Genetic Screening Reveals an Essential Role of p27kip1 in Restriction of Breast Cancer Progression

Abstract

The genetic changes and mechanisms underlying the progression of estrogen-dependent breast cancers to estrogenindependent, antiestrogen-resistant, and metastatic breast cancers are unclear despite being a major problem in endocrine therapy. To identify genes responsible for this progression, we carried out a genetic screening by an enhanced retroviral mutagen (ERM)-mediated random mutagenesis in the estrogen-dependent T47D breast cancer cells. We found that T47D cells contain only one p27kip1 (p27) allele coding for the p27 cyclin-dependent kinase (CDK) inhibitor. An ERM insertion into the p27 locus of T47D cells disrupted the p27 gene and created estrogen-independent and antiestrogenresistant breast cancer cells that still maintained functional estrogen receptors. Disruption of p27 in T47D cells resulted in several changes, and most of these changes could be rescued by p27 restoration. First, CDK2 activity was increased in the absence of estrogen or in the presence of estrogen antagonists tamoxifen or ICI 182780; second, amplified in breast cancer 1 (AIB1), a cancer overexpressed transcriptional coactivator, was hyperphosphorylated, which made AIB1 a better coactivator for E2F1; and third, growth factor receptor binding protein 2-associated binder 2 (Gab2) and Akt activity were increased following E2F1 overactivation, leading to a significant enhancement of cell migration and invasion. Furthermore, the p27-deficient cells, but not T47D control cells, developed lung metastasis in an ovarian hormone-independent manner when they were i.v. injected into nude mice. In sum, loss of p27 activated AIB1, E2F1, Gab2, and Akt; increased cell migration and invasion; caused antiestrogen insensitivity; and promoted metastasis of breast cancer cells. These findings suggest that p27 plays an essential role in restriction of breast cancer progression. [Cancer Res 2007; 67(17):8032-42]

Siah1/SIP regulates p27kip1stability and cell migration under metabolic stress

“…Moreover, accumulation of cytoplasmic p27 protein has been recognized to be associated with tumor malignancies, suggesting a role in tumor invasion. 16,17,43,44 Therefore, we explored the effect of SIP on cell motility. Initially, cell migration was monitored using transwell chambers that were seeded with MEFs derived from wild-type or SIP -/-embryos using low or high serum that was supplemented (or not) with glucose.…”

The TWIST/Mi2/NuRD protein complex and its essential role in cancer metastasis

“…8-to 12-week-old female BALB/cJ mice from Jackson Laboratory were anesthetized with 2.5% Avertin (0.1 ml/10 g, i.p.). For each recipient mouse, 4T1 mammary tumor cells (2 × 10 5 ) in 200 µl of DME-10 medium were injected into the 4th pair of mammary glands (100 µl for each gland) as described [51]. Tumor growth was measured weekly.…”

“…At the experimental endpoint, the weights of local mammary tumors were measured. The lung metastasis was carefully examined as described [51]. To determine the number of tumor cells in the blood, 150 µl of blood sample was collected from the heart of each mouse and cultured for 3 weeks in a selective medium for growth of puromycin-resistant 4T1 cells.…”

Effect of arenobufagin on human pancreatic carcinoma cells

“…Total protein (50 µg per well) was separated using 8-12% SDS-PAGE and transferred to nitrocellulose transfer membranes. The membranes were blocked in TBST (10 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.1% Tween-20) with 5% non-fat milk for 2 h. The membranes were then incubated with specific primary antibodies (all antibodies were diluted at a ratio of 1:1,000) overnight at 4˚C, followed by treatment with HRP-linked secondary antibodies (24). The protein bands were visualized using ECL agent (Thermo Fisher Scientific, Inc.) and detected using a Gel imaging system (Bio-Rad Laboratories, Inc., Hercules, California, USA).…”

“…Samples were analyzed using a FACSC flow cytometer. Additional analyses were processed by Flow Jo software (Tree Star, Inc., Ashland, OR, USA) (24).…”