BACKGROUND: Progesterone receptors (PR) are important prognostic biomarkers of estrogen receptor (ER) action in breast cancer (BC). More recently, PRs have emerged as independent (from ER) mediators of early BC progression. In advanced BC phase II trials, the antiprogestin (antiPg) onapristone demonstrated a 10% and 56% OR in second line and first line therapies respectively, while mifepristone demonstrated 10% and 12%. Upon ligand binding, transcriptionally active PR in normal tissues form a discrete focal subnuclear distribution pattern (FDP) that can be visualized by immunofluorescence (Arnett-Mansfield, 2004, 2007). Importantly, PR nuclear foci are indicative of transcriptional activity. FDP is also observed in breast and endometrial cancers independently of hormonal status. The goal of this study was to devise a method to identify patients that are more likely to benefit from antiPg treatment. METHOD: 12 PR [+] BC paraffin-embedded blocks were analyzed. Standard IHC was used with 4 Abs: anti-PR-A (PRA), PR-B (PRB), anti-PR-A+B, anti-PR A and B (PRAB). Samples were analyzed for each Ab, and a control for the primary Ab, with and without background staining. After standard histologic evaluation subnuclear structures were analyzed at 100X. IHC was performed using Abs directed to the following PR phosphorylated sites (pAb) pSer 162, 190, 294, 400 and 554. Six samples were selected for IHF using a secondary fluorescent Ab. RESULTS: All cases were PR [+]. At high magnification (100X), 2 PR nuclear distribution patterns were observed: a diffuse finely granular staining (D) or an aggregated pattern (A). This resulted in 3 tumor phenotypes (PR B Ab): A cells only in 2 cases, D cells only in 6, and a mixture of both A and D cells (AD) in 4. PR [−] malignant cells were present in various proportions. The IHF results were consistent with IHC studies. Only pAb to pSER190 was specific and sensitive, with a smaller number of [+] malignant cells relative to the standard PRB. CONCLUSION: We have developed an IHC method that utilizes formalin-fixed paraffin-embedded tissue allowing the technique to be applied on a routine basis. Three classes of PR nuclear distribution have been defined: D, A and AD. D is consistent with the expression of PR that is weakly transcriptionally active or inactive and thus potentially predictive of poor patient response to antiPg. The A or AD pattern of PR nuclear distribution indicates active PR; patients with this pattern may exhibit a increased probability of benefiting from an antiPg. Preclinical studies have demonstrated that activated PR [+] cells can in turn stimulate PR [−] (stem or progenitor) mammary epithelial cells via secreted factors. In light of these findings, we are expanding the cohort to refine and confirm this biomarker technique. Additional preclinical work is ongoing to expand the biological rationale and to evaluate this technique alongside other technologies such as PR gene expression analysis. We anticipate that approximately 50% of PR+ BC cases contain the A/AD phenotye and therefore may benefit from antiPg. The method of IHC described herein is a clinically applicable tool that may allow for selection of patients most likely to respond to antiPg treatment. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-11.
While advances in science and technology have increased options for treating breast cancer, current social trends have changed the way people deal with this disease. Women in the United States are no longer simply passive patients, but rather they are survivors, advocates and activists who are speaking up for themselves and speaking out for issues relevant to the treatment and prevention of breast cancer.As the discoveries of basic science have been translated to better clinical treatment, a new sense of hope has emerged. Quality of life now shares the spotlight with quantity of life as breast cancer has shifted from an acute to a chronic condition and as the numbers of long-term survivors increase. While this new population tends to have more optimistic expectations for survival, they are also expressing concerns about issues affecting their lives through and beyond treatment. These issues include, but are not limited to, such concerns as efficient and accurate diagnosis, the complexity of treatment decisions, access to quality cancer care, informed consent, privacy issues, availability of supportive care treatments, and effective communication skills, especially with their physicians. Survivors are also concerned about the impact of their disease on spouses and family, on fertility and sexuality issues, on their employment and (in the USA) insurability, and on their long-term survival. The identification of these increasing issues has given rise to a consumer movement that encourages a shift away from powerless victim to empowered survivor.Historically, breast cancer advocates asked for increased educational and supportive care resources. As the survivorship movement matured, new responsibilities and differing agendas arose amongst these groups. Some organizations defined their mission as one that would raise funds to support scientific research. Others felt compelled to raise awareness about early detection and treatment, controversial environmental issues, and prevention or risk reduction. A few organizations later entered the more political arenas and began lobbying for issues related to health care delivery, clinical trials access, and quality cancer care. Meanwhile, these many and varied missions are all helping to define an international agenda for breast cancer research and care, to guarantee the inclusion of consumer voices in most levels of decision-making, and to create partnerships between patients with breast cancer and the professionals who care for them.
Luminal breast cancers account for ˜75% of cases and are positive for progesterone receptor (PR) and estrogen receptor (ER) expression. PR is a classical ER-target gene and is used as a biomarker of ER activity; however, a growing body of evidence supports the role of PR as an important modifier of ER actions and a key driver of luminal breast cancer progression. Progesterone signaling is mediated by two PR isoforms: full-length PR-B and truncated PR-A, which lacks the N-terminal 164 amino acids. Little is known about PR isoform-specific actions in PR-expressing breast tumors, given that total PR expression is measured clinically. Herein, we sought to identify phenotypic differences in luminal breast cancer cells (T47D) overexpressing PR-A (T47D-YA) or PR-B (T47D-YB). We demonstrate that PR-B expression is required for anchorage-independent colony formation, while PR-A expressing cells fail to proliferate in soft agar. PR-B driven proliferation has been mapped to PR phosphorylation events, which include MAPK or CDK consensus sites such as Ser294. We demonstrate that in contrast to previous reports, PR-A is well phosphorylated at Ser294 in response to progestins (e.g. R5020) using our custom phospho-PR (Ser294) polyclonal antibody. Interestingly, Ser294 phosphorylation of PR-A occurs more rapidly and robustly following hormone treatment compared to PR-B expressing cells. Our findings indicate that PR-A is a dominant driver of stem-like expansion in breast cancer cells. PR-A tumorspheres exhibited enriched ALDH+ and CD44+/CD24- populations compared to PR-B and promoted heightened gene expression of stem-like genes (e.g. FOXO1). We demonstrate that the PR target gene and co-activator FOXO1 promotes both PR-A and PR-B phosphorylation at Ser294 and augments tumorsphere formation. Direct inhibition of FOXO1 levels abrogates phospho-PR (Ser294) levels and tumorsphere formation in PR expressing cells. Finally, we show that Ser294 is required for PR-A induced expression of stem-like genes (e.g. FOXO1) and stem-like behavior as measured by ALDH+/CD44+ tumorspheres. Taken together, our data reveal unique functions of PR isoforms as modulators of distinct and opposing pathways (i.e. proliferation versus stem-like expansion) in luminal breast cancer models. A clear understanding of PR isoform-specific actions, phosphorylation events, and the role of co-factors such as FOXO1 may lead to novel biomarkers of advanced tumor behavior and reveal new approaches to pharmacologically target PR isoforms in luminal breast cancers. Citation Format: Truong TH, Dwyer AR, Diep CH, Lange CA. Progesterone receptor (PR) isoform-specific expansion of breast cancer stem-like cells occurs through a phospho-PR/FOXO1 driven signaling axis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-06.
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