Parathyroid Hormone-related Protein Expression Research Articles

High endogenous expression of parathyroid hormone-related protein (PTHrP) supports osteogenic differentiation in human dental follicle cells

Dental follicle cells (DFCs) are progenitor cells for mineralizing cells such as alveolar osteoblasts, but little is known about the mechanisms of the differentiation. Interestingly, different cell lines sometimes have different potentials to differentiate into mineralizing cells. In this study, we compared two different DFC lines, with one cell line (DFC_B) showing a high alkaline phosphatase (ALP) activity in long-term cultures with standard medium and a reliable mineralizing potential. However, the other cell line DFC_A shows low ALP activity in standard medium and almost no mineralization. Known osteogenic markers such as RUNX2 were similarly expressed in both cell lines. However, the proosteogenic signaling pathway of the bone morphogenetic protein (BMP) is induced in DFC_B, and the parathyroid hormone-related protein (PTHrP), which is involved in tooth root development, was also expressed more strongly. Previous studies have shown that the secreted PTHrP negatively regulate the transition from pre-osteoblastic progenitors to osteoblasts, but we showed that an inhibition of PTHrP gene expression reduced the ALP activity and the BMP-signaling pathway. In addition, endogenously expressed PTHrP is located in the cell nucleus. In contrast, supplementation of PTHrP or an inhibitor for the PTHrP receptor did not affect the ALP activity of DFC_B. In conclusion, our data suggest that a high endogenous expression of PTHrP in DFCs supports the induction of osteogenic differentiation via an intracrine mode.

SAT-LB23 Paraneoplastic Hypercalcemia in a PTH Producing Adrenocortical Carcinoma - a Rare and Deadly Condition

Background: Hypercalcemia is a commonly encountered paraneoplastic manifestation of certain cancers with or without endocrine differentiation. However, the association between adrenocortical carcinoma (ACC) with paraneoplastic hypercalcemia is very rare, and therefore little is known about the cause and its relevance in the disease. Clinical Case: A 40-year-old woman presented in the hospital with a 5-month history of progressive flank pain with unintentional weight loss of 6 kg. MRI revealed a mass of 9x8.1x4.8 cm of the right adrenal gland with inhomogeneous contrast enhancement. Biochemical investigations provided evidence of endogenous hypercortisolism (24-hour urinary cortisol excretion [490 µg, n<236 µg/l], 1mg dexamethasone suppression test [199 nmol/l, n<50 nmol/l], ACTH [28 ng/l, n<61 ng/l]) although the patient did not show any specific clinical sign of overt hypercortisolism. In addition, laboratory testing revealed an exceptionally high plasma level of calcium [max 3.67 mmol/l (albumin-corrected)] and low phosphate [min 0.26 mmol/l] in the setting of low PTH [6.4 ng/l, n>15 ng/l] and PTHrP levels [<0.50 pmol/l]. However, subsequent dilution unmasked a highly elevated PTH concentration of 2171.5 ng/l with persistent low PTHrP levels, indicating false low values due to a hook effect in the initial measurement. Levels of 1,25-dihydroxy vitamin D and 25-hydroxy vitamin D were in the normal range. A PET-CT provided no indications of metabolically active (osseous) metastases. After correction of the serum calcium towards tolerable values, the tumor was removed by open en bloc adrenalectomy. Histologic evaluation confirmed an ACC (TNM pT4 pN1 (2/3), L1, V1, high grade) despite missing immunohistochemically expression of classical adrenal markers (diagnosis of exclusion). Supplemental quantitative RT-PCR studies support the diagnosis of ACC by detecting significant SF-1 and CYP11B2 expression in the tumor cells. Further analyses provided evidence that the mRNA expression of PTH, but not PTHrP, was moderately increased in the ACC sample compared to NCI H295R cells. Upon tumor resection, serum calcium levels swiftly normalized indicating the tumor as the sole source of PTH secretion. Despite initiation of adjuvant mitotane- and salvage chemo-therapy, the patient died 3 months later upon of a massive tumor relapse with a recurrence of severe hypercalcemia. Conclusion: This case demonstrates paraneoplastic hypercalcemia in a PTH producing ACC. PTH may induce hypercalcemia, impair adrenal steroid synthesis and act as an autocrine growth factor in ACC, as described in few individual cases for PTHrp producing ACC [1]. This suggests a poor prognosis for this rare entity.1. Rizk-Rabin, M., et al., Differential Expression of Parathyroid Hormone-Related Protein in Adrenocortical Tumors: Autocrine/Paracrine Effects on the Growth and Signaling Pathways in H295R Cells. 2008.

SAT-009 SSRI Use in the Peripartum Period Regulates Mammary Gland Parathyroid Hormone Related Protein (PTHrP) by a Serotonylation-Dependent Mechanism

During lactation, a woman experiences a considerable amount of bone loss and recent studies suggest bone deficits persist years postpartum. Furthermore, selective serotonin uptake inhibitors (SSRIs), which are often prescribed to women experiencing peripartum depression, have been linked to osteopenia. Serotonin signaling can increase parathyroid hormone related protein (PTHrP), a bone remodeling protein which liberates calcium for the milk. Additionally, fluoxetine (a common SSRI) results in increased mammary gland serotonin content and PTHrP, and treatment during the peripartal period reduced maternal bone mineral density. One proposed mechanism of serotonin action is by its covalent addition to proteins by transglutaminase (TG2), termed serotonylation. We therefore investigated whether the combination of fluoxetine and lactation can exacerbate maternal bone loss and the underlying mechanism. We hypothesized that SSRI-induced serotonin signaling in the lactating mammary gland increases PTHrP through a serotonylation-dependent mechanism. Treatment of mouse mammary epithelial cells (HC11) with fluoxetine significantly upregulates PTHrP gene expression and the concentration of its downstream effector, cAMP, over control (P < 0.0004). Furthermore, treatment of the HC11 cells with fluoxetine in addition to a TG2 inhibitor, monodansylcadaverine, restores PTHrP mRNA expression to levels observed in the control. Small g-proteins have emerged as a common target protein for serotonylation. Currently, our data suggest that the g-proteins, RhoA and Rab4, are potential serotonylation targets in the mammary gland. Together these data suggest that the molecular process of serotonyation in HC11 cells links serotonin signaling to increased PTHrP expression. Future work is directed at using the cre-lox system to genetically ablate serotonylation using a WAPCre/TG2Flox transgenic mouse to determine whether decreasing serotonylation in vivo in the mammary gland during lactation improves maternal bone mass.

Effects of stocking density on growth performance, growth regulatory factors, and endocrine hormones in broilers under appropriate environments

Stocking density is an important environment factor that affects the development of poultry farming, which has caused widespread concern. This study was carried out to determine the effects of stocking density on growth performance, growth regulatory factors, and endocrine hormones in broilers under appropriate environments. A total of 144 Arbor Acres male broilers (BW 1000 ± 70 g) were randomly divided into low stocking density (LSD; 6.25 birds/m2), medium stocking density (MSD; 12.50 birds/m2), and high stocking density (HSD; 18.75 birds/m2) groups, with 6 replicates in each group, and raised in 3 environmental chambers (same size) from 29-day-old to 42-day-old, respectively. The trial period lasted for 14 D with 21 ± 1°C and 60 ± 7% relative humidity, wind speed < 0.5 m/s, ammonia level<5 ppm. The results indicated that average daily food intake and average daily gain in HSD group showed significantly lower than other 2 groups (P < 0.05). Besides, the HSD group significantly reduced breast muscle yield, tibial length, tibial width, and tibial weight of broilers (P < 0.05). The HSD group increased the mRNA expression level of myostatin, and reduced the mRNA expression levels of insulin-like growth factor 1 (IGF-1) and myogenic determination factor 1 (P < 0.05). The HSD group significantly reduced the expression of parathyroid hormone-related protein in tibial growth plate (P < 0.05). The HSD group increased the serum corticosterone levels of broilers (P < 0.05), and decreased the serum IGF-1 and thyroxine (T4) levels of broiler chickens (P < 0.05) than other stocking density groups. Moreover, the serum alkaline phosphatase levels were decreased (P < 0.05) with increasing stocking density, whereas there were no significant effects on the serum 3,5,3′-triiodothyronine (T3) concentrations in 3 groups (P > 0.05). In conclusion, under appropriate environments HSD reduced the growth performance of broilers and this negative effect was likely associated with decreased growth of muscle and bone.

Hedgehog proteins and parathyroid hormone-related protein are involved in intervertebral disc maturation, degeneration, and calcification.

Parathyroid hormone‐related protein (PTHrP) and hedgehog signaling play an important role in chondrocyte development, (hypertrophic) differentiation, and/or calcification, but their role in intervertebral disc (IVD) degeneration is unknown. Better understanding their involvement may provide therapeutic clues for low back pain due to IVD degeneration. Therefore, this study aimed to explore the role of PTHrP and hedgehog proteins in postnatal canine and human IVDs during the aging/degenerative process. The expression of PTHrP, hedgehog proteins and related receptors was studied during the natural loss of the notochordal cell (NC) phenotype during IVD maturation using tissue samples and de‐differentiation in vitro and degeneration by real‐time quantitative polymerase chain reaction (RT‐qPCR) and immunohistochemistry. Correlations between their expression and calcification levels (Alizarin Red S staining) were determined. In addition, the effect of PTHrP and hedgehog proteins on canine and human chondrocyte‐like cells (CLCs) was determined in vitro focusing on the propensity to induce calcification. The expression of PTHrP, its receptor (PTHR1) and hedgehog receptors decreased during loss of the NC phenotype. N‐terminal (active) hedgehog (Indian hedgehog/Sonic hedgehog) protein expression did not change during maturation or degeneration, whereas expression of PTHrP, PTHR1 and hedgehog receptors increased during IVD degeneration. Hedgehog and PTHR1 immunopositivity were increased in nucleus pulposus tissue with abundant vs no/low calcification. In vitro, hedgehog proteins facilitated calcification in CLCs, whereas PTHrP did not affect calcification levels. In conclusion, hedgehog and PTHrP expression is present in healthy and degenerated IVDs. Hedgehog proteins had the propensity to induce calcification in CLCs from degenerated IVDs, indicating that in the future, inhibiting hedgehog signaling could be an approach to inhibit calcification during IVD degeneration.

Parathyroid hormone related protein (PTHrP)-mediated hypercalcemia in malignancy associated with anti-PD-1 immune checkpoint inhibitor treatment and related inflammatory reactions

Over the last decade, the breakthrough of immune checkpoint inhibitors has revolutionized cancer therapeutics, an enterprise not devoid of a novel constellation of unique immune-related adverse events. In this article, we present the first two patients, one with metastatic urothelial bladder cancer and another one with inoperable non-small squamous cell lung carcinoma, with immune-related parathyroid hormone related protein (PTHrP)-mediated hypercalcemia concurrent with immune-related pneumonitis following administration of anti-PD-1 monoclonal antibody nivolumab. The second patient present immune-related colitis as well. In both patients the hypercalcemia developed when cancer was in remission, rendering unlikely the diagnosis of humoral hypercalcemia of malignancy. The time of onset of PTHrP-mediated hypercalcemia–11 weeks and 15 weeks after initiation of nivolumab for the first and second patient respectively– insinuated the immune-related origin of PTHrP. The concurrent immune-related pneumonitis raised the question of whether the immune-related inflammatory milieu in the context of pneumonitis could be the source of the immune-related PTHrP. In conclusion, increased awareness of nivolumab-related hypercalcemia –an extremely rare immune-related adverse event– could enable the identification of immune-related elevation of PTHrP. Moreover, our cases provide the rationale for further research in pursuit of not only the source of immune-related PTHrP expression, but also of a causative link connecting the inflammatory milieu of immune-related pneumonitis and/or immune-related colitis with PTHrP-mediated hypercalcemia. Finally, the correlation of immune-related adverse events observed herein with response to nivolumab is in line with previous reports, necessitating further consolidation.

Systemic delivery of a Gli inhibitor via polymeric nanocarriers inhibits tumor-induced bone disease

Solid tumors frequently metastasize to bone and induce bone destruction leading to severe pain, fractures, and other skeletal-related events (SREs). Osteoclast inhibitors such as bisphosphonates delay SREs but do not prevent skeletal complications or improve overall survival. Because bisphosphonates can cause adverse side effects and are contraindicated for some patients, we sought an alternative therapy to reduce tumor-associated bone destruction. Our previous studies identified the transcription factor Gli2 as a key regulator of parathyroid hormone-related protein (PTHrP), which is produced by bone metastatic tumor cells to promote osteoclast-mediated bone destruction. In this study, we tested the treatment effect of a Gli antagonist GANT58, which inhibits Gli2 nuclear translocation and PTHrP expression in tumor cells. In initial testing, GANT58 did not have efficacy in vivo due to its low water solubility and poor bioavailability. We therefore developed a micellar nanoparticle (NP) to encapsulate and colloidally stabilize GANT58, providing a fully aqueous, intravenously injectable formulation based on the polymer poly(propylene sulfide)135-b-poly[(oligoethylene glycol)9 methyl ether acrylate]17 (PPS135-b-POEGA17). POEGA forms the hydrophilic NP surface while PPS forms the hydrophobic NP core that sequesters GANT58. In response to reactive oxygen species (ROS), PPS becomes hydrophilic and degrades to enable drug release. In an intratibial model of breast cancer bone metastasis, treatment with GANT58-NPs decreased bone lesion area by 49% (p<.01) and lesion number by 38% (p<.05) and resulted in a 2.5-fold increase in trabecular bone volume (p<.001). Similar results were observed in intracardiac and intratibial models of breast and lung cancer bone metastasis, respectively. Importantly, GANT58-NPs reduced tumor cell proliferation but did not alter mesenchymal stem cell proliferation or osteoblast mineralization in vitro, nor was there evidence of cytotoxicity after repeated in vivo treatment. Thus, inhibition of Gli2 using GANT58-NPs is a potential therapy to reduce bone destruction that should be considered for further testing and development toward clinical translation.

PTHrP, A Biomarker for CNS Metastasis in Triple-Negative Breast Cancer and Selection for Adjuvant Chemotherapy in Node-Negative Disease.

BackgroundTriple-negative breast cancer (TNBC) is characterized by poor prognosis and lack of targeted therapies and biomarkers to guide decisions on adjuvant chemotherapy. Parathyroid hormone-related protein (PTHrP) is frequently overexpressed in breast cancer and involved in proliferation and metastasis, two hallmarks of poor prognosis for node-negative breast cancer. We investigated the prognostic value of PTHrP with respect to organ-specific metastasis and nodal status in TNBC.MethodsWe assessed PTHrP expression using immunohistochemistry in a clinically annotated tissue microarray for a population-based study of 314 patients newly diagnosed with TNBC, then analyzed its correlation to progression and survival using Kaplan-Meier and Cox regression analyses. The Cancer Genome Atlas (TCGA) validation analysis was performed through Bioconductor. All statistical tests were two-sided.ResultsPTHrP overexpression (160 of 290 scorable cases, 55.2%) was statistically significantly associated in univariate analysis with decreased overall survival (OS) in our cohort (P = .0055) and The Cancer Genome Atlas (P = .0018) and decreased central nervous system (CNS)-progression-free survival (P = .0029). In multivariate analysis, PTHrP was a statistically significant independent prognostic factor for CNS-progression-free survival in TNBC (hazard ratio [HR] = 5.014, 95% confidence interval [CI] = 1.421 to 17.692, P = .0122) and for OS selectively in node-negative TNBC (HR = 2.423, 95% CI = 1.129 to 5.197, P = .0231). Strikingly, PTHrP emerged as the only statistically significant prognostic factor (HR = 2.576, 95% CI = 1.019 to 6.513, P = .0456) for OS of low-clinical risk node-negative patients who did not receive adjuvant chemotherapy.ConclusionsPTHrP is a novel independent prognostic factor for CNS metastasis and adjuvant chemotherapy selection of low-clinical risk node-negative TNBC. Its predictive value needs to be prospectively assessed in clinical trials.

The Effects of Fluvastatin on Indian Hedgehog Pathway in Endochondral Ossification.

Statins have demonstrated to be effective for treating chondrodysplasia and its effects were believed to be associated with the fibroblast growth factor receptor 3 (FGFR3). Statins promoted the degradation of FGFR3 in studies using disease-specific induced pluripotent stem cells and model mice, however, recent studies using normal chondrocytes reported that statins did not degrade FGFR3. In order to further investigate the effects of statins in endochondral ossification, this study examined the influence of statins on Indian hedgehog (Ihh), another important component of endochondral ossification, and its related pathways. The chondrocyte cell line ATDC5 was used to investigate changes in cell proliferation, mRNA, and protein expression levels. In addition, an organ culture of a mouse metatarsal bone was performed followed by hematoxylin-eosin staining and fluorescent immunostaining. Results indicated that expression level of Ihh increased with the addition of statins, which activated the Ihh pathway and altered the localization of Ihh. Changes in cholesterol modification may have affected Ihh diffusibility; however, further experiments are necessary. A reactive increase in parathyroid hormone-related protein (PTHrP) was observed in addition to changes in the Wnt pathway through secreted-related protein 2/3 and low-density lipoprotein 5/6. This led to the promotion of cell proliferation, increase of the hypertrophic chondrocyte layer, inhibition of apoptosis, and decrease in mineralization. This study demonstrated that statins had an influence on Ihh, and that the hyperfunction of Ihh may prevent premature cell death caused by FGFR3-related chondrodysplasia through an indirect increase in the expression of PTHrP.

MicroRNA-15a-5p Regulates the Development of Osteoarthritis by Targeting PTHrP in Chondrocytes.

Background and Aims A growing body of research has demonstrated that the degeneration of chondrocytes is the primary cause of osteoarthritis (OA). Parathyroid hormone-related protein (PTHrP) can alleviate the degeneration of chondrocytes via promotion of chondrocyte proliferation and inhibition of terminal differentiation, but the underlying mechanism remains unknown. This study aimed to identify the microRNAs (miRNAs) that may target PTHrP and regulate the proliferation and terminal differentiation of chondrocytes. Methods Bioinformatic analysis was used to predict which miRNAs target PTHrP. We collected human knee cartilage specimens to acquire the primary chondrocytes, which we then used to test the expression and function of the targeted miRNAs. To explore the effects of miR-15a-5p on the putative binding sites, specific mimics or inhibitors were transfected into the chondrocytes. Furthermore, a dual-luciferase reporter gene assay and chondrocyte degeneration-related factors were used to verify the possible mechanism. Results The expression of PTHrP was upregulated in the OA chondrocytes, whilst miR-15a-5p was downregulated in the OA chondrocytes. A negative correlation was observed between PTHrP and miR-15a-5p. The knockdown of miR-15a-5p promoted the growth of chondrocytes and inhibited calcium deposition, whilst overexpression of miR-15a-5p reversed this trend. The effect of miR-15a-5p overexpression was neutralised by PTHrP. Dual-luciferase reporter assays revealed that PTHrP can be used as a novel targeting molecule for miR-15a-5p. Conclusions miR-15a-5p promotes the degeneration of chondrocytes by targeting PTHrP and, in addition to helping us understand the development of OA, may be a potential biomarker of OA.

Mechanisms Underpinning Adaptations in Placental Calcium Transport in Normal Mice and Those With Fetal Growth Restriction.

Fetal delivery of calcium, via the placenta, is crucial for appropriate skeletal mineralization. We have previously demonstrated that maternofetal calcium transport, per gram placenta, is increased in the placental specific insulin-like growth factor 2 knockout mouse (P0) model of fetal growth restriction (FGR) compared to wild type littermates (WTL). This effect was mirrored in wild-type (WT) mice comparing lightest vs. heaviest (LvH) placentas in a litter. In both models increased placental calcium transport was associated with normalization of fetal calcium content. Despite this adaptation being observed in small normal (WT), and small dysfunctional (P0) placentas, mechanisms underpinning these changes remain unknown. Parathyroid hormone-related protein (PTHrP), elevated in cord blood in FGR and known to stimulate plasma membrane calcium ATPase, might be important. We hypothesized that PTHrP expression would be increased in LvH WT placentas, and in P0 vs. WTL. We used calcium pathway-focused PCR arrays to assess whether mechanisms underpinning these adaptations in LvH WT placentas, and in P0 vs. WTL, were similar. PTHrP protein expression was not different between LvH WT placentas at E18.5 but trended toward increased expression (139%; P = 0.06) in P0 vs. WTL. PCR arrays demonstrated that four genes were differentially expressed in LvH WT placentas including increased expression of the calcium-binding protein calmodulin 1 (1.6-fold; P < 0.05). Twenty-four genes were differentially expressed in placentas of P0 vs. WTL; significant reductions were observed in expression of S100 calcium binding protein G (2-fold; P < 0.01), parathyroid hormone 1 receptor (1.7-fold; P < 0.01) and PTHrP (2-fold; P < 0.05), whilst serum/glucocorticoid-regulated kinase 1 (SGK1), a regulator of nutrient transporters, was increased (1.4 fold; P < 0.05). Tartrate resistant acid phosphatase 5 (TRAP5 encoded by Acp5) was reduced in placentas of both LvH WT and P0 vs. WTL (1.6- and 1.7-fold, respectively; P < 0.05). Signaling events underpinning adaptations in calcium transport are distinct between LvH placentas of WT mice and those in P0 vs. WTL. Calcium binding proteins appear important in functional adaptations in the former whilst PTHrP and SGK1 are also implicated in the latter. These data facilitate understanding of mechanisms underpinning placental calcium transport adaptation in normal and growth restricted fetuses.

Icariin Reduces Cartilage Degeneration in a Mouse Model of Osteoarthritis and is Associated with the Changes in Expression of Indian Hedgehog and Parathyroid Hormone-Related Protein.

BackgroundThe aim of this study was to determine the role of icariin, a Chinese traditional herbal medicine extracted from Epimedium, in osteoarthritis (OA), using the murine anterior cruciate ligament transection (ACLT)-induced model of OA and micromass culture of murine chondrocytes.Material/MethodsTwenty-four three-month-old C57/6J mice were randomly divided into three groups: the sham group (no surgery and joint injection with normal saline) (N=8); the ACLT + ICA group (ACLT surgery and icariin treatment) (N=8); and the ACLT group (ACLT surgery and joint injection with normal saline) (N=8). At 12 weeks after ACLT surgery, murine articular cartilage was harvested from all mice for histological evaluation of any differences in cartilage degeneration. In vitro micromass culture of mouse chondrocytes was used to study the effects of icariin on chondrocyte differentiation and growth from the three mouse groups.ResultsIcariin treatment (mice in the ACLT + ICA group) significantly reduced degeneration of cartilage in OA with increased cartilage thickness, associated with increased expression of collagen type II alpha 1 (COL2A1), decreased chondrocyte hypertrophy, and decreased expression of collagen type X (ColX) and matrix metalloproteinase 13 (MMP13). In vitro, icariin promoted chondrocyte differentiation by upregulating the expression of agrrecan, Sox9 and parathyroid hormone-related protein (PHrP) and down-regulation of Indian hedgehog (Ihh) and genes regulated by Ihh.ConclusionsIn a mouse model of OA icariin treatment reduced destruction of cartilage, promoted chondrocyte differentiation, upregulated expression of PHrP and down-regulated the expression of Ihh.

Effect of free running wheel exercise on renal expression of parathyroid hormone receptor type 1 in spontaneously hypertensive rats.

An active lifestyle is generally recommended for hypertensive patients to prevent subsequent end‐organ damage. However, experimental data on long‐term effects of exercise on hypertension are insufficient and underlying mechanisms are not well understood. This study was aimed to investigate the effect of exercise on renal expression of parathyroid hormone‐related protein (PTHrP) and parathyroid hormone receptor type 1 (PTHR1) in spontaneously hypertensive rats (SHR). Twenty‐four rats started free running wheel exercise at the age of 1.5 months (pre‐hypertensive state) and proceeded for 1.5, 3.0, 6.0, and 10.0 months. Thirty rats kept under standard housing conditions were used as sedentary controls. Kidney function was assessed by measuring plasma creatinine levels and urine albumin‐to‐creatinine ratios. Renal expression of PTHrP and PTHR1 was analyzed by qRT‐PCR and western blot. Renal expression of PTHR1 was markedly increased between the 6th and 10th months in sedentary rats and this increase was significantly lower in SHRs with high physical activity on mRNA (−30%) and protein level (−27%). At the same time, urine albumin‐to‐creatinine ratio increased (from 65 to 231 mg/g) but somehow lower in exercise performing SHRs (48–196 mg/g). Our data suggest that enhanced exercise, stimulated by allocation of a free running wheel, is associated with lower PTHR1 expression in SHRs and this may contribute to preserved kidney function.

Abstract 2632: Identification of PTHrP as a biomarker of short survival &amp; brain metastasis in a tissue microarray retrospective analysis of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) represents 10-20% of all BC cases, and is characterized by aggressive clinical course, frequent relapse, poor patient outcome, and lack of targeted therapy due to the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2). Identifying TNBC molecular drivers and biomarkers would thus be highly beneficial to develop efficient targeted therapies. The parathyroid hormone-related protein (PTHrP) is known for its role in mammary gland and bone development, in addition to breast cancer progression. The aim of this study is to investigate the role of PTHrP as a potential prognostic biomarker in human TNBC. We assessed PTHrP expression using immunohistochemical analysis of a tissue microarray (TMA) constructed for 523 patients newly diagnosed with TNBC between January 1998 and December 2008 in a single-center series with centralized ER, PR and HER-2 testing and standardized treatment and follow-up. We evaluated the correlation between PTHrP expression and TNBC patients' clinico-pathological features as well as progression and survival outcomes for a subset of 314 patients with available clinical data. We show that PTHrP is overexpressed in 55.2% of TNBC tumors and high PTHrP expression is significantly associated with higher propensity for brain progression compared to other sites of distal progression (p=0.0458). Univariate analysis revealed that high PTHrP expression is significantly associated with decreased overall survival (OS) (p=0.0055), but not with progression-free survival (PFS) (p=0.1270). To further investigate the prognostic value of PTHrP with respect to different TNBC molecular subtypes, we analysed expression of markers known to stratify different TNBC subtypes. Multivariate analysis of PTHrP as an independent prognostic factor of survival with respect to different TNBC molecular subtypes is currently underway. In conclusion, we provide for the first time evidence that increased PTHrP expression is significantly associated with shorter OS and higher propensity of brain progression in patients diagnosed with TNBC. Consequently, stratification of this disease based on PTHrP expression might identify patients with relatively higher risk of aggressive disease with brain progression. Additional studies investigating the role of PTHrP in organ-specific metastasis and using PTHrP-targeting strategies are warranted to improve the therapeutic outcome for patients diagnosed with TNBC. Funding: Alberta Cancer Research Institute (ACRI) grant and Department of Defense (DoD, USA) Award No. W81XWH-15-1-0723 Citation Format: Gloria Assaker, Anne Camirand, Bassam Abdulkarim, Atilla Omeroglu, Jean Deschenes, Leon Van Kempen, Richard Kremer, Siham Sabri. Identification of PTHrP as a biomarker of short survival &amp; brain metastasis in a tissue microarray retrospective analysis of triple-negative breast cancer [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 2632.

Abstract 2598: In silico gene expression analysis of PTHrP and its association with molecular subtypes and organ-specific metastasis in human triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) represents 10-20% of all BC cases, and is characterized by an aggressive clinical course with high risk of metastasis and lack of targeted therapy. Gene expression profiling revealed the heterogeneity of TNBC enabling its classification into distinct molecular subtypes with distinct susceptibilities to chemotherapies; including basal-like (BL), mesenchymal (M), and luminal androgen receptor (LAR) subtypes. Overexpression of the parathyroid hormone-related protein (PTHrP) in breast cancer has been extensively linked to its progression with increased propensity for bone metastasis. However, its expression and implication in organ-specific metastasis in the TNBC subtype remains largely unknown. In this study, we conducted in silico analyses to examine the association of PTHrP with various molecular BC subgroups and with organ-specific metastasis in TNBC. Breast Cancer Gene-Expression Miner Version 4.0 (bc-GenExMiner v4.0) online database was used to evaluate the relative mRNA expression levels of the PTHLH gene (which encodes PTHrP) with respect to other markers. This microarray-based tool uses 36 public datasets and 5861 patients, and allows the expression, correlation, and prognostic analyses of genes in BC. Using the gene expression correlation analysis module of bc-GenExMiner v4.0, we assessed Pearson's correlation coefficient between PTHLH expression and gene signatures characteristic of TNBC molecular subtypes or representative of organ-specific metastasis in BC. We found that PTHLH expression displays significant positive correlations with components of signalling pathways enriched in the mesenchymal subtype, and with key luminal markers and AR signalling genes characteristic of the LAR subtype. While PTHLH expression presents significant positive correlations with signature genes involved in bone and lung metastases in all BC subtypes, we identified for the first time a correlation between PTHLH expression and brain metastasis specifically in TNBC patients. Interestingly, PTHLH correlates with the brain metastatic genes HBEGF (Heparin-binding EGF-like growth factor) and ANGPTL4 (Angiopoietin-like 4) selectively in both TNBC and BL subtypes as opposed to other BC subtypes, which is in line with studies reporting their common morphological and genetic features and increased rate of brain metastasis. In conclusion, our in silico analysis reveals for the first time a strong association between PTHrP expression and specific TNBC molecular subtypes' markers, as well as its potential role in the mechanisms of TNBC brain metastasis and the identification of a novel gene signature with a prognostic value for brain progression in TNBC. Funding: Department of Defense (DoD, USA) Award No. W81XWH-15-1-0723 Citation Format: Gloria Assaker, Anne Camirand, Siham Sabri, Richard Kremer. In silico gene expression analysis of PTHrP and its association with molecular subtypes and organ-specific metastasis in human triple-negative breast cancer [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 2598.

Parathyroid Hormone-Related Protein Negatively Regulates Tumor Cell Dormancy Genes in a PTHR1/Cyclic AMP-Independent Manner.

Parathyroid hormone-related protein (PTHrP) expression in breast cancer is enriched in bone metastases compared to primary tumors. Human MCF7 breast cancer cells “home” to the bones of immune deficient mice following intracardiac inoculation, but do not grow well and stain negatively for Ki67, thus serving as a model of breast cancer dormancy in vivo. We have previously shown that PTHrP overexpression in MCF7 cells overcomes this dormant phenotype, causing them to grow as osteolytic deposits, and that PTHrP-overexpressing MCF7 cells showed significantly lower expression of genes associated with dormancy compared to vector controls. Since early work showed a lack of cyclic AMP (cAMP) response to parathyroid hormone (PTH) in MCF7 cells, and cAMP is activated by PTH/PTHrP receptor (PTHR1) signaling, we hypothesized that the effects of PTHrP on dormancy in MCF7 cells occur through non-canonical (i.e., PTHR1/cAMP-independent) signaling. The data presented here demonstrate the lack of cAMP response in MCF7 cells to full length PTHrP(1–141) and PTH(1–34) in a wide range of doses, while maintaining a response to three known activators of adenylyl cyclase: calcitonin, prostaglandin E2 (PGE2), and forskolin. PTHR1 mRNA was detectable in MCF7 cells and was found in eight other human breast and murine mammary carcinoma cell lines. Although PTHrP overexpression in MCF7 cells changed expression levels of many genes, RNAseq analysis revealed that PTHR1 was unaltered, and only 2/32 previous PTHR1/cAMP responsive genes were significantly upregulated. Instead, PTHrP overexpression in MCF7 cells resulted in significant enrichment of the calcium signaling pathway. We conclude that PTHR1 in MCF7 breast cancer cells is not functionally linked to activation of the cAMP pathway. Gene expression responses to PTHrP overexpression must, therefore, result from autocrine or intracrine actions of PTHrP independent of PTHR1, through signals emanating from other domains within the PTHrP molecule.

Establishment and characterization of a C57BL/6 mouse model of bone metastasis of breast cancer.

Bone is one of the most common sites of metastasis in patients with advanced breast cancer; however, the mechanisms of bone metastasis remain to be fully elucidated. Animal models are essential research tools for investigating the mechanisms of diseases and drug actions. To date, there have only been a few reports in which C57BL/6 mice were used for the study of bone metastases of breast cancer. In the current study, we found that intracardiac inoculation of C57BL/6 mouse-derived parental E0771 breast cancer cells (E0771/Pa) frequently lead to bone metastases in C57BL/6 mice within 2weeks. The bone-metastatic clone of E0771 (E0771/Bone) established by sequential in vivo selection demonstrated a higher bone-metastatic potential. Although there were no apparent differences in cell morphology or proliferation in monolayer cultures, E0771/Bone showed increased tumorsphere formation in suspension cultures and tumor formation in the orthotopic mammary fat pad in C57BL/6 mice compared with E0771/Pa. Furthermore, E0771/Bone expressed breast cancer stem-like cell surface markers CD24-/CD44+. These findings suggest that E0771/Bone possesses cancer stem-like properties. Quantitative PCR analysis revealed that mRNA expression of parathyroid hormone-related protein (PTHrP), the most common mediator of osteolytic bone metastases of breast cancer, was significantly upregulated in E0771/Bone. Thus, cancer stem-like properties and elevated PTHrP expression likely contribute to the enhanced metastatic potential of E0771/Bone. We believe that this new mouse model is a useful tool for in vivo studies of bone metastases of breast cancer, especially for those using genetically engineered mice with a C57BL/6 background.

Serotonin induces parathyroid hormone-related protein in goat mammary gland.

During lactation, large amounts of calcium are exported from the mammary gland into milk to ensure skeletal growth of the offspring. Recent studies revealed that serotonin (5-HT) is essential to stimulate skeletal calcium resorption for milk synthesis. Our objective was to explore the correlation between circulating 5-HT and serum calcium and parathyroid hormone-related protein (PTHrP) concentrations around parturition in dairy goats. We also investigated the effect of 5-HT on PTHrP expression in cultured primary goat mammary epithelial cells (GMEC). Blood samples of multiparous Guanzhong dairy goats were collected on day -5 to 3 postpartum for analysis of serum concentrations of calcium, 5-HT, and PTHrP. Results revealed that from day -3 to 0 postpartum serum calcium and 5-HT concentrations decreased progressively, but serum PTHrP concentration only had a sharp drop in the postpartum period sampled. Correlation analysis of circulating 5-HT and serum calcium and PTHrP concentrations on day 1 and 2 postpartum revealed that low serum 5-HT concentration was positively correlated with serum total calcium or PTHrP concentration. By knocking down tryptophan hydroxylase-1 (TPH1) or adding 5-hydroxytryptophan (5-HTP) to decrease or increase the levels of 5-HT in GMEC, we observed that 5-HTP increased PTHrP expression in a dose-dependent manner and siTPH1 decreased PTHrP protein expression. Furthermore, 5-HT increased mRNA abundance of calcium-sensing receptor (CaSR) in a dose-dependent manner and decreased the expression of plasma membrane Ca2+ ATPase-1 (PMCA1). Taken together, 5-HT seems to induce PTHrP expression in goat mammary cells during and after parturition. These findings suggest that increasing 5-HT biosynthesis could be a potential therapeutic target for prevention of hypocalcemia in dairy goats.

Inhibition effects of total flavonoids from Sculellaria barbata D. Don on human breast carcinoma bone metastasis via downregulating PTHrP pathway.

It is abundantly clear that tumor-derived parathyroid hormone-related protein (PTHrP), receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) are central contributors in promoting osteolytic process of breast carcinoma bone metastasis. Forcusing on this molecular basis, the study was undertaken to explore the inhibition effects of total flavonoids from Scutellaria barbata D. Don (TF-SB) on human breast carcinoma bone metastasis. MDA-MB-231 cells and nude mouse models of breast cancer bone metastasis were given TF-SB in different concentrations. The proliferation, migration and invasion potentials of MDA-MB-231 cells were respectively tested. The effects of TF-SB on tumor weights and bone destruction were investigated. The mRNA and protein expression of PTHrP, OPG and RANKL were assessed by qPCR and western blot analysis. In vitro, TF-SB inhibited the proliferation, migration and invasion of MDA-MB-231 cells in a dose-dependent manner. In vivo, TF-SB prevented bone metastasis of breast cancer by decreasing the number of osteoclast cells per field in a dose-dependent manner, but not affecting tumor growth or mouse survival. Molecular analysis revealed that TF-SB controled the secretion of osteolysis-related factors PTHrP and its downstream RANKL/OPG. Together, by controlling the expression of PTHrP and its downstream OPG/RANKL, TF-SB has significant inhibition effects on breast cancer bone metastasis, which indicates a new therapeutic method.

Decreased expression of PTH1R is a poor prognosis in hepatocellular carcinoma.

Hypercalcemia is a potentially fatal and not rare complication of hepatocellular carcinoma (HCC), and its underlying mechanism remains unclear. Parathyroid hormone (PTH) is the most important regulator of the concentrations of calcium and phosphate in blood; parathyroid hormone-related protein (PTHrP) was the most frequent cause of humoral hypercalcemia of malignancy; parathyroid hormone 1 receptor (PTH1R) is the common receptor for PTH and PTHrP. The aim of this study is to investigate the expression of PTH, PTHrP, and PTH1R in HCC tissues, and their relationship with clinical pathological characters in HCC. First, a meta-analysis based on online Oncomine Expression Array database was conducted to compare the different mRNA expression of PTH1R, PTH and PTHrP between hepatocellular carcinoma and normal tissues. Then, the protein expression level of differentially expressed gene was examined by immunohistochemistry staining in 223 HCC tissues and 102 non-tumorous liver tissues controls. The relationship between the protein expression and clinicopathological parameters was analyzed by χ2 test, and overall survival analysis was performed using Kaplan-Meier survival analysis. PTH1R mRNA expression was significantly lower in HCC tissues compared with normal tissues, while the expression of PTH and PTHrP showed no significant difference between HCC tissues and normal tissues. High PTH1R protein expression was found in 90/102 cases of adjacent non-tumorous liver tissues, and in 91 of 223 cases of HCC tissues. PTH1R expression was significantly related to tumor size, Edmondson Grade, AFP, and overall survival. PTH1R may be the major cause of hypercalcemia in HCC, and the decreased PTH1R expression was a poor prognosis in HCC.