Richard Kremer

Parathyroid Hormone Related Protein (PTHrP) in Tumor Progression

Parathyroid hormone-related protein (PTHrP) is widely expressed in fetal and adult tissues and is a key regulator for cellular calcium transport and smooth muscle cell contractility, as well as a crucial control factor in cell proliferation, development and differentiation. PTHrP stimulates or inhibits apoptosis in an autocrine/paracrine and intracrine fashion, and is particularly important for hair follicle and bone development, mammary epithelial development and tooth eruption. PTHrPs dysregulated expression has traditionally been associated with oncogenic pathologies as the major causative agent of malignancy-associated hypercalcemia, but recent evidence revealed a driving role in skeletal metastasis progression. Here, we demonstrate that PTHrP is also closely involved in breast cancer initiation, growth and metastasis through mechanisms separate from its bone turnover action, and we suggest that PTHrP as a facilitator of oncogenes would be a novel target for therapeutic purposes.

Studies of the effects of 1,25-dihydroxyvitamin D on skeletal and calcium homeostasis and on inhibition of tumor cell growth.

Vitamin D, parathyroid hormone (PTH), and parathyroid hormone-related peptide (PTHrP) are major regulators of calcium metabolism and vitamin D can also reduce the growth of normal cells and tumor cells. PTHrP and PTH act via a common membrane receptor (PTHR). The mouse PTHR is regulated by a kidney-selective upstream promoter P(1) and ubiquitous downstream promoter P(2). In vitro and in vivo 1,25(OH)(2)D can inhibit PTHR expression in bone but not cartilage by downregulating transcription via P(2). Gene transcription of PTHrP per se can also be downregulated by 1,25(OH)(2)D and by low calcemic vitamin D analogs. This inhibitory effect may reduce the hypercalcemia caused by overproduction of PTHrP by tumor cells. In a malignant keratinoctye cell line, phosphorylation of the retinoid X receptor alpha occurs through the activated Ras-MAP kinase pathway and results in attenuated trans-activation by the vitamin D receptor, its heterodimeric partner. This decreases the growth-inhibitory efficacy of 1,25(OH)(2)D. Studies of the capacity of vitamin D to alter PTHrP production and action and of its anti-proliferative effects can, therefore, shed important light on basic mechanisms controlling these events, and may also have major implications for clinical medicine and therapeutics.

Chemoprevention activity of 25-hydroxyvitamin D in the MMTV-PyMT mouse model of breast cancer.

Development of oncologic conditions is often accompanied by inadequate vitamin D status. The chemoprevention ability of this molecule is of high interest for breast cancer, the most common malignancy in women worldwide. Because current effective vitamin D analogues, including the naturally occurring active metabolite 1,25-dihydroxycholecalciferol (1,25(OH)2D), frequently cause hypercalcemia at pharmacologic doses, the development of safer molecules for clinical chemopreventive use is essential. This study examines whether exogenously supplied prohormone 25-hydroxycholecalciferol (25(OH)D) can delay tumor progression in vivo without hypercalcemic effects. A low vitamin D diet (25 IU/kg) in the non-immunodeficient MMTV-PyMT mouse model of metastatic breast cancer revealed a significant acceleration of mammary neoplasia compared with normal diet (1,000 IU/kg). Systemic perfusion of MMTV-PyMT mice with 25(OH)D or 1,25(OH)2D delayed tumor appearance and significantly decreased lung metastasis, and both metabolites reduced Ki-67, cyclin D1, and ErbB2 levels in tumors. Perfusion with 25(OH)D caused a 50% raise in tumor 1,25(OH)2D levels, indicating good tumor penetration and effective activation. Importantly, in contrast with 1,25(OH)2D, perfusion with 25(OH)D did not cause hypercalcemia. In vitro treatment of cultured MMTV-PyMT mammary tumor cells with 25(OH)D inhibited proliferation, confirming local activation of the prohormone in this system. This study provides an in vivo demonstration in a non-immunodeficient model of spontaneous breast cancer that exogenous 25(OH)D delays neoplasia, tumor growth, and metastasis, and that its chemoprevention efficacy is not accompanied by hypercalcemia. Cancer Prev Res; 8(2); 120–8. ©2014 AACR.

Response of Postpoliomyelitis Patients to Bisphosphonate Treatment

To evaluate (1) the rate of change of bone mineral density (BMD) at the hip in postpolio patients treated with bisphosphonates compared with the rate of change in BMD in (a) postpolio patients not treated with bisphosphonates and (b) non‐postpolio patients treated with bisphosphonates; and (2) to compare the fracture rate in postpolio patients before and after treatment.

Vitamin D deficiency/insufficiency from childhood to adulthood: Insights from a sunny country

Vitamin D is known to be a key regulator of bone metabolism and is associated with muscle strength. Vitamin D deficiency is widely prevalent worldwide. In adults, vitamin D deficiency has been implicated in numerous health conditions including osteoporosis, cancer, diabetes, and autoimmune diseases. Considerable changes have occurred in lifestyles and childhood activities in the past years. Studies have shown that the children population is at high risks of vitamin D deficiency. The objective of this study was to learn about the extent of vitamin D deficiency in children worldwide and especially in sunny country like Israel. In this article we reviewed the extent and severity of vitamin D deficiency worldwide and especially in Israel, through a very comprehensive review of previous reports and research studies done during the last years. We found reports on vitamin D deficiency in children, which was associated with metabolic syndromes and obesity. It was more prevalent in children who spend less time on outdoor activities, in obese children, and in cases when there was imbalance between nutritional intakes and requirements. Vitamin D deficiency is common even in children living in sunny places like Israel. Health professionals should be aware of the fact that although vitamin D deficiency is prevalent in the elderly population, it is also common in children, and can be associated with different illnesses. We encourage supplementation of vitamin D to special populations (pregnant and lactating women, infants, and high risk groups). We also encourage implementation of international food fortification programs.

Vitamin D prevents lipid accumulation in murine muscle through regulation of PPARγ and perilipin-2 expression

Vitamin D plays an important role in regulation of skeletal muscle tone and contraction. Serum vitamin D status is linked to muscle power and force in adolescent girls, and vitamin D deficiency is associated with myopathies in children and poorer physical performance in the elderly. We previously reported that vitamin D deficiency is linked to a significant increase in muscle fatty infiltration in healthy young women, and studies in patients with neuromuscular disorders also associate muscle weakening and lipid content. In order to better understand the link between vitamin D status and skeletal muscle lipid metabolism, we compared the effect of a low (25IU/kg) or normal (1000IU/kg) vitamin D3 diet on muscle fat in female FVB mice maintained in a room without UVB lighting to minimize endogenous vitamin D production. Animals on low vitamin D diet displayed lower circulating 25(OH)D levels and a dramatic increase (287±52% compared to normal diet, p<0.0001) in lipid deposition in skeletal muscle accompanied by muscle fiber disorganization. Lipid droplet staining increased by 242±23% (p<0.0001) in low vitamin D diet, and lipid droplet coat protein perilipin-2 and nuclear receptor transcription factor PPARγ expression levels were increased compared to mice fed the normal vitamin D diet: average staining for PLIN2: 0.22±0.08 (25IU/kg diet) vs 0.10±0.02 (1000IU/kg). Average staining for PPARγ: 0.24±0.06 (25IU/kg diet) vs 0.07±0.04 (1000IU/kg) p<0.0001. Tissue mass spectrometry imaging revealed major differences in muscle phospholipids profile depending on diet. In vitro, 1,25(OH)2D3 treatment of 3T3-L1 pre-adipocytes inhibited appearance of lipid droplets by 79±9.3%, and caused a 80±10% and 25±8% (p=0.001) reduction in PPARγ and perilipin-2 mRNA levels (by qPCR) compared to control cells. In summary, we report here the first in vivo model illustrating the important structural muscle fiber disorganization and fat accumulation inside and outside muscle fibers that accompany vitamin D deficiency. Furthermore, we show that the underlying mechanisms involve PPARγ and perilipin-2.

Examination of VDR/RXR/DRIP205 Interaction, Intranuclear Localization, and DNA Binding in Ras-Transformed Keratinocytes and Its Implication for Designing Optimal Vitamin D Therapy in Cancer

Retinoid X receptor (RXR) occupies a central position within the nuclear receptor superfamily, serving as an obligatory partner to numerous other nuclear receptors, including vitamin D receptor (VDR). In the current study, we examined whether phosphorylation of RXRα at serine 260 affects VDR/RXR and VDR interacting protein (DRIP) 205 coactivator recruitment, interactions, and binding of the VDR/human RXRα (hRXRα)/DRIP205 complex to chromatin. Serine 260 is a critical amino acid on the hRXRα that is located in close spatial proximity to regions of coactivator and corepressor interactions. Using fluorescence resonance energy transfer and immunofluorescence studies, we showed that the physical interaction between hRXRα and DRIP205 coactivator was impaired in human keratinocytes with the ras oncogene (HPK1Aras) or transfected with the wild-type hRXRα. Furthermore, the nuclear colocalization of VDR/DRIP205, hRXRα/DRIP205, and VDR/hRXRα/DRIP205 complex binding to chromatin is impaired in the HPK1Aras cells when compared with the normal human keratinocytes (HPK1A cells). However, transfection with the nonphosphorylatable hRXRα (S260A) mutant or treatment with the mitogen-activated protein kinase (MAPK) inhibitor UO126 rescued their nuclear localization, interaction, and binding of the complex to chromatin in the HPK1Aras cells. In summary, we have demonstrated, using highly specific intracellular tagging methods in live and fixed cells, important alterations of the vitamin D signaling system in cancer cells in which the ras-raf-MAPK system is activated, suggesting that specific inhibition of this commonly activated pathway could be targeted therapeutically to enhance vitamin D efficacy.

P I – 3–1 Vitamin d deficiency/insufficiency- from childhood to adulthood: insights from a sunny country

Background/aim Vitamin D deficiency is widely prevalent worldwide. Vitamin D is known to be a key regulator of bone metabolism and is associated with muscle strength. The objective of this study was to learn also about additional implications of vitamin D deficiency in adults and to investigate the extent of vitamin D deficiency in children worldwide, especially in a sunny country like Israel. Methods The extent and severity of vitamin D deficiency, in adults and in children, were surveyed worldwide and especially in Israel, through a comprehensive review of previous reports and research studies done during the last 30 years in Israel and worldwide. Results In adults, vitamin D deficiency has also been implicated in numerous health conditions including osteoporosis, cancer, diabetes, and autoimmune diseases. In children, vitamin D deficiency was associated with metabolic syndromes and obesity. It was more prevalent in children who spend less time on outdoor activities, in obese children, and in cases when there was an imbalance between nutritional intakes and requirements. Vitamin D deficiency is common even in children living in sunny places like Israel. Conclusion Vitamin D deficiency has many previously unknown implications.The doctors should be aware of the fact that although vitamin D deficiency is prevalent in the elderly population, it can also appear in the children, and can be associated with different illnesses. We encourage supplementation of vitamin D to special populations and encourage implementation of international food fortification programs.

Fasting serum blood measures of bone and lipid metabolism in children with myelomeningocele for early detection of cardiovascular and bone fragility risk factors

Objective: This study examined serum levels in children with myelomeningocele to identify the prevalence of pre-clinical signs of disease. Design: A prospective, cross-sectional study. Setting: Patients were actively recruited from multidisciplinary care clinics at tertiary childrens hospitals from 2010–2012. The control comparison group was recruited by word-of-mouth. Patients: Twenty-eight children with myelomeningocele (93% Hispanic; 17 males; 10.0 ± 2.1 years) and 58 controls (84% Hispanic; 30 males; 10.4 ± 2.4 years) provided ≥ 8-hour fasting blood samples with concomitant dual-energy x-ray absorptiometry measurements of body fat. Interventions: Not applicable. Main Outcome Measures: The serum analysis included a lipid panel (cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein), insulin, glucose, leptin, aspartate aminotransferase, alanine transaminase, alkaline phosphatase, albumin, creatinine, calcium, phosphatase, parathyroid hormone, and vitamin D. Results: Children with myelomeningocele had higher body fat (35.2% versus 29.9%, p=0.01) and altered lipid profiles (lower high-density lipoprotein levels, 43.9 mg/dL versus 51.6 mg/dL, P = 0.03) suggesting elevated risk of metabolic syndrome. They also had a higher prevalence of vitamin D deficiency (43% versus 17%, p=0.02) and significantly lower levels of calcium (9.4 mg/dL versus 9.7 mg/dL, P = 0.003) and alkaline phosphatase (187.0 U/L versus 237.0 U/L, P = 0.003). Unexpectedly children with myelomeningocele had lower parathyroid hormone levels (14.5 pg/mL versus 18.4 pg/mL, P = 0.02) than controls despite lower calcium, vitamin D and alkaline phosphatase levels. This suggests an alteration in the sensing mechanism or response of the parathyroid gland to normal physiological stimuli in patients with myelomeningocele. Conclusions: Children with myelomeningocele have abnormal biochemical markers for cardiovascular disease, insulin resistance and bone and mineral metabolism. Early recognition and monitoring of these risk factors in patients with myelomeningocele may help prevent later complications.

Osteoporosis treatment may benefit breast cancer patients

The fascinating story of bisphosphonates started almost 50 years ago with the pioneer work of H Fleisch, a Swiss physician who found that plasma and urine contained inorganic pyrophosphate that inhibits calcium phosphate precipitation [1]. However, pyrophosphate had limited clinical application because of rapid hydrolysis after oral administration. G Russell joined Dr Fleisch’s Medical Research Institute in Davos, Switzerland, and together they collaborated with D Francis, a chemist at Procter and Gamble, that led to the discovery of bisphosphonates that were shown to resist enzymatic hydrolysis but also turned out to be powerful inhibitors of bone resorption [2]. Chemically the bisphosphonates are synthetic compounds that resemble pyrophosphate, in which the P-O-P structure is replaced by a P-C-P bond that is resistant to chemical and enzymatic hydrolysis. The basic P-C-P structure can be modified at the two lateral chains on the carbon atom or by esterifying the phosphate groups, leading to unique members of the family with distinct effects on bone metabolism. The ability of the compound to bind to bone surface seems to be dependent on the P-C-P bond while the side chain in combination with the P-C-P bond defines ability to inhibit bone resorption. The major biological effect of bisphosphonates is to reduce osteoclast numbers by inhibiting the recruitment of preosteoclasts and by promoting their apoptosis. During the 1990s, M Rogers and others within his group in Sheffield elucidated the molecular mechanisms of action of the bisphosphonates, and showed that nitrogen-containing bisphosphonates act as inhibitors of mevalonate metabolism resulting in inhibition of protein prenylation, the same pathway used by statins to block cholesterol synthesis [3]. The first clinical use of bisphosphonates taking advantage of their strong osteoclastic inhibitory activity was reported by G Russell and R Smith in patients with Paget’s disease of bone [4]. However, it was not until the 1990s that the first clinical report on the efficacy of bisphosphonates on fracture prevention in osteoporosis using the first-generation compound, etidronate, was published [5], followed by several fracture prevention trials in postmenopausal osteoporosis using the more potent aminobisphosphonates alendronate [6] and risedronate [7]. The oncology field benefited from the bisphosphonates discovery during the same period, reporting clinical usefulness in malignancy-associated hypercalcemia in patients with solid tumors by intravenous (iv.) administration [8]. Perhaps the most fascinating development in oncology came from studies testing the ‘seed and soil’ hypothesis of S Paget first proposed in 1889 [9]. An Austrian physician by the name of E Fuchs had proposed a similar hypothesis 7 years earlier but was never credited with this discovery [10]. Nevertheless, after reviewing over 700 pathological reports of cancer patients, Paget speculated that tumor cells (seed) “can only live and grow Osteoporosis treatment may benefit breast cancer patients