Progesterone receptor isoform-dependent cross-talk between prolactin and fatty acid synthase in breast cancer

Results

Expression of FASN and PRLR mRNAs is significantly elevated in PR-positive breast cancer

FASN is an endogenous PR-responsive gene [64–67], which might explain, at least in part, the simultaneous increase in expression of FASN and PR proteins early during human mammary carcinogenesis [68]. We interrogated transcriptional data from the METABRIC [69, 70] to explore the association between FASN and PR in breast cancer (Figure 1). FASN mRNA expression was significantly higher in PR-positive breast tumors than in PR-negative tumors (p < 0.0001; n=1,700), as determined by PR gene expression profiles. PRLR expression was also significantly elevated in PR-positive tumors (p < 0.0001), whereas prolactin expression was not significantly different between the two subgroups (p = 0.5755). It was therefore of interest to evaluate whether FASN mRNA expression correlated with that of PRLR. PRLR was among the top-100 genes positively correlated with FASN gene expression (r = 0.31, p < 0.0001) in the METABRIC breast cancer dataset. Indeed, when the data set was classified according to the five intrinsic subtypes (luminal-A, luminal-B, HER2-enriched, claudin-low, and basal-like) using the research-based 50-gene prediction analysis of microarray (PAM50) classifier [71], both FASN and PRLR (but not prolactin) mRNAs were found to be significantly higher in HER2-enriched and luminal subtypes than in highly aggressive/poor prognosis basal-like and claudin-low subtypes (Figure 1). The highest levels of FASN and PRLR mRNAs were detected in HER2-enriched and luminal-A subtypes, respectively.

Figure 1. Differential enrichment of FASN, prolactin, and PRLR genes in breast cancer subtypes. FASN, prolactin, and PRLR mRNA expression levels in primary breast tumors from the METABRIC project classified into distinct subtypes using different classifiers. (PR+, n=903 versus PR-, n=797, Mann Whitney test; 3-genes signature, estrogen receptor (ER)-/HER2-, n=290, ER+/HER2- high proliferation, n=603, ER+/HER2+ low proliferation, n=619, HER2+, n=188, ANOVA with Dunn’s multiple comparison test; PAM50: basal, n=161, claudin-low, n=186, HER2 enriched, n=190, luminal A, n=631, luminal B, n= 412, ANOVA with Dunn’s multiple comparison test).

Maximum prolactin secretion and PRLR expression requires both PR-A and PR-B isoforms

To evaluate the relevance of PR isoform expression and ratio on the regulatory activity of prolactin for FASN gene expression, we used the PR-A/PR-B-positive (T47D_co) and PR-null (T47D-Y) variants of the estrogen receptor (ER)/PR-positive breast cancer cell line T47D. T47D_co cells endogenously express equimolar levels of PR-A and PR-B in a constitutive and estrogen-independent manner, thereby allowing the study of the functional relevance of PR without the confounding effects of estrogen [44, 62, 72–75]. T47D-Y cells can also be used to determine the effect of PR isoform variants or mutants by stably reintroducing PR-A or PR-B on expression vectors. The resulting cell lines, termed T47D-YA (stably expressing the full-length PR-A isoform) and T47D-YB (stably expressing the full-length PR-B isoform), have been widely employed to evaluate the independent signaling function of each PR isoform [43, 62, 72–75]. Here, we employed all four T47D cell lines, each with a different PR content (Figure 2A), to test for isoform-specific prolactin gene regulation and functional interactions with the FASN gene.

Figure 2. Exogenous prolactin activates the FASN gene promoter in a PR-B/SBREP-dependent manner. (A). Immunoblotting of baseline expression status of PR-A, PR-B, PRLR, and FASN proteins in T47D_co, T47D-Y, T47D-YA and T47D-YB breast cancer cell lines. β-actin was used to control for protein loading and transfer. (B). Immunoassay-based quantification of baseline autocrine prolactin secretion into the extracellular milieu of T47D_co, T47D-Y, T47D-YA and T47D-YB breast cancer cell lines. (C, D). Estradiol-depleted cells were transiently transfected with a plasmid containing a luciferase gene driven by a 178-bp FASN gene promoter fragment harboring a SREBP-binding site, flanked by auxiliary NF-Y and Sp-1 sites or with a similar construct in which the SREBP domain was deleted. The next day, cells were treated with graded concentrations of recombinant prolactin (PRL) in 0.5% CCS. After 24 h, cells were lysed and luciferase activity was measured. Luciferase activity was expressed as relative (fold) change in transcriptional activities of FASN promoter-transfected cells in response to prolactin treatment after normalization to pRL-CMV activity. Each experimental value represents the mean fold increase (columns) ± S.D. (bars) from at least three separate experiments in which triplicate wells were measured. Luciferase activity in prolactin-treated cells was compared with that in vehicle-treated control cells (* P < 0.05; ** P < 0.005).

ELISA-based quantification of prolactin content in the conditioned medium of T47D_co, T47D-Y, T47D-YA, and T47D-YB cultures revealed considerably lower amounts (~85%) in PR-null T47D-Y cultures than in T47D_co cultures (Figure 2B). Although the individual re-expression of each PR isoform was not sufficient to reach the levels of prolactin found in T47D_co cultures, the stable re-expression of PR-A and PR-B notably augmented by 3.7- and 4.8-fold, respectively, the extracellular amounts of prolactin in T47D-Y cultures. We also compared the four cell types in terms of PRLR abundance by immunoblotting equivalent cell extracts using an antibody against PRL4 (Figure 2A). Analysis showed that T47D_co cells likewise harbored an abundant amount of PRLR, whereas a notably decreased amount of PRLR was detected in PR-null T47D-Y cells. T47D-YB cells expressed PRLR at slightly higher levels than T47D-YA cells. These findings, altogether, reveal not only a close correlation between the status of prolactin secretion and PRLR expression, but also that both PR-A and PR-B are required to achieve maximum levels of prolactin secretion and PRLR expression.

Progesterone receptor isoforms differentially impact baseline FASN expression

To assess how the PR status might alter baseline FASN promoter activity, T47D_co, T47DY, T47D-YA, and T47D-YB cells were transfected with a luciferase reporter vector encoding a sterol regulatory element-containing FASN promoter sequence fused with firefly luciferase. As shown in Figure 2C, the absence of both PR isoforms failed to alter luciferase expression in T47D-Y cells with respect to baseline levels in T47D_co cells. Intriguingly, FASN promoter activity was reduced by approximately 50% in cells exclusively expressing the PR-A isoform (T47D-YA), but increased by more than 90% in cells exclusively expressing the PR-B isoform (T47D-YB). At the protein level, immunoblotting confirmed the lower amounts of FASN in T47D-YA cells and the slightly higher FASN levels in T47D-YB cells (Figure 2A).

Prolactin-induced up-regulation of FASN gene expression is PR-B isoform-specific

Transient transfection experiments with the FASN reporter demonstrated the ability of graded concentrations of prolactin (50, 100, and 200 ng/mL) to dose-dependently up-regulate (up to 2.1-fold at 200 ng/mL prolactin) promoter activity in T47D_co cells (Figure 2D, top). PR-null T47D-Y cells, however, remained mostly insensitive to the regulatory effects of prolactin on the FASN reporter. We examined PR isoform-dependent, prolactin-driven FASN promoter activity, finding that T47D cells containing PR-A (T47D-YA cells) were completely unresponsive to prolactin. By contrast, the sole presence of PR-B preserved the ability of prolactin to increase FASN promoter activity, as previously observed in T47D_co cells (Figure 2D, top). Indeed, the presence of PR-B in the absence of PR-A significantly boosted the response of the FASN promoter to prolactin concentrations as low as 50 ng/mL, suggesting that prolactin-induced activation of the FASN reporter is largely mediated through PR-B. We further found that prolactin-induced FASN promoter activity was blocked in cells transiently transfected with a truncated version of the proximal FASN gene promoter in which the region responsible for SREBP binding was deleted (FASNΔSRE; Figure 2D, bottom), overall suggesting that prolactin activates the FASN promoter primarily through the SREBP1 regulatory site [76–78].

The PRLR-specific antagonist hPRL-G129R impedes prolactin-driven activation of FASN gene expression

To substantiate that prolactin activates FASN gene expression by its engagement with PRLR, we used the PRLR-specific antagonist hPRL-G129R [20–25, 79–82]. Prolactin-induced activation of FASN gene expression (at 200 ng/mL) in T47D_co and T47D-YB cells was completely inhibited by co-incubation with hPRL-G129R at a relatively low concentration of 1000 ng/mL (i.e., 5-fold-excess of prolactin) (Figure 3, top). Further, PRLP-specific blockade with hPRL-G129R sufficed to return the overactive FASN promoter in T47D-YB cells to the baseline state seen in T47D_co cells. Immunoblotting assays failed to demonstrate any significant changes in PRLR expression in response to prolactin, hPRL-G129R, or their combination (Figure 3, bottom); however, hPRL-G129R co-treatment prevented the ability of prolactin to marginally down-regulate PR-A and PR-B expression in T47D-YA and T47D-YB cells, respectively. These findings, altogether, imply that the prolactin-FASN signaling axis acts through PRLR in T47D cells, and suggest the specific functional engagement of the PR-B isoform as the driver of FASN gene responsiveness to the regulatory effects of prolactin/PRLR.

Figure 3. Exogenous prolactin activates FASN gene promoter activity by engaging PRLR.Top. Estradiol-depleted cells were transiently transfected with a plasmid containing a luciferase gene driven by a 178-bp FASN gene promoter fragment harboring a SREBP-binding site, flanked by auxiliary NF-Y and Sp-1 sites as described in Figure 2C, 2D. The next day, cells were treated with 200 ng/mL prolactin (PRL) in the absence or presence of a 5-fold-excess of the prolactin antagonist hPRL-G129R (1000 ng/mL) in 0.5% CCS. After ~24 h of incubation, cells were lysed, luciferase activity was measured and relative (fold) changes in transcriptional activities of FASN promoter-luciferase-transfected cells were calculated. The data are shown as the means (columns) ± S.D. (bars) from three separate experiments (performed in duplicate). Luciferase activity in prolactin- and/or hPRL-G129R-treated cells was compared with that in vehicle-treated control cells (* P < 0.05; ** P < 0.005). Bottom. Estradiol-depleted cells were treated with 200 ng/mL PRL in the absence or presence of a 5-fold excess of hPRL-G129R in 0.5% CCS for 48 h. Immunoreactive bands for PR-A, PR-B, and PRLR proteins were analyzed by immunoblotting as described in Figure 2A. β-actin was used to control for protein loading and transfer. Figure shows a representative immunoblot analysis. Similar results were obtained in 3 independent experiments.

FASN inhibition suppresses PR-A and PR-B expression

We previously demonstrated that FASN blockade suppresses the well-documented capacity of estradiol to up-regulate PR expression in endometrial cancer cells [83, 84]. We therefore explored the possibility that FASN signaling regulates the constitutive expression of both PR isoforms irrespective of estradiol stimulation. As shown in Supplementary Figure 1, the expression of PR-A and PR-B in T47D_co cells was dose-dependently suppressed in the presence of graded concentrations of the FASN inhibitor C75. Indeed, PR-A and PR-B expression was very low to undetectable in the presence of 10 μg/mL C75 not only in T47D_co cells but also in T47D-YA and T47D-YB cells (Supplementary Figure 1).

FASN inhibition modifies prolactin secretion and PRLR expression in a PR isoform-dependent manner

We next tested the hypothesis that FASN signaling regulates prolactin secretion in a PR isoform-dependent manner (Figure 4A). Prolactin secretion in T47D_co cells was reduced by C75 in a dose-dependent manner (up to approximately 80% suppression at 10 μg/mL C75; Figure 4A, top). In T47D-YA cells, however, prolactin secretion was significantly reduced by only 50% at the same concentration of C75. Conversely, C75 concentrations as low as 2.5 μg/mL sufficed to decrease prolactin secretion by 50% in T47-YB cells, whereas the suppression of secretion as high as 80% was observed at 10 μg/mL C75 (Figure 4A, top). A significant, dose-dependent up-regulation of prolactin secretion occurred in PR-null T47D-Y cells exposed to graded concentrations of C75 (up to approximately 170%). We then explored the ability of FASN signaling to regulate PRLR expression. Remarkably, we observed the complete loss of PRLR expression in T47D_co, TD47Y-A, and T47D-YB cells grown in the presence of C75 (Figure 4A, bottom). By contrast, PRLR protein expression in PR-null Y47D-Y cells was conspicuously up-regulated in response to C75 (Figure 4A, bottom.

Figure 4. Pharmacological blockade of FASN activity modifies autocrine prolactin secretion in a PR-dependent manner. (A). Top. Autocrine prolactin secretion levels in the extracellular milieu of estradiol-depleted cells cultured in the absence or presence of graded concentrations of C75 in 0.5% CCS for 48 h were determined by a commercially available EASIA kit. Data are means (columns) ± S.D. (bars) from three independent experiments performed in duplicate. Secreted amounts of prolactin in C75-treated cells were compared with those in vehicle-treated control cells (* P < 0.05; ** P < 0.005). Bottom. Cell lysates strictly obtained from the same experimental replicates employed in A were subjected to immunoblotting for PRLR protein expression. β-actin was used to control for protein loading and transfer. Figure shows a representative immunoblot analysis. Similar results were obtained in 3 independent experiments. (B). Autocrine IL-6 levels in the extracellular milieu of estradiol-depleted cells cultured in the absence or presence of 10 μg/mL C75 in 0.5% CCS for 48 h were determined by a commercially available ELISA kit. Secreted amounts of IL-6 in C75-treated cells were compared with those in vehicle-treated control cells (** P < 0.005).

FASN inhibition suppresses epithelial-to-mesenchymal transition-related aggressiveness in PR-null T47DY cells

Both PR and PRLR have been suggested to act as as promoters of more differentiated phenotypes via suppression of the epithelial-to-mesenchymal (EMT) program. Indeed, loss of PR in T47D-Y cells has been associated with a change in cell morphology to a more mesenchymal-like phenotype, accompanied by increased cell motility, and up-regulation of EMT-associated genes [85, 86]. Similarly, prolactin blockade in epithelial-like breast cancer cells has been shown to induce mesenchymal-like phenotypic changes and enhance invasiveness, whereas activation of PRLR in mesenchymal-like breast cancer cells suppresses mesenchymal properties and reduces invasive behaviors [87, 88]. A robust surrogate marker of aggressive breast cancer phenotype via regulation of EMT is interleukin-6 (IL-6) [89–92]. We therefore investigated whether the apparent ability of FASN blockade to restore a prolactin autocrine function in T47D-Y cells via augmented secretion of PR and re-activation of PRLR expression might impact the EMT-related status of IL-6 expression. EMT-like PR-null T47D-Y cells released extremely high levels of IL-6 into the extracellular milieu when compared with PR-A/-B-expressing T47D_co parental cells (approx. 13-fold increase; Figure 4B). The FASN inhibitor C75 not only suppressed the baseline IL-6 expression in T47D_co parental cells, but further reduced (by more 50%) the augmentation of IL-6 secretion promoted by loss of PR in T47D-Y cells (Figure 4B).

PRLR inhibition decreases FASN expression in HER2-overexpressing breast cancer cells

Several studies suggest that there is potential cooperation between PRLR and HER2 during breast cancer progression [82, 93, 94]. We speculated that, if a cross-talk between HER2 and autocrine prolactin/PRLR signaling is actively involved in the well-known FASN overexpressing-phenotype of HER2-positive breast cancer cells [36, 95–98], blockade of PRLR should then reduce the ability of HER2 to constitutively up-regulate FASN gene expression. We found that whereas exogenous stimulation with prolactin dose-dependently increased FASN promoter activity in HER2-negative MCF-7/neo control cells, it failed to promote any further increase in activity of the already hyperactive FASN gene promoter in MCF-7/HER2 cells (Figure 5). hPRL-G129R fully prevented the positive regulatory effects of prolactin on the FASN gene in MCF-7/neo parental cells and partially reduced the overactive FASN gene promoter in MCF-7/HER2 cells, thus suggesting that PRLR signaling actively engages HER2 signaling to fully hyperactivate the FASN gene promoter in these cells (Figure 5).

Figure 5. HER2 overexpression prevents prolactin-induced activation of the FASN gene promoter. Estradiol-depleted MCF-7/neo and MCF-7/Her2-18 cells were transiently transfected with a plasmid containing a luciferase gene driven by a 178-bp FASN gene promoter fragment harboring a SREBP-binding site, flanked by auxiliary NF-Y and Sp-1 sites or with a similar construct in which the SREBP domain was deleted. The next day, cells were treated with recombinant prolactin (PRL) in the presence or absence of hPRL-G129R in 0.5% CCS. After ~24 h of incubation, cells were lysed, luciferase activity was measured and relative (fold) changes in transcriptional activities of FASN promoter-luciferase-transfected cells were calculated. The data are shown as the means (columns) ± S.D. (bars) from three separate experiments (performed in duplicate). Luciferase activity in PRL- and/or G129R-treated cells was compared with that in vehicle-treated control cells (* P < 0.05; ** P < 0.005).

Discussion

Prolactin has a central role in mammary gland development and terminal differentiation of mammary epithelial cells. The prolactin/PRLR-signaling axis has been consistently shown to play a permissive role in the development of primary breast carcinomas and distant metastatic lesions [3, 5, 7, 12, 13, 18, 20, 22, 99, 100]. Recent studies have, however, questioned the role of prolactin in breast cancer development/progression and have highlighted a putative suppressor role in breast tumorigenesis [87, 101]. This latter role is supported by the association between prolactin/PRLR down-regulation and significantly better survival outcome in patients with breast cancer [88, 102–105], which is consistent with the lack of anti-tumorigenic effects and therapeutic benefits observed in clinical trials with PRLR antagonists [106]. Accordingly, the restoration/activation of prolactin/PRLR signaling has been shown to promote cell differentiation and reverse highly proliferative, invasive, mesenchymal and tumorigenic phenotypes, such as those of the ER/PR double-negative MDA-MB-231 breast cancer model [107]. How might we reconcile these apparently conflicting pro- and anti-tumorigenic roles of prolactin?

We took advantage of the T47D_co luminal breast cancer cell line, which constitutively express high levels of ER and PR, and allows the study of the unliganded, progesterone-independent regulatory effects of human PRs in ER-positive luminal-like breast cancer without the confounding requirement of estradiol to stimulate PR expression. In addition to this, we employed the PR-negative T47D subline T47D-Y, in which cloning approaches restored either PR-A or PR-B expression, and used these cells to study the regulatory roles of each isoform in isolation [72, 108]. Because all of these models share the natural background of PR+ luminal breast cancer, in which we detected a positive correlation between PRLR and FASN genes in the METABRIC repository, they are expected to contain the appropriate ancillary coregulatory factors needed for faithful regulation of prolactin/PR-dependent genes. Our results lead us to propose that the PR isoform-specific regulation of distinct regulatory responses in the cross-talk between prolactin and FASN might be involved in the diverse phenotypic outcomes arising from the prolactin/PRLR signaling axis in luminal breast cancer.

We first assessed how PR-B and PR-A isoforms could modify the ability of prolactin to regulate the expression of the FASN gene in PR+ breast cancer cells. Historically, PR-B has been characterized as a strong “positive” regulator of the effects of progesterone, whereas PR-A is often regarded as a ligand-independent mediator of gene repression. Also, the isoforms differ with regard to the positive versus negative regulatory direction and also the type of regulated genes. Thus, whereas PR-B mostly regulates the expression of genes required for cell proliferation [43], PR-A mainly controls the expression of genes involved in cell adherence, cell morphology, and resistance to apoptosis [50, 109, 110]. We found that the ability of the prolactin/PRLR signaling to transcriptionally up-regulate FASN gene expression was specifically dictated by: a.) the obligatory presence of PR-B to enable prolactin-driven FASN gene activation, and b.) the necessary lack of PR-A to facilitate maximum FASN gene activation in response to prolactin. Accordingly, whereas PR-B+ T47-YB cells were exquisitely responsive to exogenous prolactin in terms of FASN gene up-regulation, both PR-null T47D-Y and PR-A+ T47D-YA cells remained completely unresponsive to the FASN regulatory effects of prolactin. When both PR isoforms are co-expressed (i.e., T47D_co), prolactin-induced FASN activation is considerably dampened relative to that of cells exclusively expressing PR-B (i.e., T47D-YB). PR-A trans-repression of PR-B might explain this observation and likely implicates a negative effect of PR-A within PR heterodimers, as has been reported in multiple models [110–115]. In this case, however, how do PR isoform-specific signaling events impact the ability of the same “classical” prolactin/PRLR/JAK2/STAT5 pathway to differentially mediate prolactin-induced FASN gene transcription? Of note, the ability of PR-B to facilitate prolactin-driven FASN activation required an SREBP-binding site in the FASN gene promoter. Therefore, PR isoform-specific actions might not simply be explained in terms of STAT5-driven transcriptional regulation of the FASN gene promoter, but instead suggest that the distinct ability of each PR isoform to cooperate with STAT5 would differentially impact SREBP-1c expression. The prolactin/PRLR/JAK2/STAT5 signaling pathway drives fat synthesis and proliferation via augmented expression of SREBP-1c [116, 117]. STAT5 modulates the expression and nuclear distribution of SREBP1, thereby regulating its biological functions including the regulation of lipogenic genes such as FASN. In fact, downregulation of (phospho-active) STAT5 decreases, whereas its overexpression increases, the activation of the SREBP1 promoter [118]. Intriguingly, the ability of PR to drive JAK/STAT-dependent transcriptional responses requires the so-called CD domain of PR-B, which is located in the N-terminal B-upstream segment (BUS) region of full-length PR-B – and absent in PR-A – and is essential for proliferative signaling in breast cancer cells [119, 120]. The CD domain of PR-B is required for PR-B-dependent expression of STAT5 which, in turn, then complexes with PR-B on a specific subset of PR-target genes. Indeed, STAT5 appears to operate as a pioneer transcription factor that “opens” sites in chromatin for subsequent PR-B-driven transcriptional activation of target genes [120]. It is therefore tempting to speculate that the BUS region of PR-B might function as the shared signaling hub of the prolactin/PRLR-driven STAT5 and PR-B (but not PR-A) signaling axes. By placing the lipogenic master regulator SREBP-1c as one of the select target genes coregulated by STAT5 and PR-B, we can explain the PR-B isoform-specific regulatory actions on the cross-talk between prolactin and FASN signaling in luminal breast cancer cells (Figure 6). In a scenario in which constitutive activation of PRLR and HER2 (e.g., MCF-7/HER2 cells) leads to a dissociation between prolactin/PRLR and inducible STAT5 activation, which becomes constitutive via hyperactivation not only of JAK2 but also of PI3K/AKT/mTOR and MAPK transducers, maximal transcriptional activation of the FASN gene is no longer responsive to exogenous stimulus with prolactin, but can be partially prevented by PRLR antagonists such as a hPRL-G129R [82] (Figure 6).

Figure 6. A PR isoform-dependent cross-talk between prolactin and FASN in breast cancer: a working model. The convergence of prolactin/PRLR and PR signaling interactions on STAT5 [130, 131] might explain, at least in part, the PR-B-driven capacity of prolactin to activate FASN in luminal breast cancer cells (A). Similar inputs, likely involving yet-to-be explored phospho-modifications of PR isoforms, might underlie also the ability of FASN signaling to regulate autocrine prolactin secretion (B).

We envisaged that prolactin secretion and/or PRLR expression might be affected by pharmacological blockade of FASN activity. The achievement of higher levels of autocrine prolactin secretion (and PRLR expression) obligatorily required the presence of the PR-B isoform. Thus, PR-A+ T47-YA cells and, more notably, PR-null T47D-Y cells, secreted less prolactin and downregulated PRLR expression in comparison with PR-A+/PR-B+ T47Dco cells. With the sole exception of sexual hormone (estradiol and androgen)-induced transcriptional up-regulation of autocrine prolactin in rat trigeminal neurons, pituitary lactotrophs, and rat prostate [121, 122], the pathway(s) that regulate the synthesis and secretion of autocrine prolactin in breast cancer cells are largely unknown. The PTEN/PI3K-AKT pathway – one of the most commonly activated metabolic drivers of cancer – is an upstream regulator of autocrine prolactin production in the normal mammary gland; moreover, autocrine prolactin production is a direct mechanism by which PI3K-AKT activation results in PRLR/JAK2/STAT5 pathway activation [123]. Importantly, whereas AKT-induced up-regulation of autocrine prolactin does not require intact PRLR/JAK/STAT5 signaling, the PRLR/JAK/STAT5 pathway is required to mediate the effects of AKT on lipid synthesis in the normal mammary gland [123]. We found that the ability of the FASN inhibitor C75 to reduce prolactin secretion in PR-A+/PR-B+ T47Dco parental cells was either exacerbated in PR-B+ T47D-YB cells or partially dampened in PR-A+ T47-YA cells. Because AKT is rapidly repressed in response to C75-induced blockade of FASN activity [124, 125], our findings suggest a positive feedback regulation between FASN and autocrine prolactin expression via AKT-driven activation of the PRLR/JAK/STAT5 pathways in PR+ breast cancer cells. This positive feedback, however, is apparently fine-tuned by PR signaling in a PR isoform-specific manner, which might reflect, at least in part, the ability of PR-B to cause down-regulation of the PI3K/AKT signal via upregulation of PTEN [126]. In T47D-Y cells lacking PRs and exhibiting a more aggressive undifferentiated phenotype, the suppression of FASN signaling was accompanied by a partial recovery of prolactin secretory activation, up-regulation of PRLR expression, and down-regulation of EMT/cancer stem cell/pro-inflammatory markers such as IL-6. These findings, overall, suggest that the ability of FASN signaling to enable secretory activation of endocrine prolactin is a PR-dependent event that might dictate the level of luminal cell differentiation [38]. Further characterization of the FASN-centered relationship between PRs, prolactin/PRLR/JAK/STAT5, and PI3K/AKT pathways will be required to determine whether therapeutic approaches directed at blocking the interaction between these pathways would be more beneficial to therapeutically manage prolactin and/or FASN signaling in breast cancer cells. Nonetheless, it is important to acknowledge that we employed the FASN inhibitor C75 in the form of a racemic mixture of (-) and (+) enantiomers, which differ in their regulation of FASN and carnitine palmitoyltransferase-I (CPT-1) [127–129]. An evaluation of clinical-grade FASN inhibitors devoid of CPT-I inhibitory activity should definitely clarify the mechanistic role of FASN as a therapeutic target for differentiation therapy in certain subsets of ER+/PR- breast carcinomas [38]

In summary, our data reveal an unforeseen PR-B isoform-specific regulatory action on the cross-talk between prolactin and FASN signaling in luminal breast cancer cells. Our data suggest that the lipogenic FASN might be incorporated into the group of metabolic markers specifically enriched by PR-B (but not with those related to the malignant metabolism of cancer stem cells enriched by PR-A) in luminal A-like PR+ breast cancer cells [130–133], likely promoting survival, proliferation, and differentiation. In ER-positive/PR-negative luminal B-like breast cancer cells, however, FASN signaling might be co-opted as a negative regulator of the epithelial cell phenotype and, accordingly, its blockade might promote the restoration and activation of prolactin/PRLR-driven differentiation programs. Nonetheless, because the PR isoform ratio is a proxy for the molecular signature and endocrine therapy responsiveness of PR+ breast cancer cells, our findings might illuminate new PR-B/FASN-centered predictive and therapeutic modalities in luminal breast cancer intrinsic subtypes.

Author Contributions

J.A.M. and R. L. conceived the idea, directed the project, and wrote the manuscript. S.K.P., A.P., E.C., T.V.S, S.V., and L.V. were involved in the design, development of all the cell-based experiments, and analyzed the data. W.Y.C. provided essential materials necessary for the study.

Acknowledgments

The authors would like to thank Dr. Kenneth McCreath for editorial support.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics approval was not required for the study as per the local legislation.

Funding

This work was supported by the National Institute of Health/National Cancer Institute Grant R01 CA116623 (Ruth Lupu) and by the Department of Defense (DOD)-Breakthrough 3 Grants BC151072 and BC151072P1 (Ruth Lupu). Work in the Menendez laboratory is supported by the Spanish Ministry of Science and Innovation (Grants SAF2016-80639-P and PID2019-10455GB-I00, Plan Nacional de l+D+I, founded by the European Regional Development Fund, Spain) and by an unrestricted research grant from the Fundació Oncolliga Girona (Lliga catalana d’ajuda al malalt de càncer, Girona).

References

• 1. Nicoll CS. Ontogeny and evolution of prolactin’s functions. Fed Proc. 1980; 39:2563–66. [PubMed]
• 2. Naylor MJ, Lockefeer JA, Horseman ND, Ormandy CJ. Prolactin regulates mammary epithelial cell proliferation via autocrine/paracrine mechanism. Endocrine. 2003; 20:111–14. https://doi.org/10.1385/ENDO:20:1-2:111 [PubMed]
• 3. Oakes SR, Rogers RL, Naylor MJ, Ormandy CJ. Prolactin regulation of mammary gland development. J Mammary Gland Biol Neoplasia. 2008; 13:13–28. https://doi.org/10.1007/s10911-008-9069-5 [PubMed]
• 4. Fuh G, Wells JA. Prolactin receptor antagonists that inhibit the growth of breast cancer cell lines. J Biol Chem. 1995; 270:13133–37. https://doi.org/10.1074/jbc.270.22.13133 [PubMed]
• 5. Vonderhaar BK. Prolactin: the forgotten hormone of human breast cancer. Pharmacol Ther. 1998; 79:169–78. https://doi.org/10.1016/s0163-7258(98)00017-5 [PubMed]
• 6. Ben-Jonathan N, Liby K, McFarland M, Zinger M. Prolactin as an autocrine/paracrine growth factor in human cancer. Trends Endocrinol Metab. 2002; 13:245–50. https://doi.org/10.1016/s1043-2760(02)00603-3 [PubMed]
• 7. Clevenger CV, Furth PA, Hankinson SE, Schuler LA. The role of prolactin in mammary carcinoma. Endocr Rev. 2003; 24:1–27. https://doi.org/10.1210/er.2001-0036 [PubMed]
• 8. Perks CM, Keith AJ, Goodhew KL, Savage PB, Winters ZE, Holly JM. Prolactin acts as a potent survival factor for human breast cancer cell lines. Br J Cancer. 2004; 91:305–11. https://doi.org/10.1038/sj.bjc.6601947 [PubMed]
• 9. McHale K, Tomaszewski JE, Puthiyaveettil R, Livolsi VA, Clevenger CV. Altered expression of prolactin receptor-associated signaling proteins in human breast carcinoma. Mod Pathol. 2008; 21:565–71. https://doi.org/10.1038/modpathol.2008.7 [PubMed]
• 10. Bernichtein S, Touraine P, Goffin V. New concepts in prolactin biology. J Endocrinol. 2010; 206:1–11. https://doi.org/10.1677/JOE-10-0069 [PubMed]
• 11. Chen WY. The many faces of prolactin in breast cancer. Adv Exp Med Biol. 2015; 846:61–81. https://doi.org/10.1007/978-3-319-12114-7_3 [PubMed]
• 12. Wennbo H, Gebre-Medhin M, Gritli-Linde A, Ohlsson C, Isaksson OG, Törnell J. Activation of the prolactin receptor but not the growth hormone receptor is important for induction of mammary tumors in transgenic mice. J Clin Invest. 1997; 100:2744–51. https://doi.org/10.1172/JCI119820 [PubMed]
• 13. Rose-Hellekant TA, Arendt LM, Schroeder MD, Gilchrist K, Sandgren EP, Schuler LA. Prolactin induces ERalpha-positive and ERalpha-negative mammary cancer in transgenic mice. Oncogene. 2003; 22:4664–74. https://doi.org/10.1038/sj.onc.1206619 [PubMed]
• 14. Tworoger SS, Eliassen AH, Sluss P, Hankinson SE. A prospective study of plasma prolactin concentrations and risk of premenopausal and postmenopausal breast cancer. J Clin Oncol. 2007; 25:1482–88. https://doi.org/10.1200/JCO.2006.07.6356 [PubMed]
• 15. Tworoger SS, Hankinson SE. Prolactin and breast cancer etiology: an epidemiologic perspective. J Mammary Gland Biol Neoplasia. 2008; 13:41–53. https://doi.org/10.1007/s10911-008-9063-y [PubMed]
• 16. Tworoger SS, Eliassen AH, Zhang X, Qian J, Sluss PM, Rosner BA, Hankinson SE. A 20-year prospective study of plasma prolactin as a risk marker of breast cancer development. Cancer Res. 2013; 73:4810–19. https://doi.org/10.1158/0008-5472.CAN-13-0665 [PubMed]
• 17. Faupel-Badger JM, Duggan MA, Sherman ME, Garcia-Closas M, Yang XR, Lissowska J, Brinton LA, Peplonska B, Vonderhaar BK, Figueroa JD. Prolactin receptor expression and breast cancer: relationships with tumor characteristics among pre- and post-menopausal women in a population-based case-control study from Poland. Horm Cancer. 2014; 5:42–50. https://doi.org/10.1007/s12672-013-0165-7 [PubMed]
• 18. Sutherland A, Forsyth A, Cong Y, Grant L, Juan TH, Lee JK, Klimowicz A, Petrillo SK, Hu J, Chan A, Boutillon F, Goffin V, Egan C, et al. The role of prolactin in bone metastasis and breast cancer cell-mediated osteoclast differentiation. J Natl Cancer Inst. 2015; 108:djv338. https://doi.org/10.1093/jnci/djv338 [PubMed]
• 19. Shemanko CS. Prolactin receptor in breast cancer: marker for metastatic risk. J Mol Endocrinol. 2016; 57:R153–65. https://doi.org/10.1530/JME-16-0150 [PubMed]
• 20. Chen WY, Ramamoorthy P, Chen N, Sticca R, Wagner TE. A human prolactin antagonist, hPRL-G129R, inhibits breast cancer cell proliferation through induction of apoptosis. Clin Cancer Res. 1999; 5:3583–93. [PubMed]
• 21. Llovera M, Pichard C, Bernichtein S, Jeay S, Touraine P, Kelly PA, Goffin V. Human prolactin (hPRL) antagonists inhibit hPRL-activated signaling pathways involved in breast cancer cell proliferation. Oncogene. 2000; 19:4695–705. https://doi.org/10.1038/sj.onc.1203846 [PubMed]
• 22. Chen NY, Holle L, Li W, Peirce SK, Beck MT, Chen WY. In vivo studies of the anti-tumor effects of a human prolactin antagonist, hPRL-G129R. Int J Oncol. 2002; 20:813–18. https://doi.org/10.3892/ijo.20.4.813 [PubMed]
• 23. Schroeder MD, Brockman JL, Walker AM, Schuler LA. Inhibition of prolactin (PRL)-induced proliferative signals in breast cancer cells by a molecular mimic of phosphorylated PRL, S179D-PRL. Endocrinology. 2003; 144:5300–07. https://doi.org/10.1210/en.2003-0826 [PubMed]
• 24. Peirce SK, Chen WY. Human prolactin and its antagonist, hPRL-G129R, regulate bax and bcl-2 gene expression in human breast cancer cells and transgenic mice. Oncogene. 2004; 23:1248–55. https://doi.org/10.1038/sj.onc.1207245 [PubMed]
• 25. Wen Y, Zand B, Ozpolat B, Szczepanski MJ, Lu C, Yuca E, Carroll AR, Alpay N, Bartholomeusz C, Tekedereli I, Kang Y, Rupaimoole R, Pecot CV, et al. Antagonism of tumoral prolactin receptor promotes autophagy-related cell death. Cell Rep. 2014; 7:488–500. https://doi.org/10.1016/j.celrep.2014.03.009 [PubMed]
• 26. Binart N, Ormandy CJ, Kelly PA. Mammary gland development and the prolactin receptor. Adv Exp Med Biol. 2000; 480:85–92. https://doi.org/10.1007/0-306-46832-8_10 [PubMed]
• 27. Das R, Vonderhaar BK. Prolactin as a mitogen in mammary cells. J Mammary Gland Biol Neoplasia. 1997; 2:29–39. https://doi.org/10.1023/a:1026369412612 [PubMed]
• 28. Waters SB, Rillema JA. Effect of prolactin on enzymes of lipid biosynthesis in mammary gland explants. Am J Physiol. 1988; 255:E567–71. https://doi.org/10.1152/ajpendo.1988.255.4.E567 [PubMed]
• 29. Rudolph MC, Monks J, Burns V, Phistry M, Marians R, Foote MR, Bauman DE, Anderson SM, Neville MC. Sterol regulatory element binding protein and dietary lipid regulation of fatty acid synthesis in the mammary epithelium. Am J Physiol Endocrinol Metab. 2010; 299:E918–27. https://doi.org/10.1152/ajpendo.00376.2010 [PubMed]
• 30. Rudolph MC, Russell TD, Webb P, Neville MC, Anderson SM. Prolactin-mediated regulation of lipid biosynthesis genes in vivo in the lactating mammary epithelial cell. Am J Physiol Endocrinol Metab. 2011; 300:E1059–68. https://doi.org/10.1152/ajpendo.00083.2011 [PubMed]
• 31. Rudolph MC, Wellberg EA, Lewis AS, Terrell KL, Merz AL, Maluf NK, Serkova NJ, Anderson SM. Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium. J Lipid Res. 2014; 55:1052–65. https://doi.org/10.1194/jlr.M044487 [PubMed]
• 32. Suburu J, Shi L, Wu J, Wang S, Samuel M, Thomas MJ, Kock ND, Yang G, Kridel S, Chen YQ. Fatty acid synthase is required for mammary gland development and milk production during lactation. Am J Physiol Endocrinol Metab. 2014; 306:E1132–43. https://doi.org/10.1152/ajpendo.00514.2013 [PubMed]
• 33. Menendez JA, Lupu R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis. Nat Rev Cancer. 2007; 7:763–77. https://doi.org/10.1038/nrc2222 [PubMed]
• 34. Röhrig F, Schulze A. The multifaceted roles of fatty acid synthesis in cancer. Nat Rev Cancer. 2016; 16:732–49. https://doi.org/10.1038/nrc.2016.89 [PubMed]
• 35. Menendez JA, Lupu R. Fatty acid synthase (FASN) as a therapeutic target in breast cancer. Expert Opin Ther Targets. 2017; 21:1001–16. https://doi.org/10.1080/14728222.2017.1381087 [PubMed]
• 36. Menendez JA, Vellon L, Mehmi I, Oza BP, Ropero S, Colomer R, Lupu R. Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells. Proc Natl Acad Sci USA. 2004; 101:10715–20. https://doi.org/10.1073/pnas.0403390101 [PubMed]
• 37. Bueno MJ, Jimenez-Renard V, Samino S, Capellades J, Junza A, López-Rodríguez ML, Garcia-Carceles J, Lopez-Fabuel I, Bolaños JP, Chandel NS, Yanes O, Colomer R, Quintela-Fandino M. Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells. Nat Commun. 2019; 10:5011. https://doi.org/10.1038/s41467-019-13028-1 [PubMed]
• 38. Gonzalez-Guerrico AM, Espinoza I, Schroeder B, Park CH, Kvp CM, Khurana A, Corominas-Faja B, Cuyàs E, Alarcón T, Kleer C, Menendez JA, Lupu R. Suppression of endogenous lipogenesis induces reversion of the Malignant phenotype and normalized differentiation in breast cancer. Oncotarget. 2016; 7:71151–68. https://doi.org/10.18632/oncotarget.9463 [PubMed]
• 39. Sounni NE, Cimino J, Blacher S, Primac I, Truong A, Mazzucchelli G, Paye A, Calligaris D, Debois D, De Tullio P, Mari B, De Pauw E, Noel A. Blocking lipid synthesis overcomes tumor regrowth and metastasis after antiangiogenic therapy withdrawal. Cell Metab. 2014; 20:280–94. https://doi.org/10.1016/j.cmet.2014.05.022 [PubMed]
• 40. Hovey RC, Trott JF, Ginsburg E, Goldhar A, Sasaki MM, Fountain SJ, Sundararajan K, Vonderhaar BK. Transcriptional and spatiotemporal regulation of prolactin receptor mRNA and cooperativity with progesterone receptor function during ductal branch growth in the mammary gland. Dev Dyn. 2001; 222:192–205. https://doi.org/10.1002/dvdy.1179 [PubMed]
• 41. Goldhar AS, Duan R, Ginsburg E, Vonderhaar BK. Progesterone induces expression of the prolactin receptor gene through cooperative action of Sp1 and C/EBP. Mol Cell Endocrinol. 2011; 335:148–57. https://doi.org/10.1016/j.mce.2011.01.004 [PubMed]
• 42. Buser AC, Gass-Handel EK, Wyszomierski SL, Doppler W, Leonhardt SA, Schaack J, Rosen JM, Watkin H, Anderson SM, Edwards DP. Progesterone receptor repression of prolactin/signal transducer and activator of transcription 5-mediated transcription of the beta-casein gene in mammary epithelial cells. Mol Endocrinol. 2007; 21:106–25. https://doi.org/10.1210/me.2006-0297 [PubMed]
• 43. Richer JK, Jacobsen BM, Manning NG, Abel MG, Wolf DM, Horwitz KB. Differential gene regulation by the two progesterone receptor isoforms in human breast cancer cells. J Biol Chem. 2002; 277:5209–18. https://doi.org/10.1074/jbc.M110090200 [PubMed]
• 44. Singhal H, Greene ME, Zarnke AL, Laine M, Al Abosy R, Chang YF, Dembo AG, Schoenfelt K, Vadhi R, Qiu X, Rao P, Santhamma B, Nair HB, et al. Progesterone receptor isoforms, agonists and antagonists differentially reprogram estrogen signaling. Oncotarget. 2017; 9:4282–300. https://doi.org/10.18632/oncotarget.21378 [PubMed]
• 45. Diep CH, Knutson TP, Lange CA. Active FOXO1 is a key determinant of isoform-specific progesterone receptor transactivation and senescence programming. Mol Cancer Res. 2016; 14:141–62. https://doi.org/10.1158/1541-7786.MCR-15-0431 [PubMed]
• 46. Conneely OM, Mulac-Jericevic B, Lydon JP, De Mayo FJ. Reproductive functions of the progesterone receptor isoforms: lessons from knock-out mice. Mol Cell Endocrinol. 2001; 179:97–103. https://doi.org/10.1016/s0303-7207(01)00465-8 [PubMed]
• 47. Soyal S, Ismail PM, Li J, Mulac-Jericevic B, Conneely OM, Lydon JP. Progesterone’s role in mammary gland development and tumorigenesis as disclosed by experimental mouse genetics. Breast Cancer Res. 2002; 4:191–96. https://doi.org/10.1186/bcr451 [PubMed]
• 48. Conneely OM, Mulac-Jericevic B, Lydon JP. Progesterone-dependent regulation of female reproductive activity by two distinct progesterone receptor isoforms. Steroids. 2003; 68:771–78. https://doi.org/10.1016/s0039-128x(03)00126-0 [PubMed]
• 49. Mulac-Jericevic B, Lydon JP, DeMayo FJ, Conneely OM. Defective mammary gland morphogenesis in mice lacking the progesterone receptor B isoform. Proc Natl Acad Sci USA. 2003; 100:9744–49. https://doi.org/10.1073/pnas.1732707100 [PubMed]
• 50. McGowan EM, Clarke CL. Effect of overexpression of progesterone receptor A on endogenous progestin-sensitive endpoints in breast cancer cells. Mol Endocrinol. 1999; 13:1657–71. https://doi.org/10.1210/mend.13.10.0356 [PubMed]
• 51. Sartorius CA, Shen T, Horwitz KB. Progesterone receptors a and B differentially affect the growth of estrogen-dependent human breast tumor xenografts. Breast Cancer Res Treat. 2003; 79:287–99. https://doi.org/10.1023/a:1024031731269 [PubMed]
• 52. Tung L, Abdel-Hafiz H, Shen T, Harvell DM, Nitao LK, Richer JK, Sartorius CA, Takimoto GS, Horwitz KB. Progesterone receptors (PR)-B and -A regulate transcription by different mechanisms: AF-3 exerts regulatory control over coactivator binding to PR-B. Mol Endocrinol. 2006; 20:2656–70. https://doi.org/10.1210/me.2006-0105 [PubMed]
• 53. Faivre EJ, Daniel AR, Hillard CJ, Lange CA. Progesterone receptor rapid signaling mediates serine 345 phosphorylation and tethering to specificity protein 1 transcription factors. Mol Endocrinol. 2008; 22:823–37. https://doi.org/10.1210/me.2007-0437 [PubMed]
• 54. Wargon V, Fernandez SV, Goin M, Giulianelli S, Russo J, Lanari C. Hypermethylation of the progesterone receptor a in constitutive antiprogestin-resistant mouse mammary carcinomas. Breast Cancer Res Treat. 2011; 126:319–32. https://doi.org/10.1007/s10549-010-0908-x [PubMed]
• 55. Knutson TP, Daniel AR, Fan D, Silverstein KA, Covington KR, Fuqua SA, Lange CA. Phosphorylated and sumoylation-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression. Breast Cancer Res. 2012; 14:R95. https://doi.org/10.1186/bcr3211 [PubMed]
• 56. Khan JA, Bellance C, Guiochon-Mantel A, Lombès M, Loosfelt H. Differential regulation of breast cancer-associated genes by progesterone receptor isoforms PRA and PRB in a new bi-inducible breast cancer cell line. PLoS One. 2012; 7:e45993. https://doi.org/10.1371/journal.pone.0045993 [PubMed]
• 57. Daniel AR, Gaviglio AL, Knutson TP, Ostrander JH, D’Assoro AB, Ravindranathan P, Peng Y, Raj GV, Yee D, Lange CA. Progesterone receptor-B enhances estrogen responsiveness of breast cancer cells via scaffolding PELP1- and estrogen receptor-containing transcription complexes. Oncogene. 2015; 34:506–15. https://doi.org/10.1038/onc.2013.579 [PubMed]
• 58. Wargon V, Riggio M, Giulianelli S, Sequeira GR, Rojas P, May M, Polo ML, Gorostiaga MA, Jacobsen B, Molinolo A, Novaro V, Lanari C. Progestin and antiprogestin responsiveness in breast cancer is driven by the PRA/PRB ratio via AIB1 or SMRT recruitment to the CCND1 and MYC promoters. Int J Cancer. 2015; 136:2680–92. https://doi.org/10.1002/ijc.29304 [PubMed]
• 59. Rojas PA, May M, Sequeira GR, Elia A, Alvarez M, Martínez P, Gonzalez P, Hewitt S, He X, Perou CM, Molinolo A, Gibbons L, Abba MC, et al. Progesterone Receptor Isoform Ratio: A Breast Cancer Prognostic and Predictive Factor for Antiprogestin Responsiveness. J Natl Cancer Inst. 2017; 109:djw317. https://doi.org/10.1093/jnci/djw317 [PubMed]
• 60. Knutson TP, Truong TH, Ma S, Brady NJ, Sullivan ME, Raj G, Schwertfeger KL, Lange CA. Posttranslationally modified progesterone receptors direct ligand-specific expression of breast cancer stem cell-associated gene programs. J Hematol Oncol. 2017; 10:89. https://doi.org/10.1186/s13045-017-0462-7 [PubMed]
• 61. Lamb CA, Fabris VT, Jacobsen B, Molinolo AA, Lanari C. Biological and clinical impact of imbalanced progesterone receptor isoform ratios in breast cancer. Endocr Relat Cancer. 2018. [Epub ahead of print]. https://doi.org/10.1530/ERC-18-0179 [PubMed]
• 62. Truong TH, Dwyer AR, Diep CH, Hu H, Hagen KM, Lange CA. Phosphorylated progesterone receptor isoforms mediate opposing stem cell and proliferative breast cancer cell fates. Endocrinology. 2019; 160:430–46. https://doi.org/10.1210/en.2018-00990 [PubMed]
• 63. Bole-Feysot C, Goffin V, Edery M, Binart N, Kelly PA. Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr Rev. 1998; 19:225–68. https://doi.org/10.1210/edrv.19.3.0334 [PubMed]
• 64. Joyeux C, Chalbos D, Rochefort H. Effects of progestins and menstrual cycle on fatty acid synthetase and progesterone receptor in human mammary glands. J Clin Endocrinol Metab. 1990; 70:1438–44. https://doi.org/10.1210/jcem-70-5-1438 [PubMed]
• 65. Chalbos D, Joyeux C, Galtier F, Rochefort H. Progestin-induced fatty acid synthetase in human mammary tumors: from molecular to clinical studies. J Steroid Biochem Mol Biol. 1992; 43:223–28. https://doi.org/10.1016/0960-0760(92)90211-z [PubMed]
• 66. Chalbos D, Escot C, Joyeux C, Tissot-Carayon MJ, Pages A, Rochefort H. Expression of the progestin-induced fatty acid synthetase in benign mastopathies and breast cancer as measured by RNA in situ hybridization. J Natl Cancer Inst. 1990; 82:602–06. https://doi.org/10.1093/jnci/82.7.602 [PubMed]
• 67. Menendez JA, Oza BP, Colomer R, Lupu R. The estrogenic activity of synthetic progestins used in oral contraceptives enhances fatty acid synthase-dependent breast cancer cell proliferation and survival. Int J Oncol. 2005; 26:1507–15. [PubMed]
• 68. Esslimani-Sahla M, Thezenas S, Simony-Lafontaine J, Kramar A, Lavaill R, Chalbos D, Rochefort H. Increased expression of fatty acid synthase and progesterone receptor in early steps of human mammary carcinogenesis. Int J Cancer. 2007; 120:224–29. https://doi.org/10.1002/ijc.22202 [PubMed]
• 69. Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, Speed D, Lynch AG, Samarajiwa S, Yuan Y, Gräf S, Ha G, Haffari G, et al, and METABRIC Group. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature. 2012; 486:346–52. https://doi.org/10.1038/nature10983 [PubMed]
• 70. Pereira B, Chin SF, Rueda OM, Vollan HK, Provenzano E, Bardwell HA, Pugh M, Jones L, Russell R, Sammut SJ, Tsui DW, Liu B, Dawson SJ, et al. The somatic mutation profiles of 2,433 breast cancers refines their genomic and transcriptomic landscapes. Nat Commun. 2016; 7:11479. https://doi.org/10.1038/ncomms11479 [PubMed]
• 71. Tibshirani R, Hastie T, Narasimhan B, Chu G. Diagnosis of multiple cancer types by shrunken centroids of gene expression. Proc Natl Acad Sci USA. 2002; 99:6567–72. https://doi.org/10.1073/pnas.082099299 [PubMed]
• 72. Sartorius CA, Groshong SD, Miller LA, Powell RL, Tung L, Takimoto GS, Horwitz KB. New T47D breast cancer cell lines for the independent study of progesterone B- and A-receptors: only antiprogestin-occupied B-receptors are switched to transcriptional agonists by cAMP. Cancer Res. 1994; 54:3868–77. [PubMed]
• 73. Lange CA, Sartorius CA, Abdel-Hafiz H, Spillman MA, Horwitz KB, Jacobsen BM. Progesterone receptor action: translating studies in breast cancer models to clinical insights. Adv Exp Med Biol. 2008; 630:94–111. [PubMed]
• 74. Jacobsen BM, Schittone SA, Richer JK, Horwitz KB. Progesterone-independent effects of human progesterone receptors (PRs) in estrogen receptor-positive breast cancer: PR isoform-specific gene regulation and tumor biology. Mol Endocrinol. 2005; 19:574–87. https://doi.org/10.1210/me.2004-0287 [PubMed]
• 75. Jacobsen BM, Horwitz KB. Progesterone receptors, their isoforms and progesterone regulated transcription. Mol Cell Endocrinol. 2012; 357:18–29. https://doi.org/10.1016/j.mce.2011.09.016 [PubMed]
• 76. Swinnen JV, Ulrix W, Heyns W, Verhoeven G. Coordinate regulation of lipogenic gene expression by androgens: evidence for a cascade mechanism involving sterol regulatory element binding proteins. Proc Natl Acad Sci USA. 1997; 94:12975–80. https://doi.org/10.1073/pnas.94.24.12975 [PubMed]
• 77. Swinnen JV, Heemers H, Deboel L, Foufelle F, Heyns W, Verhoeven G. Stimulation of tumor-associated fatty acid synthase expression by growth factor activation of the sterol regulatory element-binding protein pathway. Oncogene. 2000; 19:5173–81. https://doi.org/10.1038/sj.onc.1203889 [PubMed]
• 78. Martel PM, Bingham CM, McGraw CJ, Baker CL, Morganelli PM, Meng ML, Armstrong JM, Moncur JT, Kinlaw WB. S14 protein in breast cancer cells: direct evidence of regulation by SREBP-1c, superinduction with progestin, and effects on cell growth. Exp Cell Res. 2006; 312:278–88. https://doi.org/10.1016/j.yexcr.2005.10.022 [PubMed]
• 79. Goffin V, Touraine P, Pichard C, Bernichtein S, Kelly PA. Should prolactin be reconsidered as a therapeutic target in human breast cancer? Mol Cell Endocrinol. 1999; 151:79–87. https://doi.org/10.1016/s0303-7207(99)00023-4 [PubMed]
• 80. Goffin V, Bernichtein S, Touraine P, Kelly PA. Development and potential clinical uses of human prolactin receptor antagonists. Endocr Rev. 2005; 26:400–22. https://doi.org/10.1210/er.2004-0016 [PubMed]
• 81. Goffin V, Touraine P, Culler MD, Kelly PA. Drug insight: prolactin-receptor antagonists, a novel approach to treatment of unresolved systemic and local hyperprolactinemia? Nat Clin Pract Endocrinol Metab. 2006; 2:571–81. https://doi.org/10.1038/ncpendmet0270 [PubMed]
• 82. Scotti ML, Langenheim JF, Tomblyn S, Springs AE, Chen WY. Additive effects of a prolactin receptor antagonist, G129R, and herceptin on inhibition of HER2-overexpressing breast cancer cells. Breast Cancer Res Treat. 2008; 111:241–50. https://doi.org/10.1007/s10549-007-9789-z [PubMed]
• 83. Menendez JA, Oza BP, Atlas E, Verma VA, Mehmi I, Lupu R. Inhibition of tumor-associated fatty acid synthase activity antagonizes estradiol- and tamoxifen-induced agonist transactivation of estrogen receptor (ER) in human endometrial adenocarcinoma cells. Oncogene. 2004; 23:4945–58. https://doi.org/10.1038/sj.onc.1207476 [PubMed]
• 84. Lupu R, Menendez JA. Targeting fatty acid synthase in breast and endometrial cancer: an alternative to selective estrogen receptor modulators? Endocrinology. 2006; 147:4056–66. https://doi.org/10.1210/en.2006-0486 [PubMed]
• 85. Saito-Reis C, Visbal A, Ashcroft F, Edwards D. Progesterone receptor effects on cell morphology and EMT associated genes in breast cancer cells. Conference: 2011 Society for Advancement of Hispanics/Chicanos and Native Americans in Science National Conference.
• 86. Visbal A, Boonyaratanakornkit V, Edwards DP. The role of progesterone receptor as a suppressor of epithelial-mesenchymal transition in breast cancer. The 3rd Annual REACH IRACDA Symposium.
• 87. Nouhi Z, Chughtai N, Hartley S, Cocolakis E, Lebrun JJ, Ali S. Defining the role of prolactin as an invasion suppressor hormone in breast cancer cells. Cancer Res. 2006; 66:1824–32. https://doi.org/10.1158/0008-5472.CAN-05-2292 [PubMed]
• 88. Hachim IY, López-Ozuna VM, Hachim MY, Lebrun JJ, Ali S. Concomitant expression of prolactin receptor and TGFβ receptors in breast cancer: association with less aggressive phenotype and favorable patient outcome. Int J Mol Sci. 2019; 20:1640. https://doi.org/10.3390/ijms20071640 [PubMed]
• 89. Sullivan NJ, Sasser AK, Axel AE, Vesuna F, Raman V, Ramirez N, Oberyszyn TM, Hall BM. Interleukin-6 induces an epithelial-mesenchymal transition phenotype in human breast cancer cells. Oncogene. 2009; 28:2940–47. https://doi.org/10.1038/onc.2009.180 [PubMed]
• 90. Xie G, Yao Q, Liu Y, Du S, Liu A, Guo Z, Sun A, Ruan J, Chen L, Ye C, Yuan Y. IL-6-induced epithelial-mesenchymal transition promotes the generation of breast cancer stem-like cells analogous to mammosphere cultures. Int J Oncol. 2012; 40:1171–79. https://doi.org/10.3892/ijo.2011.1275 [PubMed]
• 91. Korkaya H, Kim GI, Davis A, Malik F, Henry NL, Ithimakin S, Quraishi AA, Tawakkol N, D’Angelo R, Paulson AK, Chung S, Luther T, Paholak HJ, et al. Activation of an IL6 inflammatory loop mediates trastuzumab resistance in HER2+ breast cancer by expanding the cancer stem cell population. Mol Cell. 2012; 47:570–84. https://doi.org/10.1016/j.molcel.2012.06.014 [PubMed]
• 92. Yang L, Han S, Sun Y. An IL6-STAT3 loop mediates resistance to PI3K inhibitors by inducing epithelial-mesenchymal transition and cancer stem cell expansion in human breast cancer cells. Biochem Biophys Res Commun. 2014; 453:582–87. https://doi.org/10.1016/j.bbrc.2014.09.129 [PubMed]
• 93. Xu C, Langenheim JF, Chen WY. Stromal-epithelial interactions modulate cross-talk between prolactin receptor and HER2/Neu in breast cancer. Breast Cancer Res Treat. 2012; 134:157–69. https://doi.org/10.1007/s10549-012-1954-3 [PubMed]
• 94. Tomblyn S, Springs AE, Langenheim JF, Chen WY. Combination therapy using three novel prolactin receptor antagonist-based fusion proteins effectively inhibits tumor recurrence and metastasis in HER2/neu transgenic mice. Int J Oncol. 2009; 34:1139–46. https://doi.org/10.3892/ijo_00000242 [PubMed]
• 95. Menendez JA, Mehmi I, Verma VA, Teng PK, Lupu R. Pharmacological inhibition of fatty acid synthase (FAS): a novel therapeutic approach for breast cancer chemoprevention through its ability to suppress her-2/neu (erbB-2) oncogene-induced Malignant transformation. Mol Carcinog. 2004; 41:164–78. https://doi.org/10.1002/mc.20054 [PubMed]
• 96. Vazquez-Martin A, Colomer R, Brunet J, Lupu R, Menendez JA. Overexpression of fatty acid synthase gene activates HER1/HER2 tyrosine kinase receptors in human breast epithelial cells. Cell Prolif. 2008; 41:59–85. https://doi.org/10.1111/j.1365-2184.2007.00498.x [PubMed]
• 97. Vazquez-Martin A, Fernandez-Real JM, Oliveras-Ferraros C, Navarrete JM, Martin-Castillo B, Del Barco S, Brunet J, Menendez JA. Fatty acid synthase activity regulates HER2 extracellular domain shedding into the circulation of HER2-positive metastatic breast cancer patients. Int J Oncol. 2009; 35:1369–76. https://doi.org/10.3892/ijo_00000455 [PubMed]
• 98. Corominas-Faja B, Vellon L, Cuyàs E, Buxó M, Martin-Castillo B, Serra D, García J, Lupu R, Menendez JA. Clinical and therapeutic relevance of the metabolic oncogene fatty acid synthase in HER2+ breast cancer. Histol Histopathol. 2017; 32:687–98. https://doi.org/10.14670/HH-11-830 [PubMed]
• 99. O’Leary KA, Rugowski DE, Sullivan R, Schuler LA. Prolactin cooperates with loss of p53 to promote claudin-low mammary carcinomas. Oncogene. 2014; 33:3075–82. https://doi.org/10.1038/onc.2013.278 [PubMed]
• 100. Yonezawa T, Chen KH, Ghosh MK, Rivera L, Dill R, Ma L, Villa PA, Kawaminami M, Walker AM. Anti-metastatic outcome of isoform-specific prolactin receptor targeting in breast cancer. Cancer Lett. 2015; 366:84–92. https://doi.org/10.1016/j.canlet.2015.06.010 [PubMed]
• 101. Haines E, Minoo P, Feng Z, Resalatpanah N, Nie XM, Campiglio M, Alvarez L, Cocolakis E, Ridha M, Di Fulvio M, Gomez-Cambronero J, Lebrun JJ, Ali S. Tyrosine phosphorylation of Grb2: role in prolactin/epidermal growth factor cross talk in mammary epithelial cell growth and differentiation. Mol Cell Biol. 2009; 29:2505–20. https://doi.org/10.1128/MCB.00034-09 [PubMed]
• 102. Hachim IY, Hachim MY, Lopez VM, Lebrun JJ, Ali S. Prolactin receptor expression is an independent favorable prognostic marker in human breast cancer. Appl Immunohistochem Mol Morphol. 2016; 24:238–45. https://doi.org/10.1097/PAI.0000000000000178 [PubMed]
• 103. Hachim IY, Shams A, Lebrun JJ, Ali S. A favorable role of prolactin in human breast cancer reveals novel pathway-based gene signatures indicative of tumor differentiation and favorable patient outcome. Hum Pathol. 2016; 53:142–52. https://doi.org/10.1016/j.humpath.2016.02.010 [PubMed]
• 104. Nitze LM, Galsgaard ED, Din N, Lund VL, Rasmussen BB, Berchtold MW, Christensen L, Panina S. Reevaluation of the proposed autocrine proliferative function of prolactin in breast cancer. Breast Cancer Res Treat. 2013; 142:31–44. https://doi.org/10.1007/s10549-013-2731-7 [PubMed]
• 105. Galsgaard ED, Rasmussen BB, Folkesson CG, Rasmussen LM, Berchtold MW, Christensen L, Panina S. Re-evaluation of the prolactin receptor expression in human breast cancer. J Endocrinol. 2009; 201:115–28. https://doi.org/10.1677/JOE-08-0479 [PubMed]
• 106. Agarwal N, Machiels JP, Suárez C, Lewis N, Higgins M, Wisinski K, Awada A, Maur M, Stein M, Hwang A, Mosher R, Wasserman E, Wu G, et al. Phase I study of the prolactin receptor antagonist LFA102 in metastatic breast and castration-resistant prostate cancer. Oncologist. 2016; 21:535–36. https://doi.org/10.1634/theoncologist.2015-0502 [PubMed]
• 107. López-Ozuna VM, Hachim IY, Hachim MY, Lebrun JJ, Ali S. Prolactin pro-differentiation pathway in triple negative breast cancer: impact on prognosis and potential therapy. Sci Rep. 2016; 6:30934. https://doi.org/10.1038/srep30934 [PubMed]
• 108. Jacobsen BM, Richer JK, Schittone SA, Horwitz KB. New human breast cancer cells to study progesterone receptor isoform ratio effects and ligand-independent gene regulation. J Biol Chem. 2002; 277:27793–800. https://doi.org/10.1074/jbc.M202584200 [PubMed]
• 109. McGowan EM, Weinberger RP, Graham JD, Hill HD, Hughes JA, O’Neill GM, Clarke CL. Cytoskeletal responsiveness to progestins is dependent on progesterone receptor a levels. J Mol Endocrinol. 2003; 31:241–53. https://doi.org/10.1677/jme.0.0310241 [PubMed]
• 110. Graham JD, Yager ML, Hill HD, Byth K, O’Neill GM, Clarke CL. Altered progesterone receptor isoform expression remodels progestin responsiveness of breast cancer cells. Mol Endocrinol. 2005; 19:2713–35. https://doi.org/10.1210/me.2005-0126 [PubMed]
• 111. Vegeto E, Shahbaz MM, Wen DX, Goldman ME, O’Malley BW, McDonnell DP. Human progesterone receptor a form is a cell- and promoter-specific repressor of human progesterone receptor B function. Mol Endocrinol. 1993; 7:1244–55. https://doi.org/10.1210/mend.7.10.8264658 [PubMed]
• 112. Mulac-Jericevic B, Mullinax RA, DeMayo FJ, Lydon JP, Conneely OM. Subgroup of reproductive functions of progesterone mediated by progesterone receptor-B isoform. Science. 2000; 289:1751–54. https://doi.org/10.1126/science.289.5485.1751 [PubMed]
• 113. McGowan EM, Russell AJ, Boonyaratanakornkit V, Saunders DN, Lehrbach GM, Sergio CM, Musgrove EA, Edwards DP, Sutherland RL. Progestins reinitiate cell cycle progression in antiestrogen-arrested breast cancer cells through the B-isoform of progesterone receptor. Cancer Res. 2007; 67:8942–51. https://doi.org/10.1158/0008-5472.CAN-07-1255 [PubMed]
• 114. Wang H, Lee EW, Zhou L, Leung PC, Ross DD, Unadkat JD, Mao Q. Progesterone receptor (PR) isoforms PRA and PRB differentially regulate expression of the breast cancer resistance protein in human placental choriocarcinoma BeWo cells. Mol Pharmacol. 2008; 73:845–54. https://doi.org/10.1124/mol.107.041087 [PubMed]
• 115. Mohamed MK, Tung L, Takimoto GS, Horwitz KB. The leucine zippers of c-fos and c-jun for progesterone receptor dimerization: a-dominance in the a/B heterodimer. J Steroid Biochem Mol Biol. 1994; 51:241–50. https://doi.org/10.1016/0960-0760(94)90036-1 [PubMed]
• 116. Huang YL, Zhao F, Luo CC, Zhang X, Si Y, Sun Z, Zhang L, Li QZ, Gao XJ. SOCS3-mediated blockade reveals major contribution of JAK2/STAT5 signaling pathway to lactation and proliferation of dairy cow mammary epithelial cells in vitro. Molecules. 2013; 18:12987–3002. https://doi.org/10.3390/molecules181012987 [PubMed]
• 117. Yu Y, Yuan X, Li P, Wang Y, Yu M, Gao X. Vaccarin promotes proliferation of and milk synthesis in bovine mammary epithelial cells through the prl receptor-PI3K signaling pathway. Eur J Pharmacol. 2020; 880:173190. https://doi.org/10.1016/j.ejphar.2020.173190 [PubMed]
• 118. Li T, Weng J, Zhang Y, Liang K, Fu G, Li Y, Bai X, Gao Y. mTOR direct crosstalk with STAT5 promotes de novo lipid synthesis and induces hepatocellular carcinoma. Cell Death Dis. 2019; 10:619. https://doi.org/10.1038/s41419-019-1828-2 [PubMed]
• 119. Hagan CR, Faivre EJ, Lange CA. Scaffolding actions of membrane-associated progesterone receptors. Steroids. 2009; 74:568–72. https://doi.org/10.1016/j.steroids.2008.12.004 [PubMed]
• 120. Hagan CR, Knutson TP, Lange CA. A common docking domain in progesterone receptor-B links DUSP6 and CK2 signaling to proliferative transcriptional programs in breast cancer cells. Nucleic Acids Res. 2013; 41:8926–42. https://doi.org/10.1093/nar/gkt706 [PubMed]
• 121. Diogenes A, Patwardhan AM, Jeske NA, Ruparel NB, Goffin V, Akopian AN, Hargreaves KM. Prolactin modulates TRPV1 in female rat trigeminal sensory neurons. J Neurosci. 2006; 26:8126–36. https://doi.org/10.1523/JNEUROSCI.0793-06.2006 [PubMed]
• 122. Nevalainen MT, Valve EM, Ahonen T, Yagi A, Paranko J, Härkönen PL. Androgen-dependent expression of prolactin in rat prostate epithelium in vivo and in organ culture. FASEB J. 1997; 11:1297–307. https://doi.org/10.1096/fasebj.11.14.9409549 [PubMed]
• 123. Chen CC, Stairs DB, Boxer RB, Belka GK, Horseman ND, Alvarez JV, Chodosh LA. Autocrine prolactin induced by the Pten-Akt pathway is required for lactation initiation and provides a direct link between the Akt and Stat5 pathways. Genes Dev. 2012; 26:2154–68. https://doi.org/10.1101/gad.197343.112 [PubMed]
• 124. Grunt TW, Wagner R, Grusch M, Berger W, Singer CF, Marian B, Zielinski CC, Lupu R. Interaction between fatty acid synthase- and ErbB-systems in ovarian cancer cells. Biochem Biophys Res Commun. 2009; 385:454–59. https://doi.org/10.1016/j.bbrc.2009.05.085 [PubMed]
• 125. Menendez JA, Lupu R. Fatty acid synthase regulates estrogen receptor-α signaling in breast cancer cells. Oncogenesis. 2017; 6:e299. https://doi.org/10.1038/oncsis.2017.4 [PubMed]
• 126. De Amicis F, Guido C, Santoro M, Lanzino M, Panza S, Avena P, Panno ML, Perrotta I, Aquila S, Andò S. A novel functional interplay between progesterone receptor-B and PTEN, via AKT, modulates autophagy in breast cancer cells. J Cell Mol Med. 2014; 18:2252–65. https://doi.org/10.1111/jcmm.12363 [PubMed]
• 127. Makowski K, Mera P, Paredes D, Herrero L, Ariza X, Asins G, Hegardt FG, García J, Serra D. Differential pharmacologic properties of the two C75 enantiomers: (+)-C75 is a strong anorectic drug; (-)-C75 has antitumor activity. Chirality. 2013; 25:281–87. https://doi.org/10.1002/chir.22139 [PubMed]
• 128. Rae C, Babich JW, Mairs RJ. Differential in radiosensitizing potency of enantiomers of the fatty acid synthase inhibitor C75. Chirality. 2017; 29:10–13. https://doi.org/10.1002/chir.22668 [PubMed]
• 129. Montesdeoca N, López M, Ariza X, Herrero L, Makowski K. Inhibitors of lipogenic enzymes as a potential therapy against cancer. FASEB J. 2020. [Epub ahead of print]. https://doi.org/10.1096/fj.202000705R [PubMed]
• 130. Veigel D, Wagner R, Stübiger G, Wuczkowski M, Filipits M, Horvat R, Benhamú B, López-Rodríguez ML, Leisser A, Valent P, Grusch M, Hegardt FG, García J, et al. Fatty acid synthase is a metabolic marker of cell proliferation rather than Malignancy in ovarian cancer and its precursor cells. Int J Cancer. 2015; 136:2078–90. https://doi.org/10.1002/ijc.29261 [PubMed]
• 131. Hagan CR, Lange CA. Molecular determinants of context-dependent progesterone receptor action in breast cancer. BMC Med. 2014; 12:32. https://doi.org/10.1186/1741-7015-12-32 [PubMed]
• 132. Pateetin P, Pisitkun T, McGowan E, Boonyaratanakornkit V. Differential quantitative proteomics reveals key proteins related to phenotypic changes of breast cancer cells expressing progesterone receptor a. J Steroid Biochem Mol Biol. 2020; 198:105560. https://doi.org/10.1016/j.jsbmb.2019.105560 [PubMed]
• 133. Leehy KA, Truong TH, Mauro LJ, Lange CA. Progesterone receptors (PR) mediate STAT actions: PR and prolactin receptor signaling crosstalk in breast cancer models. J Steroid Biochem Mol Biol. 2018; 176:88–93. https://doi.org/10.1016/j.jsbmb.2017.04.011 [PubMed]