The association between expression of prolactin receptor and lymph node involvement in triple-negative breast cancer

Breast cancer (BC) is the most common cancer among women. It is known that the prolactin receptor (PRLR) may play a role in breast carcinogenesis, but the available data are often contradictory. To get a more complete picture of the relationship between the receptor and mammary gland carcinogenesis, we examined the association between changes in PRLR expression level and tumor subtype (and its main characteristics). To do this, using real-time PCR, we evaluated the level of PRLR mRNA in BC tissue samples and untransformed adjoining tissue samples (89 pairs). Since the androgen receptor (AR) has begun to be seen as a prognostic marker in breast cancer, we also evaluated the association between mRNA levels of AR and PRLR. We found a significant increase in PRLR expression in luminal subtypes; the highest level of PRLR mRNA was detected in luminal A subtype. In HER2-positive ER-, PR-negative BC, the PRLR mRNA level decreases in tumor tissues compared with untransformed tissues. High PRLR expression is also associated with smaller tumor size in luminal B HER2-negative subtype. In ER-, PR-negative tumors, PRLR expression is associated with AR expression: PRLR mRNA level is increased when AR mRNA level is reduced by more than 8 times in triple-negative tumors; in contrast, in HER2-positive subtype it decreases more significantly when AR expression is reduced by more than 3 times. A tendency towards an increase in PRLR expression with an increase in the AR mRNA level was also discovered in luminal subtypes. The level of PRLR expression depends on the age of patients. In luminal A, PRLR expression is higher in patients under 65 years. In contrast, in luminal B HER2-negative and triple-negative BC, reduced PRLR expression was observed in patients under the age of 40 years and under the age of 50 years, respectively. In this group of patients under the age of 40 years with luminal B HER2-negative BC, ER expression was also reduced (0-4 score according to the IHC assay). Thus, PRLR probably plays a different role in the development and progression of BC: in luminal A and luminal B HER2-positive subtypes PRLR may act as an oncogen, and in luminal B HER2-negative and ER-, PR-negative subtypes can play a tumor suppressor role.

BackgroundProlactin (PRL) is a polypeptide hormone secreted by the anterior pituitary to stimulate growth and differentiation of the normal mammary gland. Together with its receptor, prolactin receptor (PRLR) have been shown to play a role in breast cancer. This study aimed to examine the roles of PRL and PRLR polymorphisms and expression in breast cancer risk and aggressiveness in Thai patients.MethodsPRL (rs3756824 C/G and rs2244502 T/A) and PRLR (rs37364 G/T and rs249537 A/G) polymorphisms were genotyped by real-time PCR and PRLR expression was assessed by immunohistochemistry (IHC) in breast cancer tissues. The correlations between PRL and PRLR polymorphisms and breast cancer susceptibility/aggressiveness as well as the associations between PRLR expression and clinicopathological parameters were determined.ResultsTwo hundred and twenty-seven breast cancer patients and 119 matched controls were recruited at the Division of Head Neck and Breast Surgery, Department of Surgery, Faculty of Medicine, Siriraj Hospital, Thailand from 2010–2014. PRL and PRLR polymorphisms were not correlated with breast cancer susceptibility and there was no association between PRLR polymorphisms and PRLR expression. PRLR was frequently overexpressed in breast cancer with positive hormone receptors. High expression of PRLR was significantly related to the presence of axillary nodal metastasis and lymphovascular invasion and showed a trend towards poorer outcome.ConclusionsThere was a correlation between high PRLR expression and aggressive features of breast cancer. PRLR expression might be utilized as a prognostic factor for identification of luminal breast cancer with poorer outcome.

lncRNA MIR503HG inhibits cell proliferation and promotes apoptosis in TNBC cells via the miR-224-5p/HOXA9 axis

Background: Kinases represent one of the main therapeutic targets in cancer treatment as their impairing relates to tumor growth and progression. Despite increasing evidence that the tumor microenvironment (TME) signaling influences the behavior of surrounding cancer epithelial cells, still relatively little is known about what changes in stromal cells influence tumor cells' behavior and how they affect their response to target therapy. TNBC patients are still lacking an effective therapy, as not much is known about the biology of this BC tumor subtype. Clinical trials focus mainly on targeting epithelial cancer cells by using a combination of kinase inhibitors and standard chemotherapy however these therapeutic regimens are not considering the action of stromal cells that may influence response to treatment. Aims: • Identify fibroblasts-expressed kinases that modulate tumor cells' invasion • Characterize the mechanism by which these kinases promote/reduce tumor cell invasion. Methods: Human Mammary Fibroblast (HMF) and normal lung fibroblast (MRC5) cells were used in a high-throughput siRNA kinome screening targeting 710 Kinases. 24 hours after transfection, fibroblasts were co-cultured with MDA-MB-231 in 3D for spheroid formation. Matrigel and chemoattractants were then added to promote invasion. Spheroids pictures taken at different time-points were analyzed and results were expressed as changes in spheroid surface formation (ΔRatio= ΔCtrl/ΔKinase). Results: PIK3Cδ was among the most prominent targets, whose silencing decreased TNBC invasion. PIK3Cδ is essential for regulating chemokine production in leukocytes and promotes migration during inflammation, while PIK3Cδ inhibitors (CAL -101) interfere in tumour-stroma interactions without directly killing cancer cells. Despite PIK3Cδ being expressed mainly in leucocytes, we detected high PIK3Cδ protein expression in fibroblast cell lines and primary fibroblasts derived from TNBC patients; still, PIK3Cδ was hardly detectable in breast cancer cell lines. CAL-101 treatment affected fibroblasts viability, while it had limited/no effects on breast cancer cells. Moreover, CAL-101 resulted in reduced phosphorylation of AKT, a PIK3Cδ target. Pretreatment of fibroblasts with CAL-101 significantly decreased TNBC cells' invasion in 2D & 3D co-culture experiments. Interestingly, using transwell systems we saw that TNBC co-culturing increased PIK3Cδ expression in fibroblasts, suggesting a feedback loop that ‘fuels' tumor progression. Additional proteomic/genomic studies, provided us with evidence about the PIK3Cδ-mediated paracrine signaling leading to TNBC invasion. Conclusion: Using a novel 3D co-culture invasion assay, we identified stromal PIK3Cδ as a key mediator of TNBC invasion. Our results suggest that targeting PIK3Cδ in the TME may represent a novel transformative strategy for TNBC therapy. Citation Format: Teresa Gagliano, Viviana Vella, Kamila Bienkowska, Angeliki Ditsiou, Georgios Giamas. Fibroblast-kinome siRNA screening identifies PIK3Cδ as a mediator of Triple-negative breast cancer (TNBC) invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2087.

Prolactin receptor expression as a novel prognostic biomarker for triple negative breast cancer patients

Previous studies showed that expression of prolactin (PRL) receptor is increased in numerous hypothalamic nuclei in mid-lactating rats. The increase in PRL receptor expression could be initated by neurohormonal changes during proestrus or pregnancy, or by the suckling stimulus during lactation. The present study investigated whether the PRL receptor expression in numerous hypothalamic nuclei is altered by the suckling stimulus. Three groups (n = 4) of rats on lactation day 10 were used: a continuously suckled group, a nonsuckled group (pups removed for 12 h) and a resuckled group (pups removed for 12 and then resuckled for 9 h). Animals were perfused with 2% paraformaldehyde and brains were sectioned (20 µm) for the immunofluorescence study. Immunoreactivity was semiquantitatively analyzed by counting the immunoreactive cells and measuring the immunostaining intensity in a specific area. Neurons expressing PRL receptors were observed in numerous hypothalamic areas with the highest number being in the arcuate, paraventricular and supraoptic nuclei. The PRL receptor immunofluorescence in several nuclei was significantly decreased in the nonsuckled group, and recovered in the resuckled group. These areas included the ventromedial preoptic, ventrolateral preoptic, lateroanterior hypothalamic, ventrolateral hypothalamic and ventromedial hypothalamic nuclei. PRL receptor immunoreactivity in other areas was not significantly altered by the suckling stimulus. These results demonstrate that expression of PRL receptor in hypothalamic nuclei was differentially affected by the suckling stimulus. PRL receptors in those nuclei which were significantly altered by suckling stimulus may play more critical roles during lactation than those areas which were less sensitive to the suckling stimulus.

Rationale and purpose: Triple negative breast cancers (TNBCs), characterized by lack of hormone receptors' expression in absence of HER2 amplification, partially overlap with the basal-like subtype, with frequent occurrence in BRCA1 mutation carriers (BRCA1+). TNBCs are often associated with earlier onset, interval cancer diagnosis, larger size and aggressive clinical course with peak risk of recurrence at 1-3 years and increased mortality rate in the first 5 years. Thus, when screening women at high risk of breast cancer, special attention should be paid to patient outcome for TNBCs in comparison with non-TNBCs. Our aim was to compare TNBCs and non-TNBCs diagnosed during a prospective, non-randomized, multimodality screening study – including clinical breast examination (CBE), mammography, ultrasound (US) and magnetic resonance imaging (MRI) – on women at familial/genetic high risk of breast cancer conducted in 18 centres from June 2000 to March 2008 (ISS-HIBCRIT-1; Sardanelli F et al, Invest Radiol 2011). Methods: Comparisons were performed using Mann-Whitney U, Fisher exact and χ2 tests. Results: Among the 44 patients diagnosed with invasive cancers, 14 (31%) were TNBCs and 30 (69%) non-TNBCs, the former being 13 invasive ductal (IDC) and 1 atypical medullary carcinoma, the latter also including 15/30 lobular subtypes and/or DCIS component (p=0.005). Of the 14 TNBCs, 10 (71%) were found in BRCA1+, 2 (14%) in BRCA2+ and 2 (14%) in BRCA-untested women with strong family history of breast/ovarian cancer; the same data for 30 non-TNBCs were 9 (30%), 6 (20%) and 15 (50%) respectively (p=0.028). We had only three interval cancers, all TNBCs. The median age at diagnosis was 49 years (range 36-62) for TNBCs and 53 years (range 35-72) for non-TNBCs (p=n.s). TNBCs presented a higher rate (11/14, 79%) of pathological grade 3 IDCs compared with non-TNBCs (8/30, 27%) (p=0.002). The mean tumor size was 1.6 cm for TNBCs and 1.2 cm for non-TNBCs (p=n.s). Nodal status was negative in 12/14 (86%) TNBCs and in only 16/30 (53%) non-TNBCs (p=0.038). MRI similarly outperformed CBE, mammography and US in both TNBCs and non-TNBCs. Clinical course and survival could be monitored for 40/44 patients (91%), 13 TNBCs and 27 non-TNBCs, with a follow-up of 5.8 and 6.3 years (p=n.s) respectively. The rate of disease-free patients for over 5 years was 8/13 (62%; mean disease-free interval 7.0 years, range 5.0-8.0) for TNBCs and 17/27 (63%; mean 7.2 years, range 5.2-9.9) for non-TNBCs. Death due to BC occurred for 2/13 TNBC (15%, at 3.5 and 4.2 years) and 3/27 non-TNBC patients (11%; at 2.0, 4.9 and 5.7 years). The rate of locoregional relapse was 1/13 (8%, at 4.4 years) and 5/27 (19%; mean time of 5.0 years, range 2.4-7.0) respectively. Distant recurrence was reported for only 2 non-TNBC patients. Conclusion: TNBCs showed stronger association with BRCA1+ status, lower rate of lobular subtypes or DCIS component, and less frequent nodal involvement. Despite a more frequent pathological grade 3 and the tendency to be diagnosed as interval cancers, under the current treatment protocols TNBCs showed relapse and BC-related death rates and over-5-year disease-free intervals similar to those of non-TNBCs. These data provide outcome evidence supporting the value of entering women at high risk of TNBC (in particular BRCA1+) in intensive screening programs including MRI. Citation Format: Franca Podo, Filippo Santoro, Siranoush Manoukian, Clelia de Giacomi, Laura Cortesi, Lorenzo Preda, Stefano Corcione, Francesco Sardanelli. High-risk patients found affected with breast cancer during a multimodality screening program: Triple negative versus non-triple negative breast cancers. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B13.

PurposeDecreased production of the aqueous component of the tear film is an important cause of the development of dry eye disease (DED). Tear production is influenced by hormones and hormone-like factors. Prolactin (PLR), a multifunctional pituitary gland hormone, is regularly present in the lacrimal gland of rats and rabbits. In humans, serum PLR concentration correlates with tear quality. To gain deeper insights of possible effects of PRL, prolactin receptor (PRLR) and prolactin inducible protein (PIP), we analyzed the three proteins in the human lacrimal apparatus and in reflex tears of healthy volunteers as well as patients suffering from DED.MethodsGene expression of PRLR and PIP was analyzed by RT-PCR in cadaveric human lacrimal gland and ocular surface tissues, immortalized human corneal epithelial cells (HCE and hTEPI) and human Meibomian gland epithelial cells (HMGECs). At the protein level, the expression and localization of PRL, PRLR and PIP in formalin-fixed paraffin sections of the lacrimal apparatus were studied by immunohistochemistry. In addition, tear fluid from DED patients and healthy volunteers was analyzed by ELISA to determine the concentration of PRL and PIP.ResultsRT-PCR analyses revealed gene expression of PRLR and PIP in human tissue samples of cornea, lacrimal glands, and eyelids, whereas only PIP, but not PRLR, was detectable in immortalized corneal epithelial cells. Immunohistochemistry revealed for the first time the expression and localization of PRL, PRLR, and PIP in human tissues of the lacrimal apparatus and at the ocular surface. PRL and PRLR were detectable in corneal epithelium, lacrimal glands, and Meibomian glands. Reflex tears from DED patients revealed significantly increased PIP concentrations, whereas PRL was undetectable in tears of DED patients and healthy volunteers.ConclusionPRL, PRLR, and PIP are found in the lacrimal apparatus and on the ocular surface. PIP, but not PRL, is present in human tears and appears to be involved in the physiology of tear film quality. Our clinical data revealed that PIP may affect tear quality, but further functional analyses are needed to fully elucidate the effects of PRL and PIP-associated factors in tear secretion as well as in the connection of DED.

BackgroundEndocrine mechanisms governing canine reproductive function remain still obscure. Progesterone (P4) of luteal origin is required for maintenance of pregnancy. Corpora lutea (CL) are gonadotrop-independent during the first third of dioestrus; afterwards prolactin (PRL) is the primary luteotropic factor. Interestingly, the increasing PRL levels are accompanied by decreasing P4 concentrations, thus luteal regression/luteolysis occurs in spite of an increased availability of gonadotropic support. PRL acts through its receptor (PRLr), the expression of which has not yet been thoroughly investigated at the molecular and cellular level in the dog.MethodsThe expression of PRLr was assessed in CL of non-pregnant dogs during the course of dioestrus (days 5, 15, 25, 35, 45, 65 post ovulation; p.o.) as well as in CL, the utero/placental compartments (Ut/Pl) and interplacental free polar zones (interplacental sites) from pregnant dogs during the pre-implantation, post-implantation and mid-gestation period of pregnancy and during the normal and antigestagen-induced luteolysis. Expression of PRLr was tested by Real Time PCR, immunohistochemistry and in situ hybridization.ResultsIn non-pregnant CL the PRLr expression was significantly upregulated at day 15 p.o. and decreased significantly afterwards, towards the end of dioestrus. CL of pregnancy showed elevated PRLr expression until mid gestation while prepartal downregulation was observed. Interestingly, placental but not interplacental expression of PRLr was strongly time-related; a significant upregulation was observed towards mid-gestation. Within the CL PRLr was localized to the luteal cells; in the Ut/Pl it was localized to the fetal trophoblast and epithelial cells of glandular chambers. Moreover, in mid-pregnant animals treated with an antigestagen, both the luteal and placental, but not the uterine PRLr were significantly downregulated.ConclusionsThe data presented suggest that the luteal provision of P4 in both pregnant and non-pregnant dogs may be regulated at the PRLr level. Furthermore, a role of PRL not only in maintaining the canine CL function but also in regulating the placental function is strongly suggested. A possible functional interrelationship between luteal P4 and placental and luteal PRLr expression also with respect to the prepartal luteolysis is implied.

Background: Triple negative breast cancer makes up 10-20% of all mammary tumors. It is so named because it exhibits low or no expression of both the estrogen receptor (ER) and progesterone receptor (PR), and has low or no expression of the human epidermal growth factor (HER2) receptor. With no current molecular target identified, treatments options for TNBCs are limited to conventional chemotherapy, which has been shown to be only moderately effective. Further, diagnosis is correlated with a high rate of relapse, low survival rate five years past diagnosis, and overall poor prognosis. Nm23-H1 is the most well characterized in a class of tumor suppressor genes that have received increased attention as a potential therapeutic target in breast cancers. First identified in 1988, nm23, has been shown have low levels of mRNA expression in highly metastatic tumor cells, compared to normal or non-neoplastic tissue. This expression level coincides with not only increased metastasis but poor clinical prognosis in several type cancers, including breast cancer. Further, nm23-H1 inhibits a class of Rho small GTPases including Rac1, Rho and cdc42. These proteins mediate cell-to-cell adhesion and cytoskeletal reorganization, which ultimately moderate the metastatic profile i.e. metastasis, cellular motility, and invasion. Methods: Western blotting was used to examine the basal level of expression in a panel of mesenchymal and epithelial TNBC cell lines. Transient siRNA was used to silence nm23-H1 in MDAMB231 and HCC1806 cells to determine the effects on downstream Nm23-H1 targets Rac1, Rho and cdc42. The effects of nm23-H1 inhibition on the invasive potential of TNBC cells were assessed using Matrigel chamber assays. Wound healing assays were employed to assess the effects of nm23-H1 inhibition on the migratory potential of TNBC cells. Results: Our studies indicate that nm23-H1 is expressed in all of the TNBC cell lines that we tested but to varying degrees with mesenchymal cells expressing the protein at a higher level compared to epithelial cells. Immunoblotting suggested that silencing of nm23-H1 resulted in an increase in p-Rac. Silencing also increased the invasive ability of TNBC cells compared to control cells. Conclusion: These results demonstrate the vital role that nm23-H1 plays in the inhibition of cellular invasion in TNBCs. An increase in the expression of proteins associated with motility and invasion pathways validates the role of nm23-H1 in inhibiting the metastatic profile in TNBC. As a potential molecular marker for metastatic TNBC, nm23-H1 warrants further investigation. Citation Format: Tanisha Z. McGlothen, Rachel Tobin, Tiffanie Alcaide, LaTonia Taliaferro-Smith, Tongrui Liu, Ruth O'Regan. Examining the role of nm23-H1 in the metastatic profile of triple negative breast cancer (TNBC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 56. doi:10.1158/1538-7445.AM2014-56

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Objective:The aim of this study is to analyze the different expression and function of androgen receptors (AR) and estrogen receptor (ER-α) in laryngeal squamous cell carcinoma (LSCC). In combination with the expression of prolactin receptor (PRLR), we analyzed the prognostic impact of three receptors on the laryngeal carcinoma. Method:In this study, immunohistochemistry and reverse transcription polymerase chain reaction (RT-PC) analysis were performed on the tumor tissues and adjacent normal tissues in 112 LSCC patients (95 males and 17 females). We found that hormone receptor expression is closely related to the clinical tumor lesions and overall survival data. Result:The expression of AR, ER-α and PRLR in tumor tissues were much higher than those in adjacent normal tissues (P>0.05) at both protein and mRNA levels. The higher PRLR level indicate poor survival in LSCC patients (P=0.02), while higher ER-α expression could influence the survival with considerable trend toward significance (P=0.06). Furthermore, the higher expression of ER-α in tumours were corresponding with PRLR cytoplasmic higher level expression (r=0.802, P=0.04). This mutual promoted effect between ER-α and PRLR possibly suggests potential mechanisms among those sex related hormone receptors in laryngeal cancer. Conclusion:It has become increasingly credible that the sex related hormone receptors play an important role in the development of LSCC.

Prolactin receptor expression by lymphoid tissues in normal and immunized rats

Purpose: To study clinical characteristics of different histological subtypes of triple negative breast cancer (TNC). TNC are often considered as one group, but consist of many different histological subtypes with different biology and behavior. We decided to analyze a large cohort of TNC in a single centre and focus on clinical behavior including age distribution, occurrence of lymph node invasion, and outcome in different histological subtypes. Patients and methods: We used data from the breast cancer database from UH Leuven, containing 479 newly diagnosed TNC from January 1999 till November 2009, of which 396 received upfront surgery, 64 neoadjuvant chemotherapy, and 18 with metastases at diagnosis. Univariate and multivariate relations were studied between age, lymph node status, histological subtype, treatment and outcome. Results: Apocrine and invasive lobular TNC occur more often in older patients compared to IDC-NOS (resp. P = 0.0004, P = 0.0496), the latter being by far the most common subtype (80,2%). Although TNC are considered as an aggressive subtype of breast cancer with a bad prognosis, only 35.1% have lymph nodal involvement. There were no significant differences in nodal invasion between subtypes, but most subtypes contained few patients. In contrast to previous reports, 7/14 (50%) of apocrine TNC had lymph node involvement. DFS was different in different histological subtypes, but most of the histological subgroups contained insufficient numbers to be able to draw firm conclusions. Within the histologically ‘homogeneous’ IDC-NOS group with primary surgery and outcome data (n=308), DFS with 3.5 year median FUP decreased with increasing age, but chemotherapy and also radiotherapy were much less frequently given with increasing age (in age <50, 50-69, 70+ no chemo in 2/135, 25/126, 39/44; no radiotherapy in 14/135, 22/126, 12/44 respectively). In multivariate analysis, increasing age (HR for 10 yr increase: 0.68 (0.52-0.89)), presence of lymph node involvement (HR 2.17 (1.24-3.77)), administration of chemotherapy (HR 0.28 (0.12 — 0.67)), and radiotherapy (HR 0.39 (0.20-0.73)) were significant predictors of relapse. Conclusion: TNC is not a uniform disease. Different histological subtypes have different age distribution and outcome. The prognosis of the most common group of TNC, IDA-NOS is unexpectedly better in older patients, but this is counterbalanced by the significantly decreased use of chemotherapy and radiotherapy in this population. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-05.

BackgroundMammary tumors represent the most common neoplastic disease in female dogs. Recently, the promoting role of prolactin (PRL) in the development of human breast carcinoma has been shown. Possible proliferative, anti-apoptotic, migratory and angiogenic effects of PRL on human mammary cancer cells in vitro and in vivo were suggested. The effects of PRL are mediated by its receptor, and alterations in receptor expression are likely to play a role in tumor development. Currently, not much data is available about prolactin receptor (PRLR) expression in canine mammary tumors. To set the basis for investigations on the role of PRL in mammary tumorigenesis in this species, prolactin receptor expression was evaluated by semi-quantitative real time PCR and immunohistochemistry on 10 formalin-fixed, paraffin-embedded samples each of canine non-neoplastic mammary tissue, mammary adenomas and adenocarcinomas.ResultsThe highest PRLR expression levels were found in normal mammary tissue, while adenomas, and to an even higher degree adenocarcinomas, showed a significant decrease in prolactin receptor expression. Compared to normal tissue, PRLR mRNA was reduced 2.4 fold (p = 0.0261) in adenomas and 4.8 fold (p = 0.008) in adenocarcinomas. PRLR mRNA expression was significantly lower in malignant than in benign lesions (p = 0.0165). Immunohistochemistry demonstrated PRLR expression in all three tissue types with signals mostly limited to epithelial cells.ConclusionsMalignant transformation of mammary tissue was associated with a decline in prolactin receptor expression. Further studies are warranted to address the functional significance of this finding.