Heat shock protein-90-alpha, a prolactin-STAT5 target gene identified in breast cancer cells, is involved in apoptosis regulation.
BACKGROUND
The prolactin-Janus-kinase-2-signal transducer and activator of transcription-5 (JAK2-STAT5) pathway is essential for the development and functional differentiation of the mammary gland. The pathway also has important roles in mammary tumourigenesis. Prolactin regulated target genes are not yet well defined in tumour cells, and we undertook, to the best of our knowledge, the first large genetic screen of breast cancer cells treated with or without exogenous prolactin. We hypothesise that the identification of these genes should yield insights into the mechanisms by which prolactin participates in cancer formation or progression, and possibly how it regulates normal mammary gland development.


METHODS
We used subtractive hybridisation to identify a number of prolactin-regulated genes in the human mammary carcinoma cell line SKBR3. Northern blotting analysis and luciferase assays identified the gene encoding heat shock protein 90-alpha (HSP90A) as a prolactin-JAK2-STAT5 target gene, whose function was characterised using apoptosis assays.


RESULTS
We identified a number of new prolactin-regulated genes in breast cancer cells. Focusing on HSP90A, we determined that prolactin increased HSP90A mRNA in cancerous human breast SKBR3 cells and that STAT5B preferentially activated the HSP90A promoter in reporter gene assays. Both prolactin and its downstream protein effector, HSP90alpha, promote survival, as shown by apoptosis assays and by the addition of the HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in both untransformed HC11 mammary epithelial cells and SKBR3 breast cancer cells. The constitutive expression of HSP90A, however, sensitised differentiated HC11 cells to starvation-induced wild-type p53-independent apoptosis. Interestingly, in SKBR3 breast cancer cells, HSP90alpha promoted survival in the presence of serum but appeared to have little effect during starvation.


CONCLUSIONS
In addition to identifying new prolactin-regulated genes in breast cancer cells, we found that prolactin-JAK2-STAT5 induces expression of the HSP90A gene, which encodes the master chaperone of cancer. This identifies one mechanism by which prolactin contributes to breast cancer. Increased expression of HSP90A in breast cancer is correlated with increased cell survival and poor prognosis and HSP90alpha inhibitors are being tested in clinical trials as a breast cancer treatment. Our results also indicate that HSP90alpha promotes survival depending on the cellular conditions and state of cellular transformation.
Perotti
Christian
C
Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, T2N 1N4, Canada.
Liu
Ruixuan
R
Parusel
Christine T
CT
Böcher
Nadine
N
Schultz
Jörg
J
Bork
Peer
P
Pfitzner
Edith
E
Groner
Bernd
B
Shemanko
Carrie S
CS
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D016227:Benzoquinones / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics*; D002454:Cell Differentiation; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D015723:Gene Library; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D011388:Prolactin / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; C533585:HSP90AA2P protein, human; D047029:Lactams, Macrocyclic; D012333:RNA, Messenger; D050799:STAT5 Transcription Factor; C494095:STAT5B protein, human; D016159:Tumor Suppressor Protein p53; C112765:tanespimycin; D011388:Prolactin; D008156:Luciferases; C507924:JAK2 protein, human; D053614:Janus Kinase 2