Honors & Awards

  • CHRI Postdoctoral Fellowship, Child Health Research Institute (2016)
  • ÖGES Young Investigator Award, Austrian Society for Endocrinology and Metabolism, Austria (2015)
  • Young Investigator Award, 2nd International Calcium-­ Sensing Receptor Symposium, San Diego, USA (2015)
  • ÖGKM Fellowship, Austrian Society for Bone and Mineral Research, Austria (2015)
  • EACR Fellowship, European Association for Cancer Research (2014)
  • Drabikowski Award, European Calcium Society Meeting, France (2014)
  • Young Investigator Award, Austrian Society for Endocrinology and Metabolism, Austria (2014)
  • Young Investigator Award, Rote Pfote, Austria (2014)
  • ÖGKM Fellowship, Austrian Society for Bone and Mineral Research, Austria (2014)
  • Best Poster Award, Centre of Pathophysiology and Immunology, Medical University of Vienna, Austria (2013)
  • ÖGMBT Fellowship, Austrian Association of Molecular Life Sciences and Biotechnology, Austria (2013)
  • ÖGKM Fellowship, Austrian Society for Bone and Mineral Research, Austria (2013)
  • ECS Fellowship, European Calcium Society, France (2012)
  • Science Communication Award, Rote Pfote, Austria (2012)
  • ICSCC 2012 Fellowship, International Conference on Stem Cells and Cancer, India (2012)
  • International Postgraduate Scholarship, Newcastle University, UK (2009)

Boards, Advisory Committees, Professional Organizations

  • Ambassador, European Association for Cancer Research (2014 - Present)
  • Mentor, University of Manchester (2012 - Present)
  • President, Young Scientist Association of the Medical University of Vienna (2013 - 2014)
  • Vice President, Young Scientist Association of the Medical University of Vienna (2012 - 2013)

Professional Education

  • PhD, Medical University of Vienna, Malignant Diseases (2015)
  • Master of Science, Newcastle University (2010)
  • Post Graduate Diploma, Institute of Psychotherapy and Management Sciences, Psychology and Counselling (2009)
  • Bachelor of Technology, Vellore Institute Technology (2009)

Stanford Advisors

Research & Scholarship

Lab Affiliations


All Publications

  • Identification of tumor-autonomous and indirect effects of vitamin D action that inhibit breast cancer growth and tumor progression. The Journal of steroid biochemistry and molecular biology Aggarwal, A., Feldman, D., Feldman, B. J. 2017


    Several epidemiological studies have found that low vitamin D levels are associated with worse prognosis and poorer outcomes in patients with breast cancer (BCa), although some studies have failed to find this association. In addition, prior research has found that BCa patients with vitamin D deficiency have a more aggressive molecular phenotype and worse prognostic biomarkers. As vitamin D deficiency is common in patients diagnosed with BCa, elucidating the cause of the association between poor outcomes and vitamin D deficiency promises to have a significant impact on improving care for patients with BCa including enabling the development of novel therapeutic approaches. Here we review our recent findings in this area, including our data revealing that reduction of the expression of the vitamin D receptor (Vdr) within BCa cells accelerates primary tumor growth and enables the development of metastases, demonstrating a tumor autonomous effect of vitamin D signaling to suppress BCa metastases. We believe that these findings are likely relevant to humans as we discovered evidence that a mechanism of VDR regulation identified in our mouse models is conserved in human BCa. In particular, we identified a negative correlation between serum 25(OH)D concentration and the level of expression of the tumor progression factor ID1 in primary tumors from patients with breast cancer.

    View details for DOI 10.1016/j.jsbmb.2017.07.003

    View details for PubMedID 28710021

  • Macrophage-released ADAMTS1 promotes muscle stem cell activation. Nature communications Du, H., Shih, C. H., Wosczyna, M. N., Mueller, A. A., Cho, J., Aggarwal, A., Rando, T. A., Feldman, B. J. 2017; 8 (1): 669


    Coordinated activation of muscle stem cells (known as satellite cells) is critical for postnatal muscle growth and regeneration. The muscle stem cell niche is central for regulating the activation state of satellite cells, but the specific extracellular signals that coordinate this regulation are poorly understood. Here we show that macrophages at sites of muscle injury induce activation of satellite cells via expression of Adamts1. Overexpression of Adamts1 in macrophages in vivo is sufficient to increase satellite cell activation and improve muscle regeneration in young mice. We demonstrate that NOTCH1 is a target of ADAMTS1 metalloproteinase activity, which reduces Notch signaling, leading to increased satellite cell activation. These results identify Adamts1 as a potent extracellular regulator of satellite cell activation and have significant implications for understanding the regulation of satellite cell activity and regeneration after muscle injury.Satellite cells are crucial for growth and regeneration of skeletal muscle. Here the authors show that in response to muscle injury, macrophages secrete Adamts1, which induces satellite cell activation by modulating Notch1 signaling.

    View details for DOI 10.1038/s41467-017-00522-7

    View details for PubMedID 28939843

  • The Circadian Clock Regulates Adipogenesis by a Per3 Crosstalk Pathway to Klf15. Cell reports Aggarwal, A., Costa, M. J., Rivero-Gutiérrez, B., Ji, L., Morgan, S. L., Feldman, B. J. 2017; 21 (9): 2367–75


    The generation of new adipocytes from precursor cells (adipogenesis) has implications for systemic metabolism and is a commonly used model for studying the process of cell differentiation in vitro. Previous studies from us and others suggested that the peripheral circadian clock can influence adipogenesis in vitro, but the mechanisms driving this activity and the relevance for adipogenesis in vivo are unknown. Here we reveal that mouse adipocyte precursor cells (APCs) contain a circadian clock that oscillates in vivo. We expose context-specific features of the clock in APCs: expression of the canonical core clock component Per1 does not significantly oscillate, whereas the lesser-understood paralog Per3 has a prominent rhythm. We discovered that deletion of Per3 promotes adipogenesis in vivo by a clock output pathway in which PER3 and BMAL1 directly regulate Klf15 expression. These findings demonstrate that Per3 has a major role in the APC clock and regulates adipogenesis in vivo.

    View details for DOI 10.1016/j.celrep.2017.11.004

    View details for PubMedID 29186676

  • A glucocorticoid- and diet-responsive pathway toggles adipocyte precursor cell activity in vivo. Science signaling Wong, J. C., Krueger, K. C., Costa, M. J., Aggarwal, A., Du, H., McLaughlin, T. L., Feldman, B. J. 2016; 9 (451): ra103-?


    Obesity is driven by excess caloric intake, which leads to the expansion of adipose tissue by hypertrophy and hyperplasia. Adipose tissue hyperplasia results from the differentiation of adipocyte precursor cells (APCs) that reside in adipose depots. Investigation into this process has elucidated a network of mostly transcription factors that drive APCs through the differentiation process. Using in vitro and in vivo approaches, our study revealed a signaling pathway that inhibited the initiation of the adipocyte differentiation program. Mouse adipocytes secreted the extracellular protease ADAMTS1, which triggered the production of the cytokine pleiotrophin (PTN) through the Wnt/β-catenin pathway, and promoted proliferation rather than differentiation of APCs. Glucocorticoid exposure in vitro or in vivo reduced ADAMTS1 abundance in adipocytes. In addition, mice fed a high-fat diet showed decreased Adamts1 expression in the visceral perigonadal adipose depot, which expanded by adipogenesis in response to the diet, and increased Adamts1 expression in the subcutaneous inguinal adipose depot, which did not induce adipogenesis. Similar to what occurred in mouse subcutaneous adipose tissue, diet-induced weight gain increased the expression of ADAMTS1, PTN, and certain Wnt target genes in the subcutaneous adipose depot of human volunteers, suggesting the relevance of this pathway to physiological adipose tissue homeostasis and the pathogenesis of obesity. Thus, this pathway functions as a toggle on APCs, regulating a decision between differentiation and proliferation and coordinating the response of adipose tissue to systemic cues.

    View details for PubMedID 27811141

  • Vitamin D mitigates the adverse effects of obesity on breast cancer in mice ENDOCRINE-RELATED CANCER Swami, S., Krishnan, A. V., Williams, J., Aggarwal, A., Albertelli, M. A., Horst, R. L., Feldman, B. J., Feldman, D. 2016; 23 (4): 251-264


    Obesity is an established risk factor for postmenopausal breast cancer (BCa), insulin resistance and vitamin D deficiency, and all contribute to increased synthesis of mammary estrogens, the drivers of estrogen receptor-positive (ER+) BCa growth. Since both dietary vitamin D and calcitriol treatments inhibit breast estrogen synthesis and signaling, we hypothesized that vitamin D would be especially beneficial in mitigating the adverse effects of obesity on ER+ BCa. To assess whether obesity exerted adverse effects on BCa growth and whether vitamin D compounds could reduce these unfavorable effects, we employed a diet-induced obesity (DIO) model in ovariectomized C57BL/6 mice. Breast tumor cells originally from syngeneic Mmtv-wnt1 transgenic mice were then implanted into the mammary fat pads of lean and obese mice. DIO accelerated the initiation and progression of the mammary tumors. Treatments with either calcitriol or dietary vitamin D reduced the adverse effects of obesity causing a delay in tumor appearance and inhibiting continued tumor growth. Beneficial actions of treatments with vitamin D or calcitriol on BCa and surrounding adipose tissue included: repressed Er, aromatase and Cox-2 expression, decreased tumor derived estrogen and PGE2 and reduced expression of leptin receptors and increased adiponectin receptors. We demonstrate that vitamin D treatments decreased insulin resistance, reduced leptin and increased adiponectin signaling and also regulated the LKB1/ AMPK pathway contributing to an overall decrease in local estrogen synthesis in the obese mice. We conclude that calcitriol and dietary vitamin D, acting by multiple interrelated pathways, mitigate obesity enhanced BCa growth in a postmenopausal setting.

    View details for DOI 10.1530/ERC-15-0557

    View details for Web of Science ID 000377691700007

    View details for PubMedID 26817629

  • Tumor Autonomous Effects of Vitamin D Deficiency Promote Breast Cancer Metastasis. Endocrinology Williams*, J., Aggarwal*, A., Swami, S., Krishnan, A., Ji, L., Albertelli, M., Feldman, B. J. 2016
  • Mutant mice with calcium-sensing receptor (CaSR) activation have hyperglycemia, that is rectified by calcilytic therapy. Endocrinology Babinsky, V. N., Hannan, F. M., Ramracheya, R. D., Zhang, Q., Nesbit, M. A., Hugill, A., Bentley, L., Hough, T. A., Joynson, E., Stewart, M., Aggarwal, A., Prinz-Wohlgenannt, M., Gorvin, C. M., Kallay, E., Wells, S., Cox, R. D., Richards, D., Rorsman, P., Thakker, R. V. 2017


    The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor (GPCR) that plays a pivotal role in extracellular calcium homeostasis. The CaSR is also highly expressed in pancreatic islet α- and β-cells that secrete glucagon and insulin, respectively. To determine whether the CaSR may influence systemic glucose homeostasis, we characterized a mouse model with a germline gain-of-function CaSR mutation, Leu723Gln, referred to as Nuclear flecks (Nuf). Heterozygous- (CasrNuf/+) and homozygous-affected (CasrNuf/Nuf) mice were shown to have hypocalcemia in association with impaired glucose tolerance and insulin secretion. Oral administration of a CaSR antagonist compound, known as a calcilytic, rectified the glucose intolerance and hypoinsulinemia of CasrNuf/+ mice, and ameliorated glucose intolerance in CasrNuf/Nuf mice. Ex vivo studies showed CasrNuf/+ and CasrNuf/Nuf mice to have reduced pancreatic islet mass and β-cell proliferation. Electrophysiological analysis of isolated CasrNuf/Nuf islets showed CaSR activation to increase the basal electrical activity of β-cells independently of effects on the activity of the ATP-sensitive K+ (KATP) channel. CasrNuf/Nuf mice also had impaired glucose-mediated suppression of glucagon secretion, which was associated with increased numbers of α-cells and a higher α-cell proliferation rate. Moreover, CasrNuf/Nuf islet electrophysiology demonstrated an impairment of α-cell membrane depolarization in association with attenuated α-cell basal KATP channel activity. These studies indicate that the CaSR activation impairs glucose tolerance by a combination of α- and β-cell defects and also influences pancreatic islet mass. Moreover, our findings highlight a potential application of targeted CaSR compounds for modulating glucose metabolism.

    View details for DOI 10.1210/en.2017-00111

    View details for PubMedID 28575322

  • Expression profiling of colorectal cancer cells reveals inhibition of DNA replication licensing by extracellular calcium. Biochimica et biophysica acta Aggarwal, A., Schulz, H., Manhardt, T., Bilban, M., Thakker, R. V., Kallay, E. 2017; 1864 (6): 987-996


    Colorectal cancer is one of the most common cancers in industrialised societies. Epidemiological studies, animal experiments, and randomized clinical trials have shown that dietary factors can influence all stages of colorectal carcinogenesis, from initiation through promotion to progression. Calcium is one of the factors with a chemoprophylactic effect in colorectal cancer. The aim of this study was to understand the molecular mechanisms of the anti-tumorigenic effects of extracellular calcium ([Ca(2+)]o) in colon cancer cells. Gene expression microarray analysis of colon cancer cells treated for 1, 4, and 24h with 2mM [Ca(2+)]o identified significant changes in expression of 1571 probe sets (ANOVA, p<10(-5)). The main biological processes affected by [Ca(2+)]o were DNA replication, cell division, and regulation of transcription. All factors involved in DNA replication-licensing were significantly downregulated by [Ca(2+)]o. Furthermore, we show that the calcium-sensing receptor (CaSR), a G protein-coupled receptor is a mediator involved in this process. To test whether these results were physiologically relevant, we fed mice with a standard diet containing low (0.04%), intermediate (0.1%), or high (0.9%) levels of dietary calcium. The main molecules regulating replication licensing were inhibited also in vivo, in the colon of mice fed high calcium diet. We show that among the mechanisms behind the chemopreventive effect of [Ca(2+)]o is inhibition of replication licensing, a process often deregulated in neoplastic transformation. Our data suggest that dietary calcium is effective in preventing replicative stress, one of the main drivers of cancer and this process is mediated by the calcium-sensing receptor.

    View details for DOI 10.1016/j.bbamcr.2017.01.017

    View details for PubMedID 28161520

    View details for PubMedCentralID PMC5424886

  • Cross Talk between the Calcium-Sensing Receptor and the Vitamin D System in Prevention of Cancer FRONTIERS IN PHYSIOLOGY Aggarwal, A., Kallay, E. 2016; 7


    There is epidemiological evidence for the cancer preventive effect of dietary calcium (Ca(2+)) and vitamin D. This effect is strongest in colorectal cancer (CRC). The active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25D3), bound to its receptor, the vitamin D receptor (VDR) regulates the expression of hundreds of different genes in a cell- and tissue-specific manner. While Ca(2+) acts through multiple mechanisms and pathways, some of its effects are mediated by the calcium-sensing receptor (CaSR). The joint action of Ca(2+) and 1,25D3 is due to the fact that both regulate some of the main processes involved in the development of various cancers, such as proliferation, differentiation, apoptosis, migration, and inflammation. Moreover, 1,25D3, bound to VDR can induce translation of the CaSR, while the amount and activity of the CaSR affects 1,25D3 signaling. However, the complexity of the cross-talk between the CaSR and the vitamin D system goes beyond regulating similar pathways and affecting each other's expression. Our aim was to review some of the mechanisms that drive the cross-talk between the vitamin D system and the CaSR with a special focus on the interaction in CRC cells. We evaluated the molecular evidence that supports the epidemiological observation that both vitamin D and calcium are needed for protection against malignant transformation of the colon and that their effect is modulated by the presence of a functional CaSR.

    View details for DOI 10.3389/fphys.2016.00451

    View details for Web of Science ID 000385466800001

    View details for PubMedID 27803671

  • The calcium-sensing receptor and the hallmarks of cancer BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH Tennakoon, S., Aggarwal, A., Kallay, E. 2016; 1863 (6): 1398-1407


    The calcium-sensing receptor (CaSR) plays a pivotal role in systemic calcium metabolism by regulating parathyroid hormone secretion and urinary calcium excretion. The CaSR is ubiquitously expressed, implying a wide range of functions regulated by this receptor. Abnormal CaSR function affects the development of both calciotropic disorders such as hyperparathyroidism, and non-calciotropic disorders such as cardiovascular disease and cancer, which are the leading causes of mortality worldwide. The CaSR is able to bind a plethora of ligands; it interacts with multiple G protein subtypes, and regulates highly divergent downstream signalling pathways, depending on the cellular context. The CaSR is a key regulator for such diverse processes as hormone secretion, gene expression, inflammation, proliferation, differentiation, and apoptosis. Due to this pleiotropy, the CaSR is able to regulate cell fate and is implicated in the development of many types of benign or malignant tumours of the breast, prostate, parathyroid, and colon. In cancer, the CaSR appears to have paradoxical roles, and depending on the tissue involved, it is able to prevent or promote tumour growth. In tissues like the parathyroid or colon, the CaSR inhibits proliferation and induces terminal differentiation of the cells. Therefore, loss of the receptor, as seen in colorectal or parathyroid tumours, confers malignant potential, suggestive of a tumour suppressor role. In contrast, in prostate and breast tumours the expression of the CaSR is increased and it seems that it favours metastasis to the bone, acting as an oncogene. Deciphering the molecular mechanism driving the CaSR in the different tissues could lead to development of new allosteric drug compounds that selectively target the CaSR and have therapeutic potential for cancer. This article is part of a Special Issue entitled: Calcium and Cell Fate . Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.

    View details for DOI 10.1016/j.bbamcr.2015.11.017

    View details for Web of Science ID 000375885900008

    View details for PubMedID 26608608

  • The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure. American journal of physiology. Cell physiology Schepelmann, M., Yarova, P. L., Lopez-Fernandez, I., Davies, T. S., Brennan, S. C., Edwards, P. J., Aggarwal, A., Graça, J., Rietdorf, K., Matchkov, V., Fenton, R. A., Chang, W., Krssak, M., Stewart, A., Broadley, K. J., Ward, D. T., Price, S. A., Edwards, D. H., Kemp, P. J., Riccardi, D. 2016; 310 (3): C193–204


    The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive (SM22α)CaSR(Δflox/Δflox) [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.

    View details for DOI 10.1152/ajpcell.00248.2015

    View details for PubMedID 26538090

  • The calcium-sensing receptor suppresses epithelial-to-mesenchymal transition and stem cell- like phenotype in the colon MOLECULAR CANCER Aggarwal, A., Prinz-Wohlgenannt, M., Groeschel, C., Tennakoon, S., Meshcheryakova, A., Chang, W., Brown, E. M., Mechtcheriakova, D., Kallay, E. 2015; 14


    The calcium sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is expressed also in tissues not directly involved in calcium homeostasis like the colon. We have previously reported that CaSR expression is down-regulated in colorectal cancer (CRC) and that loss of CaSR provides growth advantage to transformed cells. However, detailed mechanisms underlying these processes are largely unknown.In a cohort of 111 CRC patients, we found significant inverse correlation between CaSR expression and markers of epithelial-to-mesenchymal transition (EMT), a process involved in tumor development in CRC. The colon of CaSR/PTH double-knockout, as well as the intestine-specific CaSR knockout mice showed significantly increased expression of markers involved in the EMT process. In vitro, stable expression of the CaSR (HT29(CaSR)) gave a more epithelial-like morphology to HT29 colon cancer cells with increased levels of E-Cadherin compared with control cells (HT29(EMP)). The HT29(CaSR) cells had reduced invasive potential, which was attributed to the inhibition of the Wnt/β-catenin pathway as measured by a decrease in nuclear translocation of β-catenin and transcriptional regulation of genes like GSK-3β and Cyclin D1. Expression of a spectrum of different mesenchymal markers was significantly down-regulated in HT29(CaSR) cells. The CaSR was able to block upregulation of mesenchymal markers even in an EMT-inducing environment. Moreover, overexpression of the CaSR led to down-regulation of stem cell-like phenotype.The results from this study demonstrate that the CaSR inhibits epithelial-to-mesenchymal transition and the acquisition of a stem cell-like phenotype in the colon of mice lacking the CaSR as well as colorectal cancer cells, identifying the CaSR as a key molecule in preventing tumor progression. Our results support the rationale to develop new strategies either preventing CaSR loss or reversing its silencing.

    View details for DOI 10.1186/s12943-015-0330-4

    View details for Web of Science ID 000353324700001

    View details for PubMedID 25879211

  • The calcium-sensing receptor: A promising target for prevention of colorectal cancer. Biochimica et biophysica acta 2015


    The inverse correlation between dietary calcium intake and the risk of colorectal cancer (CRC) is well known, but poorly understood. Expression of the calcium-sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is downregulated in CRC leading us to hypothesize that the CaSR has tumor suppressive roles in the colon. The aim of this study was to understand whether restoration of CaSR expression could reduce the malignant phenotype in CRC. In human colorectal tumors, expression of the CaSR negatively correlated with proliferation markers whereas loss of CaSR correlated with poor tumor differentiation and reduced apoptotic potential. In vivo, dearth of CaSR significantly increased expression of proliferation markers and decreased levels of differentiation and apoptotic markers in the colons of CaSR/PTH double knock-out mice confirming the tumor suppressive functions of CaSR. In vitro CRC cells stably overexpressing wild-type CaSR showed significant reduction in proliferation, as well as increased differentiation and apoptotic potential. The positive allosteric modulator of CaSR, NPS R-568 further enhanced these effects, whereas treatment with the negative allosteric modulator, NPS 2143 inhibited these functions. Interestingly, the dominant-negative mutant (R185Q) was able to abrogate these effects. Our results demonstrate a critical tumor suppressive role of CaSR in the colon. Restoration of CaSR expression and function is linked to regulation of the balance between proliferation, differentiation, and apoptosis and provides a rationale for novel strategies in CRC therapy.

    View details for DOI 10.1016/j.bbamcr.2015.02.011

    View details for PubMedID 25701758

  • Impact of CYP24A1 overexpression on growth of colorectal tumor xenografts in mice fed with vitamin D and soy. International journal of cancer. Journal international du cancer 2015


    Our previous studies showed that the 1,25-dihydroxyvitamin D (1,25-D3 ) catabolizing enzyme, 1,25-dihydoxyvitamin D 24 hydroxylase (CYP24A1) was overexpressed in colorectal tumors and its level correlated with increased proliferation. We hypothesized that cells overexpressing CYP24A1 have growth advantage and a diet rich in vitamin D and soy would restore sensitivity to the antitumorigenic effects of vitamin D. Soy contains genistein, a natural CYP24A1 inhibitor. To determine causality between CYP24A1 and tumor growth, we established xenografts in male SCID mice with HT29 cells stably overexpressing either GFP-tagged CYP24A1 or GFP. Mice were fed with either high (2500 IU D3 /kg) or low vitamin D (100 IU D3 /kg) diet in the presence or absence of soy (20% diet). In vitro, cells overexpressing CYP24A1 grew faster than controls. 1,25-D3 , the active vitamin D metabolite, reduced cell number only in the presence of the CYP24A1 inhibitor VID400. Regardless of the amount of vitamin D in the diet, xenografts overexpressing CYP24A1 grew faster, were heavier and more aggressive. Soy reduced tumor volume only in the control xenografts, while the tumors overexpressing CYP24A1 were larger in the presence of dietary soy. In conclusion, we demonstrate that CYP24A1 overexpression results in increased aggressiveness and proliferative potential of colorectal tumors. Irrespective of the dietary vitamin D3 , dietary soy is able to increase tumor volume when tumors overexpress CYP24A1, suggesting that combination of vitamin D3 and soy could have an antitumorigenic effect only if CYP24A1 levels are normal. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/ijc.29717

    View details for PubMedID 26238339

  • miR-135b- and miR-146b-dependent silencing of calcium-sensing receptor expression in colorectal tumors. International journal of cancer. Journal international du cancer 2015


    Studies have shown that the calcium-sensing receptor (CaSR) mediates the antitumorigenic effects of calcium against colorectal cancer (CRC). Expression of the CaSR in colorectal tumors is often reduced. We have reported previously that silencing of CaSR in CRC is caused in part by methylation of CaSR promoter 2 and loss of histone acetylation. We investigated the impact of aberrant microRNA expression on loss of CaSR expression. A microarray study in two Caco-2 subclones (Caco2/AQ and Caco2/15) that have similar genetic background, but different CaSR expression levels (Caco2/AQ expressing more CaSR than Caco2/15), identified 22 differentially expressed microRNAs that potentially target the CaSR. We validated these results by performing gain and loss of function studies with the top candidates: miR-9, miR-27a, miR-135b, and miR-146b. Modulation of miR-135b or miR-146b expression by mimicking or inhibiting their expression regulated CaSR protein levels in two different colon cancer cell lines: Caco2/AQ (moderate endogenous CaSR expression) and HT29 (low endogenous CaSR levels). Inhibition of miR-135b and miR-146b expression led to high CaSR levels and significantly reduced proliferation. In samples of colorectal tumors we observed overexpression of miR-135b and miR-146b, and this correlated inversely with CaSR expression (miR-135b: r=-0.684, p<0.001 and miR-146b: r=-0.448, p<0.001), supporting our in vitro findings. We demonstrate that miR-135b and miR-146b target the CaSR and reduce its expression in colorectal tumors, reducing the antiproliferative and prodifferentiating actions of calcium. This provides a new approach for finding means to prevent CaSR loss, developing better treatment strategies for CRC. This article is protected by copyright. All rights reserved. © 2014 Wiley Periodicals, Inc.

    View details for DOI 10.1002/ijc.29681

    View details for PubMedID 26178670

  • Effect of 1,25-dihydroxyvitamin D3 on the Wnt pathway in non-malignant colonic cells. The Journal of steroid biochemistry and molecular biology 2015


    Epidemiological studies suggest a correlation between vitamin D deficiency and colorectal cancer (CRC) incidence. The majority of sporadic tumors develop from premalignant lesions with aberrant activation of the Wnt/β-catenin signaling pathway. The adenoma cell line LT97 harbors an adenomatous polyposis coli (APC) mutation leading to constitutively active Wnt signaling. In these cells, expression of Wnt target genes leads to increased survival capacity. We hypothesized that 1,25-dihydroyvitamin D3 (1,25-D3), the active form of vitamin D3, promotes differentiation by modulating β-catenin/T-cell factor (TCF) 4-mediated gene transcription. The effect of dietary vitamin D on colonic Wnt signaling was investigated in mice fed either with 100IU or 2500IU vitamin D/kg diet. We examined the effect of 1,25-D3 on differentiation by measuring alkaline phosphatase activity. We analyzed mRNA expression of Wnt target genes by real time qRT-PCR. The impact of 1,25-D3 on β-catenin and TCF4 protein expression was assessed by western blot and immunohistochemistry. In LT97 cells, 1,25-D3 increased cellular differentiation and reduced nuclear β-catenin levels. Further, 1,25-D3 decreased mRNA expression of the Wnt target genes BCL-2, Cyclin D1, Snail1, CD44 and LGR5. In healthy colon of mice fed with high vitamin D diet, the mRNA levels of Wnt5a and ROR2, that promote degradation of β-catenin, were upregulated whereas β-catenin and TCF4 protein expression were decreased. In conclusion, 1,25-D3 inhibits Wnt signaling even in nonmalignant cells underlining its importance in protection against colorectal tumorigenesis and early tumor progression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

    View details for DOI 10.1016/j.jsbmb.2015.02.011

    View details for PubMedID 25777538

  • Active vitamin D potentiates the anti-neoplastic effects of calcium in the colon: A cross talk through the calcium-sensing receptor. The Journal of steroid biochemistry and molecular biology 2015


    Epidemiological studies suggest an inverse correlation between dietary calcium (Ca(2+)) and vitamin D intake and the risk of colorectal cancer (CRC). It has been shown in vitro that the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3) can upregulate expression of the calcium-sensing receptor (CaSR). In the colon, CaSR has been suggested to regulate proliferation of colonocytes. However, during tumorigenesis colonic CaSR expression is downregulated and we hypothesized that the loss of CaSR could influence the anti-tumorigenic effects of Ca(2+) and vitamin D. Our aim was to assess the impact of CaSR expression and function on the anti-neoplastic effects of 1,25-D3 in colon cancer cell lines. We demonstrated that in the healthy colon of mice, high vitamin D diet (2500IU/kg diet) increased expression of differentiation and apoptosis markers, decreased expression of proliferation markers and significantly upregulated CaSR mRNA expression, compared with low vitamin D diet (100IU/kg diet). To determine the role of CaSR in this process, we transfected Caco2-15 and HT29 CRC cells with wild type CaSR (CaSR-WT) or a dominant negative CaSR mutant (CaSR-DN) and treated them with 1,25-D3 alone, or in combination with CaSR activators (Ca(2+) and NPS R-568). 1,25-D3 enhanced the anti-proliferative effects of Ca(2+) and induced differentiation and apoptosis only in cells with a functional CaSR, which were further enhanced in the presence of NPS R-568, a positive allosteric modulator of CaSR. The mutant CaSR inhibited the anti-tumorigenic effects of 1,25-D3 suggesting that the anti-neoplastic effects of 1,25-D3 are, at least in part, mediated by the CaSR. Taken together, our data provides molecular evidence to support the epidemiological observation that both, vitamin D and calcium are needed for protection against malignant transformation of the colon and that their effect is modulated by the presence of a functional CaSR. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

    View details for DOI 10.1016/j.jsbmb.2015.02.006

    View details for PubMedID 25758239

  • Calcium-sensing receptor silencing in colorectal cancer is associated with promoter hypermethylation and loss of acetylation on histone 3. International journal of cancer. Journal international du cancer Fetahu, I. S., Höbaus, J., Aggarwal, A., Hummel, D. M., Tennakoon, S., Mesteri, I., Baumgartner-Parzer, S., Kállay, E. 2014; 135 (9): 2014-2023


    The calcium-sensing receptor (CaSR) is suggested to mediate the antiproliferative effects of calcium in colon. However, in colorectal cancer (CRC) the expression of the CaSR is silenced and the underlying mechanisms leading to its loss are poorly understood. We investigated whether loss of the CaSR expression in colorectal tumors is caused by DNA hypermethylation and imbalance of transcriptionally permissive/repressive histone alterations. We observed significantly lower CaSR mRNA expression (n = 65, p < 0.001) in colorectal tumors compared with the adjacent mucosa from the same patient. Immunofluorescence staining confirmed downregulation of the CaSR protein also. The CaSR promoter was methylated to a greater extent in tumors compared with adjacent mucosa as determined by bisulfite sequencing (n = 20, p < 0.01) and by pyrosequencing (n = 45, p < 0.001), and methylation correlated inversely with mRNA expression (n = 20, ρ = -0.310, p < 0.05 and n = 45, ρ = -0.588, p < 0.001). Treatments with 5-aza-2'-deoxycytidine (DAC), a DNA methyltransferase inhibitor and/or with two different histone deacetylase inhibitors, trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) restored the expression of CaSR in colon cancer cells. Restored CaSR expression in Coga1A and HT29 cells was functional. Inhibition of lysine-specific demethylase 1 (LSD1) to prevent demethylation of mono- and dimethylated H3K4, increased CaSR expression only marginally. Our data show that hypermethylation of the CaSR promoter and H3K9 deacetylation, but not H3K4me2 demethylation are important factors that cause silencing of the CaSR in colorectal cancer.

    View details for DOI 10.1002/ijc.28856

    View details for PubMedID 24691920

  • The vitamin D system is deregulated in pancreatic diseases JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY Hummel, D., Aggarwal, A., Borka, K., Bajna, E., Kallay, E., Horvath, H. C. 2014; 144: 402-409


    The vitamin D system is deregulated during development and progression of several cancer types. Data on the expression of the vitamin D system in the diseased pancreas are missing. The aim of this study was to investigate the expression of the vitamin D receptor (VDR), 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1), and the calcium-sensing receptor (CaSR), a vitamin D target gene, in the different regions of the pancreas in patients with chronic pancreatitis (n=6) and pancreatic ductal adenocarcinomas (PDAC) (n=17). We analyzed the expression of these genes at mRNA and protein level with quantitative real-time RT-PCR and immunostaining. mRNA expression of CYP24A1 and VDR was significantly increased in tumors compared with the adjacent non-tumorous tissue (p<0.01), while CaSR mRNA expression decreased. Both the VDR and the CaSR protein were highly expressed in the endocrine compared with the exocrine pancreas. In CP the CYP24A1 expression was highest in the endocrine pancreas, while in PDACs in the transformed ducts. In the PDAC patients CYP24A1 expression in the islets was significantly lower than in CP patients. Our data suggest that during ductal adenocarcinoma development the vitamin D system in the pancreas becomes deregulated on two levels: in the islets CYP24A1 expression decreases weakening the negative feedback regulation of the vitamin D-dependent insulin synthesis/secretion. In the transformed ducts CYP24A1 expression increases, impairing the antiproliferative effect of vitamin D in these cells.

    View details for DOI 10.1016/j.jsbmb.2014.07.011

    View details for Web of Science ID 000345183000017

    View details for PubMedID 25090635

  • Regulation of the calcium-sensing receptor expression by 1,25-dihydroxyvitamin D3, interleukin-6, and tumor necrosis factor alpha in colon cancer cells. journal of steroid biochemistry and molecular biology Fetahu, I. S., Hummel, D. M., Manhardt, T., Aggarwal, A., Baumgartner-Parzer, S., Kállay, E. 2014; 144: 228-231


    Anti-proliferative effects of calcium in the colon are mediated, at least in part, via the calcium-sensing receptor (CaSR), a vitamin D target gene. The expression of CaSR decreases during colorectal tumor progression and the mechanisms regulating its expression are poorly understood. The CaSR promoter harbors vitamin D elements responsive to 1,25-dihydroxyvitamin D3 (1,25D3) and NF-κB, STAT, and SP1 binding sites accounting for responsiveness to proinflammatory cytokines. Therefore, in the current study we investigated the impact of 1,25D3, tumor necrosis factor alpha (TNFα), and interleukin (IL)-6 on CaSR expression in a differentiated (Caco2/AQ) and in a moderately differentiated (Coga1A) colon cancer cell line. 1,25D3 induced CaSR expression in both cell lines. Treatment with TNFα was accompanied by a 134-fold induction of CaSR in Coga1A (p<0.01). In Caco2/AQ cells the expression of CaSR was upregulated also by IL-6 (3.5-fold). Our data demonstrated transcriptional and translational activation of the CaSR by 1,25D3, TNFα, and IL-6 in a time- and cell line-dependent manner. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

    View details for DOI 10.1016/j.jsbmb.2013.10.015

    View details for PubMedID 24176760

  • Increased copy-number and not DNA hypomethylation causes overexpression of the candidate proto-oncogene CYP24A1 in colorectal cancer. International journal of cancer. Journal international du cancer Höbaus, J., Hummel, D. M., Thiem, U., Fetahu, I. S., Aggarwal, A., Müllauer, L., Heller, G., Egger, G., Mesteri, I., Baumgartner-Parzer, S., Kallay, E. 2013; 133 (6): 1380-1388


    In colorectal cancer (CRC) the vitamin D catabolizing enzyme 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) is overexpressed with a potentially significant, positive impact on the catabolism of 1,25-dihydroxyvitamin D3 (1,25-D3 ). However, the underlying mechanism of CYP24A1 overexpression is poorly understood. In the present study, we investigated possible causes including hypomethylation of the CYP24A1 promoter, amplification of the CYP24A1 gene locus (20q13.2), and altered expression of CYP24A1-specific transcription factors. We quantified CYP24A1 gene copy-number, performed bisulfite sequencing of the CYP24A1 promoter to assess DNA methylation, and measured mRNA expression of CYP24A1, 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1), vitamin D receptor (VDR) and retinoid X receptor (RXR). We found that 77 (60%) out of 127 colorectal tumors showed increased CYP24A1 gene copy-number and that more than 6 copies of CYP24A1 correlated positively with CYP24A1 mRNA expression suggestive of a causal relationship. No differences in CYP24A1 promoter methylation were found between tumor tissue and adjacent mucosa from the same patient or between tissues with high or low mRNA expression, thus excluding DNA hypomethylation as a possible cause of CYP24A1 overexpression in CRC. Furthermore, mRNA expression of several factors involved in replication licensing positively correlated with CYP24A1 mRNA expression, raising the possibility that CYP24A1 overexpression might favor increased proliferation in tumors by suppressing local 1,25-D3 levels. We conclude that high copy-number gain is a key determinant of CYP24A1 overexpression in CRC. Other postulated causes of CYP24A1 overexpression including promoter hypomethylation and enhanced VDR and/or RXR expression do not appear to be involved.

    View details for DOI 10.1002/ijc.28143

    View details for PubMedID 23463632

  • Calcium sensing receptor signalling in physiology and cancer BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH Brennan, S. C., Thiem, U., Roth, S., Aggarwal, A., Fetahu, I. S., Tennakoon, S., Gomes, A. R., Brandi, M. L., Bruggeman, F., Mentaverri, R., Riccardi, D., Kallay, E. 2013; 1833 (7): 1732-1744


    The calcium sensing receptor (CaSR) is a class C G-protein-coupled receptor that is crucial for the feedback regulation of extracellular free ionised calcium homeostasis. While extracellular calcium (Ca(2+)o) is considered the primary physiological ligand, the CaSR is activated physiologically by a plethora of molecules including polyamines and l-amino acids. Activation of the CaSR by different ligands has the ability to stabilise unique conformations of the receptor, which may lead to preferential coupling of different G proteins; a phenomenon termed 'ligand-biased signalling'. While mutations of the CaSR are currently not linked with any malignancies, altered CaSR expression and function are associated with cancer progression. Interestingly, the CaSR appears to act both as a tumour suppressor and an oncogene, depending on the pathophysiology involved. Reduced expression of the CaSR occurs in both parathyroid and colon cancers, leading to loss of the growth suppressing effect of high Ca(2+)o. On the other hand, activation of the CaSR might facilitate metastasis to bone in breast and prostate cancer. A deeper understanding of the mechanisms driving CaSR signalling in different tissues, aided by a systems biology approach, will be instrumental in developing novel drugs that target the CaSR or its ligands in cancer. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

    View details for DOI 10.1016/j.bbamcr.2012.12.011

    View details for Web of Science ID 000320496700019

    View details for PubMedID 23267858