Ligand-dependent down-regulation that leads to rapid and extensive loss of protein is characteristic of several nuclear steroid receptors, including human progesterone receptors (PRs). In breast cancer cells, >95% of PRs are degraded 6 h after the start of progestin treatment. The mechanism for down-regulation is unknown. We examined the role of PR phosphorylation by mitogenactivated protein kinases (MAPKs) in this process. Lactacystin and calpain inhibitor I, specific inhibitors of the 26S proteasome, blocked progestin-induced down-regulation, and ubiquitinated conjugates of PR accumulated in cells. Ligand-dependent PR degradation was also blocked by specific inhibition of p42 and p44 MAPKs. To define the targets of phosphorylation by this kinase, two serine͞proline MAPK consensus sites on PR were mutated. We demonstrate that mutation of PR serine-294 to alanine (S294A) specifically and completely prevents ligand-dependent receptor down-regulation. We also find that rapid, ligand-independent degradation of immature PR intermediates occurs by a proteasome-mediated pathway. These results demonstrate that PR destruction, by either of two alternate routes, is mediated by the 26S proteasome. Specifically, down-regulation of mature PRs occurs by a mechanism in which ligand binding activates PR phosphorylation by MAPKs at a unique serine residue, which then targets the receptors for degradation.

Breast cancers often exhibit elevated expression of tyrosine kinase growth factor receptors; these pathways influence breast cancer cell growth in part by targeting steroid hormone receptors, including progesterone receptors (PR). To mimic activation of molecules downstream of growth factor-initiated signaling pathways, we overexpressed mitogen-activated protein kinase (MAPK; also known as extracellular signal-regulated kinase) kinase kinase 1 (MEKK1) in T47D human breast cancer cells expressing the B isoform of PR. MEKK1 is a strong activator of p42 and p44 MAPKs. MEKK1 expression increased progestin-mediated transcription 8-to 10-fold above normal PR-driven transcription levels. This was dependent on the presence of a progesterone response element and functional PR. PR protein levels were unchanged by MEKK1 alone but were extensively down-regulated by MEKK1 plus the progestin R5020. MEKK1 expression resulted in phosphorylation of PR on Ser294, a MAPK consensus site known to mediate ligand-dependent PR degradation. MEK inhibitors blocked phosphorylation of Ser294 and attenuated PR transcriptional hyperactivity in response to MEKK1 plus R5020; stabilization of PR by inhibition of the 26S proteasome produced similar results. T47D cells stably expressing mutant S294A PR, in which serine 294 is replaced by alanine, fail to undergo ligand-dependent down-regulation and are resistant to MEKK1-plus-R5020-induced transcriptional synergy but respond to progestins alone. Similarly, c-myc protein levels are synergistically increased by epidermal growth factor and R5020 in cells expressing wild-type PR, but not S294A PR. Thus, highly stable mutant PR are functional in response to progestins but are incapable of cross talk with MAPK-driven pathways. These studies demonstrate a paradoxical coupling between steroid receptor down-regulation and transcriptional hyperactivity. They also suggest a link between phosphorylation of PR by MAPKs in response to peptide growth factor signaling and steroid hormone control of breast cancer cell growth.Many solid tumors, including breast cancers, exhibit elevated mitogen-activated protein kinase (MAPK) expression and/or activities (13, 40), presumably due to increased expression of growth factor receptors that couple to MAPK activation. Overexpression of type I tyrosine kinase growth factor receptors in the epidermal growth factor (EGF) receptor/cErbB family is believed to contribute to proliferative signaling in breast cancer and to be indicative of a poor prognosis. Analogous to other members of the steroid receptor superfamily, human estrogen receptors (ER) and progesterone receptors (PR) are highly phosphorylated and therefore sensitive to growth factor-initiated signaling pathways. Indeed, the same phosphorylation sites on ER and/or PR can be regulated in response to steroid hormone or growth factor treatment of cells (reviewed in references 18 and 49). Although the role of direct phosphorylation of steroid hormone receptors and the exact kinase-signaling pathways involved remain large...

During late stages of breast cancer progression, tumors frequently acquire steroid hormone resistance with concurrent amplification of growth factor receptors; this alteration predicts a poor prognosis. We show here that following treatment with the progestin, R5020, breast cancer cells undergo a "biochemical shift" in the regulation of epidermal growth factor (EGF)-stimulated signaling pathways: R5020 potentiates the effects of EGF by up-regulating EGFR, c-ErbB2 and c-ErbB3 receptors, and by enhancing EGF-stimulated tyrosine phosphorylation of signaling molecules known to associate with activated type I receptors. Independently of EGF, R5020 increases Stat5 protein levels, association of Stat5 with phosphotyrosine-containing proteins, and tyrosine phosphorylation of JAK2 and Shc. Furthermore, progestins "prime" breast cancer cells for growth signals by potentiating EGF-stimulated p42/p44 mitogenactivated protein kinase (MAPK), p38 MAP kinase, and JNK activities. Although the levels of cyclin D1, cyclin E, and p21 WAF1 , are up-regulated by R5020 alone, they are synergistically up-regulated by EGF in the presence of R5020. Up-regulation of cell cycle proteins by EGF is blocked by inhibition of p42/p44 MAPK only in the presence of R5020, supporting a shift in the regulation of these cell cycle mediators from MAPK-independent to MAPK-dependent pathways. In summary, progesterone selectively increases the sensitivity of key kinase cascades to growth factors, thereby priming cells for stimulation by latent growth signals. These data support a model in which breast cancer cell growth switches from steroid hormone to growth factor dependence.Estradiol and progesterone are involved in breast cancer development, but at the time of diagnosis only one-third of tumors are steroid hormone-dependent. As they progress, these tumors often acquire steroid hormone resistance, yet retain nuclear steroid receptors. Indeed, nuclear receptor loss or mutation accounts for only 10 -20% of steroid hormone-resistant tumors (1). Thus, it has been postulated that in the majority of resistant tumors, control over growth is assumed by locally acting autocrine or paracrine peptide growth factors. As a result, invasive cancers with the worst prognosis are those that are growth factor receptor positive and steroid hormone resistant (2). Growth factors regulate cell growth by activating intracellular signal transduction pathways after binding to high affinity tyrosine kinase receptors on the cell surface. Steroid hormone receptors are ligand-activated nuclear transcription factors. Herein, we sought to understand how these two separate pathways converge to regulate breast cancer cell growth.The mechanisms by which growth factors stimulate cell proliferation are complex. Growth factors induce receptor dimerization and activation of intrinsic tyrosine kinase activity. Autophosphorylation of tyrosine residues located in the cytoplasmic domain of the receptors stimulates binding of specific regulatory proteins via their SH2 1 and SH3 domains, which ...

BACKGROUND:In head and neck cancer (HNC), 3-month post-treatment positron emission tomography (PET)/computed tomography (CT) reliably identifies persistent/recurrent disease. However, further PET/CT surveillance has unclear benefit. The impact of posttreatment PET/CT surveillance on outcomes is assessed at 12 and 24 months. METHODS: A 10-year retrospective analysis of HNC patients was carried out with long-term serial imaging. Imaging at 3 months included either PET/CT or magnetic resonance imaging, with all subsequent imaging comprised of PET/CT. PET/CT scans at 12 and 24 months were evaluated only if preceding interval scans were negative. Of 1114 identified patients, 284 had 3-month scans, 175 had 3-and 12-month scans, and 77 had 3-, 12-, and 24-month scans. RESULTS: PET/CT detection rates in clinically occult patients were 9% (15 of 175) at 12 months, and 4% (3 of 77) at 24 months. No difference in outcomes was identified between PET/CT-detected and clinically detected recurrences, with similar 3-year diseasefree survival (41% vs 46%, P ¼ .91) and 3-year overall survival (60% vs 54%, P ¼ .70) rates. Compared with 3-month PET/CT, 12-month PET/CT demonstrated fewer equivocal reads (26% vs 10%, P < .001). Of scans deemed equivocal, 6% (5 of 89) were ultimately found to be positive. CONCLUSIONS: HNC patients with negative 3-month imaging appear to derive limited benefit from subsequent PET/ CT surveillance. No survival differences were observed between PET/CT-detected and clinically detected recurrences, although larger prospective studies are needed for further investigation.

The two, nearly identical, isoforms of human progesterone receptors (PR), PR-B and -A, share activation functions (AF) 1 and 2, yet they possess markedly different transcriptional profiles, with PR-B being much stronger transactivators. Their differences map to a unique AF3 in the B-upstream segment (BUS), at the far N terminus of PR-B, which is missing in PR-A. Combined mutation of two LXXLL motifs plus tryptophan 140 in BUS, to yield PR-BdL140, completely destroys PR-B activity, because strong AF3 synergism with downstream AF1 and AF2 is eliminated. This synergism involves cooperative interactions among receptor multimers bound at tandem hormone response elements and is transferable to AFs of other nuclear receptors. Other PR-B functions-N-/C-terminal interactions, steroid receptor coactivator-1 coactivation, ligand-dependent down-regulation-also require an intact BUS. All three are autonomous in PR-A, and map to N-terminal regions common to both PR. This suggests that the N-terminal structure adopted by the two PR is different, and that for PR-B, this is controlled by BUS. Indeed, gene expression profiling of breast cancer cells stably expressing PR-B, PR-BdL140, or PR-A shows that mutation of AF3 destroys PR-B-dependent gene transcription without converting PR-B into PR-A. In sum, AF3 in BUS plays a critical modulatory role in PR-B, and in doing so, defines a mechanism for PR-B function that is fundamentally distinct from that of PR-A.

Sixty to seventy percent of all primary human breast cancers are estrogen-dependent and express both estrogen (ER) and progesterone receptors (PR). Whereas expression of the two naturally occurring PR isoforms, PR-A and PR-B, is close to equimolar in normal human tissues, the ratio of the two receptors varies extensively in tumors. This is important since the two PR are functionally distinct and have differential repressor effects on ER. The PR isoform content may, therefore, affect the outcome of endocrine therapies targeted at ER. Study of PR isoforms is difficult because the two receptors are co-expressed in cells under estradiol stimulation. We have engineered four sets of T47D human breast cancer cells that, independent of estrogen: (i) express only PR-A; (ii) express only PR-B; (iii) are PR-negative; or (iv) contain both PR isoforms. Each of these cell lines was grown into solid tumors in nude mice in a strictly 17beta-estradiol-dependent manner. Results show, first, that PR-A expressing cells grow into tumors that are approximately half the size of PR-B expressing tumors, and second, that the reduced growth of PR-A tumors occurs in the absence of PR ligand. Tamoxifen treatment preferentially inhibited the growth of PR-A tumors, whereas PR-B tumors were unaffected. Thus, PR are not just passive markers of functional ER; the prevalence of PR-A or PR-B may differentially influence tumor phenotype.

Although long-term clinical use of progestins is associated with an increased incidence of breast cancers, their role in established cancers is unclear. Estrogens are considered to be the main mitogens in the majority of breast cancers. Whether progesterone affects proliferation and/or differentiation is under debate. To assess the role of progesterone in established breast cancers, we used T47D human breast cancer cells that are estrogen receptor (ER) positive and either progesterone receptor (PR) negative or positive for PRA, PRB, or both. These cells were grown as strictly estrogen-dependent solid tumors in ovariectomized female nude mice. Progesterone or medroxyprogesterone acetate (MPA) alone did not support tumor growth, nor did progesterone or MPA given simultaneously with estrogen significantly alter estrogen-dependent tumor growth. However, treatment of mice bearing ER+PR+ but not ER+PRÀ tumors with either progesterone or MPA increased expression of the myoepithelial cytokeratins (CK) 5 and 6 in a subpopulation of tumor cells. These CK5+/CK6+ cells had decreased expression of luminal epithelial CK8, CK18, and CK19. We conclude that progestins exert differentiative effects on tumors characterized by transition of a cell subpopulation from luminal to myoepithelial. This may not be beneficial, however, because such a phenotype is associated with poor prognosis. (Cancer Res 2005; 65(21): 9779-88)

Objective: 1) Discuss the theory of multilevel sleep surgery for obstructive sleep apnea (OSA) management. 2) Evaluate the outcomes of lateral pharyngoplasty and midline glossectomy.Method: Retrospective chart review of adult patients with polysomnographic evidence of OSA treated at a tertiary care center with multilevel surgery involving lateral pharyngoplasty and midline glossectomy between 2009 and 2010. Outcome measures included change in preoperative and postoperative AHI, RDI, and oxygen nadir. Statistical analysis performed using Wilcoxon rank test.Results: Thirty-three patients were identified that met inclusion criteria. The mean preoperative BMI was 30.4, and postoperative BMI was 29.1 (P = .04). The mean decrease in AHI was 12.5 from 32.7 to 20.2 (P = .027). The mean decrease in RDI was 16.4 from 37.2 to 20.7 (P = .17). The mean improvement in oxygen nadir was 13% from 63.5 to 76.8 (P = .09).Conclusion: Multilevel sleep surgery results in a statistically significant improvement in polysomnographic metrics, such as AHI and RDI, for obstructive sleep apnea.