Tamoxifen-stimulated growth of breast cancer due to p21 loss

Abstract

Tamoxifen is widely used for the treatment of hormonally responsive breast cancers. However, some resistant breast cancers develop a growth proliferative response to this drug, as evidenced by tumor regression upon its withdrawal. To elucidate the molecular mediators of this paradox, tissue samples from a patient with tamoxifen-stimulated breast cancer were analyzed. These studies revealed that loss of the cyclin-dependent kinase inhibitor p21 was associated with a tamoxifen growth-inducing phenotype. Immortalized human breast epithelial cells with somatic deletion of the p21 gene were then generated and displayed a growth proliferative response to tamoxifen, whereas p21 wild-type cells demonstrated growth inhibition upon tamoxifen exposure. Mutational and biochemical analyses revealed that loss of p21's cyclin-dependent kinase inhibitory property results in hyperphosphorylation of estrogen receptor-alpha, with subsequent increased gene expression of estrogen receptor-regulated genes. These data reveal a previously uncharacterized molecular mechanism of tamoxifen resistance and have potential clinical implications for the management of tamoxifen-resistant breast cancers.

Fig. 1.

Loss of p21 expression correlates with tamoxifen-induced breast cancer growth. (A) Metastatic skin nodule (arrowhead, Upper) while the patient was receiving tamoxifen (+Tam) and residual disease (arrowhead, Lower) 3 months after tamoxifen cessation (−Tam). (B) H&E staining (Left) and p21 immunohistochemical labeling (Right) of tumor samples taken from the patient's primary tumor, colon, and skin. Arrowheads (Bottom Right) denote positive p21 labeling within sweat glands serving as an internal control.

Fig. 2.

Loss of p21's CDK inhibitory function leads to growth stimulation by tamoxifen. (A) Cells were plated at equal density and cultured for 6 days with 10 nM 17-β-estradiol (E), ethanol (Vehicle), 1 μM 4-OH-tamoxifen (T), 1 μM ICI 182,780 (ICI) or combinations of these drugs as indicated and then stained with crystal violet to assess growth. The p21 and ER status of each cell line is shown. Results are representative of six independent experiments using two independently derived ERIK clones. (B) ERIK (ER-positive, p21-negative) cells were transiently transfected with parental plasmid DNA (Plasmid Control), wild-type p21 cDNA (wt p21) or p21 cDNA devoid of CDK inhibitory activity (p21 cdk−) and then grown with 10 nM 17-β-estradiol (E) or 1 μM 4-OH-tamoxifen (T) and stained with crystal violet after six days to assess growth. Results are representative of three independent experiments.

Fig. 3.

ER Serine 118 hyperphosphorylation is required for tamoxifen-induced growth in p21 null ERIK cells. (A) Cells were cultured in either assay media with vehicle control alone (A), or supplemented with 10 nM 17-β-estradiol (E), 1 μM 4-OH-tamoxifen (T), or a combination of the two (ET). Total ER, serine 118 phosphorylated ER (ER Ser118), and GAPDH protein levels were assessed by Western blotting. Results are representative of two independent clones for each ERIN and ERIK cell line. (B) MCF-10A p21 null cells were transfected with an ER cDNA containing a serine-to-alanine mutation at codon 118 as described in Materials and Methods. Growth assays were performed as in Materials and Methods except cells were cultured for 12 days. Cells were seeded at equal density and were cultured in either assay media with vehicle control alone (A), or supplemented with 10 nM 17-β-estradiol (E), 1 μM 4-OH-tamoxifen (T), or a combination of the two (ET). Results are representative of three independent experiments.

Fig. 4.

Loss of p21 leads to hyperphosphorylation of ER serine 118 in breast cancer cells growth stimulated by tamoxifen. (A) Parental MCF-7 and TAM1 cells were cultured with 1 μM 4-OH-tamoxifen or vehicle control and then subjected to Western blot analysis to assess protein levels of total ER, serine 118 phosphorylated ER (ER Ser118), total p21, and β-actin as described in Materials and Methods. Nonphosphorylated (Unbound) p21 and phosphorylated (Bound) p21 proteins were separated, and the collected fractions were further concentrated for Western blot analysis of p21 protein as described in Materials and Methods. (B) Immunohistochemical labeling for ER (Total ER) and serine 118 phosphorylated ER (ER Ser118) in metastatic recurrent lesions of the colon during which time the patient was not receiving tamoxifen (−), versus the metastatic skin lesion during which time the patient was on tamoxifen therapy (+). Arrowheads denote tumor nuclei.