. Crucially, we found PRL to mediate these effects in vivo in a pre-clinical animal xenograft of TNBC controlling tumor growth. These results reveal that the lactogenic hormone PRL may exert its anti-tumorigenic effects on TNBC through cellular reprogramming indicative of differentiation resulting in the depletion of BCSCs and restricting tumorigenesis.

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    PRL suppresses the CD44+/CD24 and ALDH+ BCSCs in TNBC cells. (A) BCSC subpopulations (%) present in MDA-MB-231 WT, MDA-MB-231/vector, MDA-MB-231/PRLR and MDA-MB-453 cells. A representative analysis of three independent experiments is shown. (B) MDA-MB-231/PRLR cells were treated or not with dox (100 ng/mL) and hPRL (250 ng/mL) for 72 h. Cells were then immunostained for CD44 and CD24 and processed for flow cytometry analysis (Material and methods). Left panel depicts representative dot plots. Middle panel incorporates fluorescent intensity of all subpopulations treated (orange) or not (blue) with hPRL. Representative histograms of CD44 and CD24 distribution levels are depicted. Right panels show quantification (%) analysis of CD44+/CD24− BCSC subpopulation. Data represent the mean ± s.e.m. of triplicates of three independent experiments. (C) MDA-MB-453 cells were treated or not with hPRL (250 ng/mL) for 5 days and ALDH positivity was assessed by ALDEFLOUR assay followed by flow cytometry analysis (Material and methods). Left panel shows a representative dot plot of ALDEFLOUR activity in the presence or absence of hPRL using DEAB-treated cells as a control. Right panel, quantification % of ALDH+ subpopulation, is depicted. Data represent the mean ± s.e.m. of triplicates of three independent experiments.

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    PRL inhibits stemness and viability of TNBC BCSCs. (A) MDA-MB-231/PRLR cells were treated or not with dox (100 ng/mL) and hPRL (250 ng/mL) for 72 h and gene expression of stem cell markers and transcription factors (CD24, CD44, Oct4, Sox2 and Nanog) were examined using RT-qPCR (P < 0.05). Results are expressed as log2 fold change of triplicates of three independent experiments. (B) MDA-MB-453 cells were treated or not with hPRL (250 ng/mL) for 5 days and the expression of stem cell markers and transcription factors (CD24, CD44, Oct4, Sox2 and Nanog) was examined using RT-qPCR (P < 0.05). Results are expressed as log2 fold change of triplicates of three independent experiments. (C) MDA-MB-231/vector and MDA-MB-231/PRLR were treated or not with dox (100 ng/mL) and hPRL (250 ng/mL) for 72 h before tumorsphere formation assay was performed for 7 days. Left and right panels depict primary and secondary tumorsphere formation capacity (Material and methods). Data represent the mean ± s.e.m. of triplicates of three independent experiments. Representative microphotographs of primary and secondary tumorspheres are shown. (D) MDA-MB-453 cells were treated or not with hPRL (250 ng/mL) for 5 days before tumorsphere formation assay was performed for 7 days. Left and right panels depict primary and secondary tumorsphere formation, respectively. Data represent the mean ± s.e.m. of triplicates of three independent experiments. Representative microphotographs of primary and secondary tumorspheres respectively are shown. (E) CD44+/CD24, CD44+/CD24+ and CD44/CD24 cell subpopulations were sorted from MDA-MB-231/PRLR and subjected to tumorsphere formation assay under hPRL stimulation. Upper panel, representative microphotographs of primary tumorspheres are shown. Lower panel, quantification data (%) represents the mean ± s.e.m. of triplicates of three independent experiments. (F) ALDH+ subpopulation isolated from MDA-MB-453 was subjected to tumorsphere formation assay under hPRL stimulation. Upper panel, representative microphotographs of primary tumorspheres are shown. Lower panel, quantification data (%) represents the mean ± s.e.m. of triplicates of three independent experiments. (G) The CD44+/CD24 cell subpopulation sorted from MDA-MB-231/PRLR were plated and treated or not with hPRL (250 ng/mL) for 24, 48 and 72 h. MTT assays were performed and the results are presented as means ± s.e.m. for triplicates of three independent experiments. (H) ALDH+ cell subpopulation was isolated from MDA-MB-453 and treated or not with hPRL (250 ng/mL) for 5 days. MTT assays were performed and the results are presented as means ± s.e.m. for triplicates of three independent experiments.

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    Prolactin induces cell cycle arrest and correlates positively with cell cycle regulators in TNBC patients. (A) MDA-MB-231/PRLR cells were synchronized and treated with dox (100 ng/mL) overnight and then treated or not with hPRL (250 ng/mL) for 72 h followed by cell cycle analysis. Data represent the mean ± s.e.m. of triplicates of one representative experiment. (B) MDA-MB-453 cells were synchronized and treated or not with hPRL (250 ng/mL) for 5 days followed by cell cycle analysis. Data represent the mean ± s.e.m. of triplicates of one representative experiment. (C) MDA-MB-231/PRLR cells were treated or not with dox (100 ng/mL) and hPRL (250 ng/mL) for 72 h and the expression of cell cycle-associated genes (RB, p21, p16, p15, INK4C and cyclin E) were examined using qRT-PCR (P < 0.05). Results are presented as means ± s.e.m. for triplicates of three independent experiments. (D) MDA-MB-453 cells were treated or not with hPRL (250 ng/ml) for 5 days and the expression of cell cycle-associated genes (RB, p21, p16, p15 and INK4C) were examined using RT-qPCR (P < 0.05). Results are presented as means ± s.e.m. for triplicates of three independent experiments. (G) The correlation between RB1 gene expression levels and PRL, PRLR, Jak2 and Stat5a in 374 TNBC samples using the correlation analysis tool of bc-GenExMiner4.1 database. (F) Correlation between PRLR gene expression levels and cyclin E in TNBC patient samples using Pearson’s pairwise correlation plot and heat map in bc-GenExMiner4.1 database.

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    PRL induces H3K9me3 heterochromatin formation and senescence-associated ultra-structural phenotype in TNBC cells. (A) H3K9me3 staining was assessed using confocal microscopy in MDA-MB-231/PRLR and MDA-MB-453 cells following hPRL stimulation. Right panels depict H3K9me3-DAPI colocalization. Data are presented as mean ± s.e.m. of three independent experiments. (B) MDA-MB-231/PRLR and MDA-MB-453 cells were stimulated or not with hPRL (250 ng/mL) for 72 h and 5 days, respectively. Cell lysates were immune detected using antibodies to H3K9me3 and β-tubulin. Data are presented as mean ± s.e.m. of three independent experiments. (C) MDA-MB-231/PRLR and MDA-MB-453 cells were treated or not with hPRL (250 ng/mL) for 72 h and 5 days, respectively. Gene expression of SUV39H1 and HP1were examined using RT-qPCR. Results are presented as means ± s.e.m. for triplicates of three independent experiments (P < 0.05). (D) MDA-MB-453 cells were treated or not with hPRL (250 ng/mL) for 5 days and processed for EM. Left panel depicted EM images of MDA-MB-453 untreated cells (2 different cells); nuclear membrane (nm), nucleolus (nl), mitochondria (mt), lysosome (ly) and rough endoplasmic reticulum (rr) can be appreciated. Right panel represents EM images of MDA-MB-453 cells treated with PRL for 5 days (3 different cells); multi-lobulated nucleus (**n), loss on continuity in the nuclear membrane (**nm), irregular mitochondria, Golgi apparatus and rough endoplasmic reticulum patterns (**mt, **go, **rr), presence of fatty vesicles (**fv). (E) MDA-MB-231/PRLR and MDA-MB-453 cells were treated or not with hPRL (250 ng/mL) for 72 h and 5 days, respectively. Lamin B1 gene expression was examined using RT-qPCR. Data are presented as mean ± s.e.m. of three independent experiments (P < 0.05). (F) MDA-MB-231/PRLR cells were treated or not with dox (100 ng/mL) and hPRL (250 ng/mL) for 72 h followed by assessment of positive SAβ-gal staining. Data are presented as mean ± s.e.m. of triplicates of three independent experiments. Representative images are shown. (G) MDA-MB-453 cells were treated or not with hPRL (250 ng/mL) for 5 days followed by assessment of positive SAβ-gal staining. Data are presented as mean ± s.e.m. of triplicates of three independent experiments. Representative images are shown. (H) MDA-MB-231/PRLR cells were plated and treated or not treated with dox (100 ng/mL) and hPRL (250 ng/mL) for 72 h. Next, cells were re-plated in full growth media (DMEM-10% FBS) for 1 week. MTT assays were performed and the results are presented as means ± s.e.m. of triplicates of three independent experiments (P = 0.0001). (I) MDA-MB-453 cells were plated and treated or not treated with hPRL (250 ng/mL) for 5 days. Next, cells were re-plated in full growth media (DMEM-10% FBS) for 1 week. MTT assays were performed and the results are presented as means ± s.e.m. of triplicates of three independent experiments (P = 0.0001).

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    PRL suppresses tumor growth and markers of proliferation and stemness while induces genomic heterochromatin hypermethylation in vivo. (A) Graph depicting tumor volume of xenografts of MDA-MB-231/Vector and MDA-MB-231/PRLR treated or untreated with hPRL. (B) Representative pictures of NOD/SCID mice bearing tumors (upper panel). Pictures are shown of dissected tumors from the different experimental groups (lower panel). Table: Indicates the number of mice injected with cancer cells and the number of mice that showed tumor development. (C) Immunohistochemical staining and quantification of PRLR expression in tumors xenografts is shown (4× and 40×). (D) Immunohistochemical staining and quantification of CD44 expression in tumors xenografts is shown (4× and 40×). (E) Immunohistochemical staining and quantification of Ki67 expression in tumors xenografts is shown (4× and 40×). (F) Immunohistochemical staining and quantification of H3K9m3 expression in tumors xenografts is shown (4× and 40×).

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