Anterior gradient-2 plays a critical role in breast cancer cell growth and survival by modulating cyclin D1, estrogen receptor-alpha and survivin.
BACKGROUND
Anterior-gradient 2 (AGR2) is an estrogen-responsive secreted protein. Its upregulation has been well documented in a number of cancers, particularly breast cancer, for which mixed data exist on the prognostic implications of AGR2 expression. Although emerging evidence indicates that AGR2 is associated with poor prognosis, its function and impact on cancer-relevant pathways have not been elucidated in breast cancer.


METHODS
To investigate the biologic role of AGR2 in breast cancer, AGR2 was transiently knocked down, by using siRNA, in T47 D and ZR-75-1 (estrogen receptor-alpha (ER)-positive) and MDA-MB-231 and SK-BR-3 (ER-negative) human breast cancer cell lines. The impact of silencing AGR2 was evaluated in both anchorage-dependent and anchorage-independent growth (soft agar, spheroid) assays. Cell-cycle profiles in ER-positive cell lines were determined with BrdU incorporation, and cell death was measured with Annexin V, JC-1, and F7-26 staining. After transiently silencing AGR2 or stimulating with recombinant AGR2, modulation of key regulators of growth and survival pathways was assessed with Western blot. Combination studies of AGR2 knockdown with the antiestrogens tamoxifen and fulvestrant were carried out and assessed at the level of anchorage-dependent growth inhibition and target modulation (cyclin D1, ER).


RESULTS
AGR2 knockdown inhibited growth in anchorage-dependent and anchorage-independent assays, with a more-pronounced effect in ER-positive cell lines. Cyclin D1 levels and BrdU incorporation were reduced with AGR2 knockdown. Conversely, cyclin D1 was induced with recombinant AGR2. AGR2 knockdown induced cell death in ZR-75-1 and T47 D cells, and also downregulated survivin and c-Myc. Evidence of AGR2-ER crosstalk was demonstrated by a reduction of ER at the protein level after transiently silencing AGR2. AGR2 knockdown in combination with fulvestrant or tamoxifen did not preclude the efficacy of the antiestrogens, but enhanced it. In addition, p-Src, implicated in tamoxifen resistance, was downregulated with AGR2 knockdown.


CONCLUSIONS
Transiently silencing AGR2 in ER-positive breast cancer cell lines inhibited cell growth and cell-cycle progression and induced cell death. Breast cancer drivers (ER and cyclin D1) as well as cancer-signaling nodes (pSrc, c-Myc, and survivin) were demonstrated to be downstream of AGR2. Collectively, the data presented support the utility of anti-AGR2 therapy in ER-positive breast cancers because of its impact on cancer-relevant pathways.
Vanderlaag
Kathryn E
KE
Schering-Plough Biopharma, 901 South California Avenue, Palo Alto, CA 94304, USA. kathy_vanderlaag@hotmail.com
Hudak
Susan
S
Bald
Laura
L
Fayadat-Dilman
Laurence
L
Sathe
Manjiri
M
Grein
Jeff
J
Janatpour
Mary J
MJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation; D003114:Colony-Forming Units Assay; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism*; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D000077267:Fulvestrant; D006801:Humans; D007114:Immunization; D051034:Inhibitor of Apoptosis Proteins; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D009088:Mucoproteins; D015513:Oncogene Proteins; D011506:Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D011917:Rats, Inbred Lew; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077022:Survivin; D013629:Tamoxifen / Q000494:pharmacology
C502296:AGR2 protein, human; C107489:BIRC5 protein, human; C530104:CCND1 protein, human; C506487:ESR1 protein, human; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009088:Mucoproteins; D015513:Oncogene Proteins; D011506:Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000077022:Survivin; D013629:Tamoxifen; D019938:Cyclin D1; D000077267:Fulvestrant; D004958:Estradiol