Building the case for the calcitonin receptor as a viable target for the treatment of glioblastoma

Abstract

Researchers are actively seeking novel targeted therapies for the brain tumour glioblastoma (GBM) as the mean survival is less than 15 months. Here we discuss the proposal that the calcitonin receptor (CT Receptor), expressed in 76–86% of patient biopsies, is expressed by both malignant glioma cells and putative glioma stem cells (GSCs), and therefore represents a potential therapeutic target. Forty-two per cent (42%) of high-grade glioma (HGG; representative of GSCs) cell lines express CT Receptor protein. CT Receptors are widely expressed throughout the life cycle of organisms and in some instances promote apoptosis. Which of the common isoforms of the CT Receptor are predominantly expressed is currently unknown, but a functional response to cell stress of the insert-positive isoform is hypothesised. A model for resistant malignancies is one in which chemotherapy plays a direct role in activating quiescent stem cells for replacement of the tumour tissue hierarchy. The putative role that the CT Receptor plays in maintenance of quiescent cancer stem cells is discussed in view of the activation of the Notch–CT Receptor–collagen V axis in quiescent muscle (satellite) stem cells. The pharmacological CT response profiles of four of the HGG cell lines were reported. Both CT responders and non-responders were sensitive to an immunotoxin based on an anti-CT Receptor antibody. The CALCR mRNA exhibits alternative splicing commonly associated with cancer cells, which could result in the atypical pharmacology exhibited by CT non-responders and an explanation of tumour suppression. Due to the inherent instability of CALCR mRNA, analysis of CT Receptor protein in patient samples will lead to improved data for the expression of CT Receptor in GBM and other cancers, and an understanding of the role and activity of the splice variants. This knowledge will aid the effective targeting of this receptor for treatment of GBM.

Keywords

antibody brain tumour calcitonin receptor CTR CT Receptor glioblastoma glioma stem cells G protein-coupled receptor

Introduction

Glioblastoma (multiforme, GBM) is the most common malignant primary brain tumour in adults. Aggressive surgical resection decreases the tumour-cell burden by 99%, leaving about 100 million cells and with cytotoxic adjuvant therapy with an agent such as temozolomide the burden is reduced to about 10 million cells, which would include cancer stem cells.¹ The mean overall survival achieved with wide-local surgical resection, followed by adjuvant therapy of radiation and temozolomide (TMZ) plus maintenance therapy with TMZ, remains only 14.6 months.²

Glioblastoma (GBM) are highly heterogeneous tumours thought to be derived from oligodendrocyte-type 2-astrocyte (O-2A) progenitors of the astroglial lineage⁴ as malignant glioma cells express glial fibrillary acidic protein (GFAP) and respond to similar mitogens and differentiation factors.⁵

The tumour architecture (illustrated in Figure 1) includes regions of necrosis and oedema (where the blood–brain barrier is compromised), pro-inflammatory micro-environments with cellular infiltrates and proliferative domains. Vascular-like structures referred to as vascular mimicry have been described.¹ Within these latter hyperplastic structures pericyte precursors^1,6 and endothelial cells^7–9 share the same genetic modifications as glioma stem-like cells (GSCs), supporting plasticity with regard to differentiation lineages and the existence of dominant GSC clones.^3,10–13 It is noteworthy that pericyte precursors are multipotent,¹⁴ are evident in the GBM vasculature in the close vicinity to the zones of proliferation, and a percentage express GFAP as noted in a stroke model.¹⁵ This raises the possibility that re-emerged GBM post-treatment might be derived from the pericyte lineage.

Figure 1.

(A) Illustration of the major anatomical features of glioblastomas (GBM); (B) Haematoxylin and eosin stain of a tissue section of GBM with an example of vascular mimicry (central vessel) and oedema; and (C) A serial section showing staining of malignant glioma cells³ with the anti-human CT Receptor antibody mAb31/01-1H10. Nuclei are stained blue with haematoxylin.

Comprehensive studies have detailed the gene expression profiles of GBM tissue samples^10–13 and this is supplemented by an anatomical transcriptional atlas.¹⁶

The current inability to improve or predict patient outcomes based on genetic profiling or histopathological features points to a deficit in our understanding of the driving forces for tumorogenesis of GBM and hence targets for tumour reduction or potential therapy.

GSCs play a role as precursor cells for GBM.¹⁷ They display inherent functional diversity,^1,3 convey relative resistance to conventional treatments such as chemo and radiotherapy,¹⁸ and provide invasive potential.^19–21 GSCs may also contribute to tumour survival and expansion in a number of hostile (hypoxic, inflammatory) micro-environments. As described for normal stem cell populations, GSCs are likely to be associated with dense vascular beds,²² which are generally located towards the periphery of GBM, and GSCs are believed to be present within the surrounding neuropil perhaps in a quiescent state. GSCs, also known as brain tumour-propagating cells¹⁹ in contrast to brain tumour-initiating cells,^20,23 can be perpetuated and expanded serum free²⁴ in vitro as high grade glioma (HGG) cell lines.^25–27 When formed as xenografts in the brains of immune-compromised mice, HGG cell lines form orthotopic, intracranial (ic) tumours that retain their invasive potential and recapitulate much of the pathology of the original tumour.^{17,19,20,23,24,27}

Some evidence has been presented to support the idea that some GSC populations are normally quiescent (G₀ phase of the cell cycle) and re-enter G₁ phase for self-renewal, but during quiescence are resistant to radiotherapy and conventional chemotherapy, designed to target proliferating cells. Furthermore, evidence suggests that dying cells targeted by chemotherapy release mitogens that stimulate quiescent stem cells to repopulate the tumour in between cycles of chemotherapy.^3,18 Thus, the biology of quiescent stem cells represents further complexity when considering the evaluation of new drugs. GSCs are currently recognised as potential targets for therapy, in which case specific molecular targets on quiescent GSCs should be validated and corresponding therapeutics developed. In support of the idea that quiescent stem cells are responsible for GBM relapse, in childhood lymphoblastic leukaemia, quiescent leukaemic (stem) cells appear to account for relapse-causing minimal residual disease (MRD).²⁸

In GBM, genetic profiles generated from patient biopsies with considerable cellular hierarchy might not provide enough definition to characterise targets on small subpopulations of quiescent GSCs which have the potential to become dominant clones following expansion after treatment cycles.

In a study that investigated CT Receptor protein expression in a small number of GBM patient biopsies, 86% showed expression that is restricted to glioma cells (example in Figure 2) and smaller cells bearing the GSC associated marker, CD-133.²⁹ The fact that the surrounding neurophil is negative for CT Receptor²⁹ in those regions of the brain in which GBM is common suggests CT Receptor is a GBM restricted biomarker. Another report described expression of CALCR mRNA found in 115/152 (76%) of primary tumours³⁰ as calculated from previous reported data.¹²

Figure 2.

An example of a Glioblastoma (GBM) tumour stained with an anti-human CT Receptor monoclonal antibody mAb30/07-9B4. Malignant glioma cells are stained brown and nuclei are stained blue with haematoxylin.²⁹

The widespread expression of the calcitonin receptor (CT Receptor)

CT Receptor isoforms are expressed in a wide range of tissues throughout the life cycle of mammals, under conditions such as cell stress, inflammation and wound healing, and in a range of diseases (Table 1).³¹ In spite of this wide expression, it is not normally expressed in the cerebrum/cortex where GBMs typically arise but is restricted to specific neuronal networks in the limbic system, and in the mid and hindbrain.

Table 1.

Updated and abbreviated list of tissues that express CTR from several mammalian species. A more comprehensive list with references is listed in Wookey et al.³¹

Life cycle entity, condition or disease	Tissue	References
Blastula		Wang et al.³²
Gastrula		Burgess³³
Foetus		Jagger et al.^34,35
Embryonic	Brain	Wookey et al.,³¹ Tolcos et al.³⁶
Perinatal	Gut (transient expression)	Wookey et al.³⁷
	Kidney	Tikellis et al.³⁸
Adult tissues	Brain	Fischer et al.,³⁹ Sexton et al.,⁴⁰ Becskei et al.,⁴¹ Paxinos et al.,⁴² Goda et al.,⁴³ Walker et al.⁴⁴
	Bone (osteoclasts, osteocytes)	Marx et al.,⁴⁵ Nicholson et al.,⁴⁶ Hattersley and Chalmers⁴⁷, Zaidi et al.,⁴⁸ Gooi et al.⁴⁹
	Kidney	Marx et al.,⁴⁵ Sexton et al.⁵⁰
	Thyroid	Hanna et al.⁵¹
	Satellite stem cells of muscle	Fukada et al.,⁵² Gnocchi et al.,⁵³ Yamaguchi et al.,⁵⁴ Baghdadi and Tajbakhsh⁵⁵
	Spermatozoa	Silvestroni et al.,⁵⁶ Adeoya-Osiguwa et al.⁵⁷
	Placenta	Nicholson et al.,⁵⁸ Kovacs et al.⁵⁹
	Prostate	Wu et al.⁶⁰
Conditions	Cell stress	Furness et al.⁶¹
	Inflammatory cytokines	Meeuwsen et al.⁶²
	Wound healing	Wookey et al.³¹
Cardiovascular disease	Blood vessels	Wookey et al.^63,64
Cancers	Glioblastoma	Wookey et al.,²⁹ Gilabert-Oriol et al.,⁶⁵ Pal et al.⁶⁶
	Prostate	Ritchie et al.,⁶⁷ Thomas et al.⁶⁸
	Medullary thyroid	Frendo et al.,^69,70 Cappagli et al.⁷¹
	Thyroid carcinomas	Boot et al.⁷²
	Bone (osteoclastoma, giant cell)	Nicholson et al.,⁷³ Gorn et al.⁷⁴
	Multiple myeloma	Silvestris et al.⁷⁵
	Leukaemia (ALL, AML), K562	Wookey et al.,³¹ Mould and Pondel⁷⁶
	Ovarian cell line BIN-67	Gorn et al.⁷⁷
	Breast	Findlay et al.,⁷⁸ Gillespie et al.⁷⁹

ALL, Acute Lymphoblastic Leukaemia; AML, Acute Myeloid Leukaemia.

There are two common isoforms, CT Receptor_a, insert-negative and CT Receptor_b insert-positive.⁸⁰ The insert positive form was originally isolated from a breast cancer cell line and has an additional 16-amino acid sequence within the first intracellular loop. The insert negative form has the more extensively characterised pharmacology and appears to be the relevant isoform for the well-defined physiology of calcitonin signaling and calcium metabolism.

Data from transfected COS (monkey kidney) cell lines that express either of the two human isoforms show CT Receptor_a is located predominantly in the plasma membrane (Figure 3) whereas CT Receptor_b is largely intracellular, located in the perinuclear domain presumably in small membranous elements and has a lower molecular weight suggesting an unglycosylated form that has not been processed for normal cell-surface expression.⁸¹ In transfected cell lines flow cytometry experiments confirm that lesser amounts of CT Receptor_b are found on the plasma membrane, but the relative distribution is cell line dependent.⁸²

Figure 3.

COS-7 stable transfectants expressing CT Receptor_a and CT Receptor_b⁸¹ stained with primary anti-human CT Receptor antibody mAb31/01-1H10, secondary goat anti-mouse IgG2a:AF568 and imaged using confocal microscopy (Zeiss LSM 800). Nuclei are stained blue with DAPI (4’,6 diamidino-2-phenylindole).

The CT Receptor_b isoform is expressed more widely than previously thought across the mammalian order. In the majority of mammalian species examined so far (exception Muroidae), the position of insertion is identical at the beginning of the second transmembrane span; however, the insertion varies in amino acid sequence and length (16–18), and would be predicted to interfere with the interaction of the receptor with its primary transducers. The recent description of the widespread expression of insert-positive isoforms throughout the mammalian species does, however, suggest a specific function of CT Receptor_b in cell physiology.⁸¹

There are no conclusive data yet that demonstrates in which normal and diseased tissues CT Receptor_b is more highly expressed and what its function might be. However, there is some evidence that this isoform is more highly expressed in ovary and placenta⁸⁰ and CT Receptor_b mRNA predominates in a group of samples of mononuclear blood cells.⁸³ The recent validation of a mouse monoclonal anti-human CT Receptor_b antibody⁸¹ will aid in the resolution of questions about expression in normal tissues, during inflammation and diseases. Unpublished data from our group on the expression of CT Receptor_b upregulated in cell stress (see also Adeoya-Osiguwa and Fraser)⁵⁷ are consistent with a cytosolic function.

Pharmacology of CT Receptor_a and CT Receptor_b

The pharmacology of these isoforms is quite different in terms of the second messenger outputs which, for CT Receptor_a, includes adenylyl cyclase activation, phosphorylation of ERK1/2 and p38 MAP kinase, as well as mobilisation of intracellular calcium (summarised in Figure 4 below). This might be expected given the location of the insert in relation to the binding of CT Receptor to its primary transducer.⁸⁴ In heterologous systems (COS-7 and HEK 293), second messenger coupling of CT Receptor_b is substantially reduced: the potency for adenylate cyclase activation is more than 100-fold weaker, ligand stimulated phosphorylation of ERK^1/2 is reduced in its maximum with some reduction in potency and CT Receptor-dependent mobilisation of intracellular calcium is undetectable.⁸² Such outputs are also cell type dependent^82,85 and mediated by different G protein complexes. However, it was noted that stimulation with salmon CT of HEK-293 transfectants (both CT Receptor isoforms compared to vector control) resulted in acidification of the media⁸⁶ suggesting possible metabolic outputs or involvement in efflux mechanisms from acidified intracellular compartments.

Figure 4.

Typical signaling pathways from CT Receptor_a and CT Receptor_b.

The CT Receptor ligand promoted conformation of Receptor bound G protein complexes influences their residency on the plasma membrane and contributes to ligand-mediated biased agonism of CT Receptor_a.⁸⁷ However, little is known about these mechanisms in the context of insert-positive CT Receptor_b and how the peptide insert might perturb residency, and thus coupling of G protein complexes.

CT Receptor protein was detected in the 5/12 (42%) of HGG cell lines (isolated from human biopsies) tested,²⁷ including JK2 (mesenchymal), PB1 (proneural/classic), SB2b (mesenchymal/classic) and WK1 (classic), using immunoblotting with the monoclonal antibody mAb31/01-1H10³⁰ which binds an intracellular epitope. In contrast to the major, broad CT Receptor band of 70–80 kD from COS-7/CT Receptor_a transfectants,⁸¹ the upper band is tight, with an apparent molecular weight (MW) of approximately 57 kD, consistent with an unglycosylated form. Unglycosylated CT Receptor is largely confined to the cytosolic domain and is characteristic of the CT Receptor_b isoform⁸¹ (Figure 3 above). However, as discussed above a small proportion of CT Receptor_b is located on the cell surface, which is cell line dependent,⁸² and in the case of HGG cell lines JK2, SB2b and WK1, CT Receptor is found in the membrane fraction determined from immunoblot.³⁰ In PB1 there is much less CT Receptor protein found in the membrane fraction. Rapid turnover of unglycosylated human CT Receptor_a protein located in intracellular compartments has been described.⁸⁸ Further studies including nanopore sequencing to identify splicing events will be important to improve our understanding of the biology and functional consequences of these observations.

The pharmacology of CT Receptor was studied in these HGG lines³⁰ and only SB2b had functional CT Receptor as determined by responses to classic ligands which stimulated adenylyl cyclase, calcium mobilisation and ERK^1/2 phosphorylation. Pharmacological studies are discussed in more detail below.

Hypothesis on the mechanism of the CT Receptor_b isoform

This hypothesis is built on several separate ideas. Firstly, intracellular CT Receptors are folded in the membrane of intracellular structures attached to the cytoskeleton.⁸⁹ Secondly, unglycosylated CT Receptor_b is confined to the cytosol (Figure 3 above) and is concentrated in a structure thought to be the microtubule organising centre (MTOC). In experiments similar to those described by Safaei et al.⁹⁰ we found cytotoxin concentrated in the exosomes harvested from MG63 cells treated with staurosporine and these exosomes were also positive for CT Receptor (unpublished results). As these exosomes originate from lysosomes (pH 3–4) their release is likely to contribute to acidification of the media as described previously⁸⁶ for CT Receptor_b.

The upregulation of CALCR mRNA in response to cytokines TNFα and IL1β by primary cultures of human astrocytes has been described,⁶² demonstrating a response to cell stress.⁶¹ Furthermore, in U87 MG cells treated with staurosporine, nanopore sequencing of long cDNA products from CT Receptor mRNA has established alternative splicing events that include exon 10 such that the CT Receptor_b isoform (insert-positive) is significantly upregulated,⁸¹ although total CALCR mRNA remains unchanged.

Taken together there is evidence for the upregulation of CT Receptor_b in the cytosol of stressed cells and the production of CT Receptor-positive exosomes laden with cytotoxins, which as hypothesised, amounts to new important mechanism together with a number of other cellular stress responses.

In the context of the expression of CT Receptor by malignant glioma cells and GSCs, targeting CT Receptor might provide an opportunity to overcome resistance to chemotherapeutics and treat the pool of GSCs thought to be responsible for relapse.

Calcitonin/CT Receptor, cell survival/apoptosis and the cell cycle

There are several reports that describe data showing CT or CT Receptor promote proliferation/survival or apoptosis resulting in decreased survival in model cancer cell lines. The effect of CT Receptor expression and calcitonin-dependent CT Receptor activation differs according to the cell line under investigation.

CT stimulates proliferation early in treatment protocol of T47D cells (derived from human breast cancer) and then later inhibits proliferation in the log phase.⁹¹ In serum deprived LLC-PK cells, derived from porcine kidney, CT reduced cell survival⁹² perhaps by induction of apoptosis.

In a study of serum-starved transfected HEK-293 cells induced to express either human hCT Receptor_a compared to hCT Receptor_b or vector alone, treatment of these lines with salmon CT resulted in decreased proliferation, in the accumulation of cells in G₂ phase of the cell cycle in the hCT Receptor_a transfectant but not the hCT Receptor_b transfectant.⁸⁶ In the hCT Receptor_a transfectant, ERK^1/2 activation mediates modulation of the cell cycle via p21^Cip1. However, G₂-arrest was unexpected as p21^Cip has been shown to inhibit CDK2 rather than CDK1 (cdc 2)⁹³ and results in a cycle block at G1.^94,95 This anomaly has never been resolved as far as we are aware and raises questions about the validity of CT Receptor transfectants to probe the real biological functions of CT Receptor.

In a subsequent report CT induced apoptosis in an hCT Receptor_a transfectant under conditions of low serum which were not observed with replete serum.⁹⁶ While one explanation might be a factor present in serum that overcomes the CT effect, another possibility is metabolic reprogramming driven by starvation and cell stress associated with autophagy which is independent of caspase 3.⁹⁶ In this regard, in a p53^–/– mouse model with thymic lymphoma, CT Receptor is essential for the transmission of the effects of amylin for metabolic reprogramming, induction of apoptosis and tumour regression.⁹⁷

SiRNA mediated knockdown of CT Receptor in TT2609-C02 cells derived from follicular thyroid carcinoma, resulted in G₁ arrest, a decrease in proliferation and an increase in caspase 3 activity.⁷² The authors conclude that CT Receptor is one of several genes important for survival of thyroid carcinomas and therefore can be classified as a putative oncogene.

In human prostate cancer cell lines and mouse models, CT and CT Receptor activate survival of cells following cytotoxic insult⁶⁸ and enhanced tumour growth⁹⁸ consistent with the induction of apoptosis following knockdown of CT Receptor.⁹⁹

Consistent with these results are further reports that CT prevents apoptosis in osteocytes and osteoblasts¹⁰⁰ and promotes survival of osteoclasts.¹⁰¹

Overall in the context of different cell lines maintained in normal serum (unstarved), CT/CT Receptor promotes cell survival and proliferation, while promotion of apoptosis is associated with P53 status, metabolic reprogramming and nutrient deprivation.

Stem cell quiescence and a role for a surrogate ligand of CT Receptor

In skeletal muscle stem (satellite) cells maintenance of quiescence is dependent on activation of CT Receptor⁵⁴ and it is now proposed that an active cell autonomous Notch–Collagen V (COL V)–CT Receptor axis maintains the quiescent muscle stem cells in their niche.⁵⁵ Notch also contributes to the stem-like character of glioma cells.¹⁰²

The mechanism of how COL V acts as a surrogate ligand of CT Receptor is yet to be described. We have modelled [unpublished] the carboxy cleaved peptide of collagen V and found similarities when arranged as a 3₁₀ helix with salmon CT. In this case the carboxyl tail of nascent COL V might act as a surrogate ligand for CT Receptor after it is cleaved. However, the researchers⁵⁵ used a commercial collagen V isolated from human placenta and it is unclear whether this is pure mature collagen V or contains pre-pro-collagen V and processed peptides.⁵⁵ If this work can be corroborated this discovery will have a profound effect on our understanding of CT Receptor as expressed in the context of many tissues. As presented in Table 1, there is a wide expression of CT Receptor throughout the life cycle in many tissues, sometimes expression is transient and sometimes persistent, and expression is found in cells that play a prominent role in a range of diseases.

For instance, this axis might well be a driving factor in atherosclerosis in which we have identified expression of CT Receptor^{63,64,103,104} and expression of collagen V is also known.^103,104

Formation of granulation tissue is a histological hallmark of wound healing following tissue injury.¹⁰⁵ COLV immunostaining predominates in the blood vessel walls of the granulation tissue and COLV transcript is expressed in the fibroblast-like cells of the granulation tissue.^105,106 Similar cells and blood vessels express CT Receptor in a mouse model of wound healing.³¹

Genotyped profiles of GBM

Extensive genetic profiles of GBM patient biopsies have been published,^10–12 including regions of chromosomal aberrations both broad and focal¹⁰ together with the somatic genomic landscape.¹²

Gliomas with broad amplification of chromosome 7 have properties different from those with overlapping focal EGFR amplification (chr7p11) and the broad events around chr7q32 (CALCR gene location chr 7q21.3) which appear to act in part through effects on MET (CXCR4) and its ligand HGF and correlate with MET dependence in vitro.¹⁰ It should be noted that CALCR maps close to MET.

Alternative splicing has also been recognised as a driving force of tumorogenesis.¹⁰⁷ Aberrant profiles observed in tumours mostly reflect the selection of endogenous alternative splicing variants with different functional properties that allow the malignant progression of initiated tumour cells. Selected functions relate, for example, to sustained proliferation, evasion of apoptosis, metabolic adaptation, or angiogenesis.¹⁰⁸ Results from our group investigating the U87 MG cell line have demonstrated increased frequency of exon 10 (insert-positive sequence) splicing-in when these cells are stressed with cytotoxin treatment.⁸¹ The possibility of other splicing events that might result in receptor inactivation are currently under investigation.

In a study on oncocytic thyroid carcinomas⁷² hemi-methylation in the region of the 5’UTR of CALCR was characterised, upregulation of CALCR mRNA was found in the majority of samples and in the cell line TT2609-C02 reduction in the expression of CTR leads to a pause in the cell cycle, upregulation of caspase 3 mRNA and enhanced cell death. Consistently, there was no loss of heterozygosity of chromosome 7 which was interpreted as a role for maternal and paternal genes imprinted on this chromosome including CALCR. These data support a role for CT Receptor as an oncogene.

Regulation of CALCR transcription and stability of mRNA

The structure of human CALCR gene has been described in BIN-67 cells (human ovarian carcinoma) cell line,⁷⁷ human osteoclasts and MCF-7 cells (human breast adenocarcinoma).¹⁰⁹ Promoters P1 and P2 were demonstrated in transfected T47D cells (human breast cancer)^110,111 and a further promoter (POc) is specifically active in human osteoclasts.¹¹² This paper also described the tissue-specific splicing of the CALCR 5’UTR and the transcripts that are generated. Analysis of the 5’UTR of human CALCR reveals multiple Sp1 binding sites¹¹³ and CpG islands. The regulation by Sp1 is of particular interest because many genes upregulated during stress are also regulated by this transcription factor which has been proposed to drive the adaptive response of cancer cells to hypoxia.¹¹⁴

The structure of the murine CALCR gene from brain has been determined¹¹⁵ and later CALCR mRNA shown to be transcribed from three promoters (P1, P2 and P3) of which P3 is osteoclast specific and the promoters become functional through alternative splicing of exons in the 5’UTR.¹¹⁶ A comparison of the exon structures in human and mouse CALCR has been published.¹¹² The structure of porcine CALCR gene has been reported from LLC-PK1 cells.¹¹⁷

In the 3’UTR of human CALCR mRNA there are eight AUUUA sequences and five in porcine CALCR.¹¹⁸ These sequences have been demonstrated to increase the instability of transcripts.^119,120 CALCR mRNA levels are generally low in tissues and HGG cell lines³⁰ perhaps reflecting the inherent instability of this mRNA due to multiple (AUUUA) sequences. This instability could explain the low representation of CALCR mRNA in some genetic studies of GBM patients.¹¹

Targeting inactivated CT Receptor in GBM

The pharmacology of responses to CT ligands was reported in these four HGG cell lines.³⁰ Only SB2b responded to CT-like ligands (human CT, salmon CT, rat amylin) indicating that CT Receptor is non-functional in the cell lines JK2, PB1 and WK1. It is likely that this inactivation results from alternative splicing common in cancers including the possibility that expression of CT Receptor_b predominates. A conclusion drawn from this study is that pharmacological intervention of CT Receptor is unlikely to provide an avenue for treatment of GBM.

Our group⁶⁵ has published a study with HGG cell lines which characterises the potency of an anti-hCT Receptor antibody conjugated to cytotoxins monomethyl-aurostatin E (MMAE) or the plant toxins diathin-30¹²¹ and gelonin (ribosome-inactivating proteins). The anti-hCT Receptor antibody deployed in the ADC or immunotoxin binds an extracellular epitope and is internalised. The potency of the immunotoxin (EC₅₀ 10–20 pM) is greatly increased with saponins (triterpene glycoside SO1861) which enhance the release of the toxin from the acidic lysosomes.⁶⁵

Three of the HGG cell lines (JK2, SB2b, WK1, all classic/mesenchymal)²⁷ were equally sensitive to the immunotoxin and expressed high levels of the CT Receptor protein in the membrane fraction as determined by immunoblot³⁰ using an anti-CT Receptor antibody that recognises an intracellular epitope. Although all four HGG cell lines displayed similar levels of CT Receptor using whole cell lysates,⁶⁵ one HGG cell line PB1 (proneural) expresses low levels of CT Receptor in the membrane fraction³⁰ and is relatively resistant to the immunotoxin.⁶⁵

These data suggest that a potential treatment of GBM stem-like cells is possible using immunotoxin directed against CT Receptor regardless of the pharmacological status of the receptor.

The proposed mechanism⁶⁵ of this therapy (refer to Figure 5) involves binding to the extracellular domain of CT Receptor on the plasma membrane, uptake of the immunotoxin via the endosomes into the lysosomes (with low pH) and cleavage of the immunotoxin (either protease or pH mediated depending on the linker). The escape of the toxin from the lysosomal compartment⁶⁵ is enhanced by the triterpene glycoside SO1861.¹²² The toxin targets the ribosomes.¹²¹

Figure 5.

Proposed mechanism of CT Receptor_b in the excretion of cell debris and cytotoxins via exosomes.

Whether the HGG cell lines express CT Receptor_a, CT Receptor_b or an alternatively spliced isoform is yet to be established. From our current analysis elevated CT Receptor expression in the membrane fraction is necessary for sensitivity to the immunotoxin as PB1 is deficient in this respect and is resistant to the immunotoxin.

Animal studies are planned to test the immunotoxins in vivo and further refinements of the prototype immunotoxin, to improve tumour penetration, are also in progress.

Evidence for CTR as a tumour suppressor and/or an oncogene

Tumour suppressor genes regulate a diverse range of cellular activities including cell cycle checkpoint responses and mitogenic signaling.¹²³ Classic tumour suppressors have three principal characteristics, firstly they are recessive and undergo biallelic inactivation in tumours, secondly inheritance of a mutant allele potentiates tumour initiation, and thirdly, the same gene is frequently inactivated in sporadic tumours.¹²³

CT Receptor has been shown to influence the cell cycle with induction of quiescence (G₀) for satellite stem cells and checkpoint G₁ for cell lines as discussed above. Two reports have identified inactivated CT Receptor expressed in GBM.^30,66 As CT Receptor is upregulated with cell stress⁶¹ and ligand activation results in apoptosis (discussed above) then inactivation of CT Receptor by mutation or alternative splicing (U87 MG, discussed above) could result in survival of tumour cells. It should be noted at this point that U87 MG might not have originated from GBM.¹²⁴ CT Receptor protein is expressed by HGG cell lines in which CT Receptor activation is either non-canonical or inhibited by mutation or deletion, or the CT Receptor_b isoform is preferentially expressed.

Pal et al.⁶⁶ describe the downregulated levels of CALCR transcripts in GBMs from data sets TCGA, GSE7696 and the Indian cohorts. However, as described above, low levels might be expected to result as the transcripts include eight repeat AUUUA sequences in the 3’UTR that are responsible for instability. The steady state levels are not an indication of the rates of transcription. They also found altered activity of mutated CT Receptor derived from a small cohort⁶⁶ of GBM patients and drew conclusions about survival compared to normal CT Receptor. While much of the data are not conclusive, the idea that CT Receptor might act as a tumour suppressor and/or oncogene warrants further exploration.

Evidence for a role as an oncogene includes knockdown of CT Receptor in tumour cell lines as outlined above, which results in apoptosis and suggests, in the case of mutant CT Receptor, a further function of ligand-insensitive CT Receptor for tumour survival as proposed in Figure 5. It remains to be demonstrated that knockdown of inactivated (mutant) CT Receptor leads to cell death in the case of GBM.

Conclusion

CT Receptor is a G protein-coupled receptor (GPCR) that is highly expressed in patient biopsies between 76% (CALCR mRNA) and 86% (CT Receptor protein). Furthermore, it is expressed by malignant glioma cells, in cells that express markers of brain tumour initiating cells and 42% of HGG cell lines that represent tumour stem cells.

Quiescent cancer stem cells represent minimal resistant disease and enter the cell cycle in response to treatment to re-establish tumour malignancy. We postulate that the maintenance of quiescence results from activation of the Notch–COL V-CT Receptor axis as has been proposed for skeletal muscle satellite (stem) cells.

There are likely to be several potential mechanisms for the upregulation of CT Receptor in GBM. Firstly, the region around chromosome 7q21.3 (CALCR gene) is frequently amplified. Secondly, the transcription factor Sp1 regulates genes important for stress responses in many tissues including GBM and Sp1 has been shown to stimulate CALCR mRNA transcription in cell lines.

The potential role of the CT Receptor_b isoform is discussed in a mechanism that provides resistance to cytotoxins and drug resistance in glioblastoma and possibly other cancers.

In HGG cell lines CT Receptor is frequently pharmacologically inactive although the protein is detected by immunoblotting. Inactivation might result either by inactivating mutations/deletions/insertions or alternative splicing resulting in inactivation or a switch to the CT Receptor_b isoform. The inactivation of CT Receptor would be consistent with its role as a tumour suppressor. Furthermore, knockdown of CT Receptor promotes apoptosis consistent with a role as an oncogene.

The antibody (mAb2C4) that binds the extracellular epitope of human CT Receptor has been developed as an immunotoxin to study its potency with HGG cell lines. The mAb2C4:dianthin immunotoxin has an effective EC₅₀ of 10–20pM as compared to an equivalent ADC (mAb2C4:MMAE) which is 250 times less potent.

Targeting CT Receptor expressed by quiescent cancer stem cells with the immunotoxin is expected to provide an additional weapon, in combination with traditional therapy that targets dividing malignant glioma cells, for the treatment of glioblastoma. This novel therapy aims to eradicate minimal residual disease associated with this cancer.

Footnotes

Acknowledgements

The authors would like to thank Apop Biosciences Pty Ltd for access to the mAb2C4 antibody (CalRexin).

Conflict of interest statement

PJW and DLH own shares in Apop Biosciences (formerly Apop Imaging) which owns intellectual property around the anti-CTR antibodies mAb2C4 and mAb9B4. The other authors declare no conflict of interest.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: SGBF is an ARC Future Fellow (FT180100543).

ORCID iD

Lucas Bittencourt

References

Das

Marsden

. Angiogenesis in glioblastoma. N Engl J Med 2013; 369: 1561–1563.

Stupp

Mason

van den Bent

, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005; 352: 987–996.

Kreso

O’Brien

van Galen

, et al. Variable clonal repopulation dynamics influence chemotherapy response in colorectal cancer. Science 2013; 339: 543–548.

Raff

Miller

Noble

. A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature 1983; 303: 390–396.

Noble

Gutowski

Bevan

, et al. From rodent glial precursor cell to human glial neoplasia in the oligodendrocyte-type-2 astrocyte lineage. Glia 1995; 15: 222–230.

Cheng

Huang

Zhou

, et al. Glioblastoma stem cells generate vascular pericytes to support vessel function and tumor growth. Cell 2013; 153: 139–152.

Wang

Chadalavada

Wilshire

, et al. Glioblastoma stem-like cells give rise to tumour endothelium. Nature 2010; 468: 829–833.

Ricci-Vitiani

Pallini

Biffoni

, et al. Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature 2010; 468: 824–828.

Soda

Marumoto

Friedmann-Morvinski

, et al. Transdifferentiation of glioblastoma cells into vascular endothelial cells. Proc Natl Acad Sci 2011; 108: 4274–4280.

10.

Beroukhim

Getz

Nghiemphu

, et al. Assessing the significance of chromosomal aberrations in cancer: methodology and application to glioma. Proc Natl Acad Sci 2007; 104: 20007–20012.

11.

Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 2008; 455: 1061–1068.

12.

Brennan

Verhaak

McKenna

, et al. The somatic genomic landscape of glioblastoma. Cell 2013; 155: 462–477.

13.

Lee

Wang

, et al. Spatiotemporal genomic architecture informs precision oncology in glioblastoma. Nat Genet 2017; 49: 594–599.

14.

Dore-Duffy

Mehedi

Wang

, et al. Immortalized CNS pericytes are quiescent smooth muscle actin-negative and pluripotent. Microvasc Res 2011; 82: 18–27.

15.

Sharma

Ling

Rewell

, et al. A novel population of alpha-smooth muscle actin-positive cells activated in a rat model of stroke: an analysis of the spatio-temporal distribution in response to ischemia. J Cereb Blood Flow Metab 2012; 32: 2055–2065.

16.

Puchalski

Shah

Miller

, et al. An anatomic transcriptional atlas of human glioblastoma. Science 2018; 360: 660–663.

17.

Singh

Clarke

Terasaki

, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res 2003; 63: 5821–5828.

18.

Chan

. Molecular pathways: targeting cancer stem cells awakened by chemotherapy to abrogate tumor repopulation. Clin Cancer Res 2016; 22: 802–806.

19.

Lathia

Heddleston

Venere

, et al. Deadly teamwork: neural cancer stem cells and the tumor microenvironment. Cell Stem Cell 2011; 8: 482–485.

20.

Singh

Hawkins

Clarke

, et al. Identification of human brain tumour initiating cells. Nature 2004; 432: 396–401.

21.

Rath

Fair

Jamal

, et al. Astrocytes enhance the invasion potential of glioblastoma stem-like cells. PLoS One 2013; 8: e54752.

22.

Calabrese

Poppleton

Kocak

, et al. A perivascular niche for brain tumor stem cells. Cancer Cell 2007; 11: 69–82.

23.

Galli

Binda

Orfanelli

, et al. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 2004; 64: 7011–7021.

24.

Lee

Kotliarova

Kotliarov

, et al. Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 2006; 9: 391–403.

25.

Verhaak

Hoadley

Purdom

, et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 2010; 17: 98–110.

26.

Day

Stringer

Al-Ejeh

, et al. EphA3 maintains tumorigenicity and is a therapeutic target in glioblastoma multiforme. Cancer Cell 2013; 23: 238–248.

27.

Stringer

Day

D’Souza

RCJ

, et al. A reference collection of patient-derived cell line and xenograft models of proneural, classical and mesenchymal glioblastoma. Scientific Rep 2019; 9: 4902.

28.

Lutz

Woll

Hall

, et al. Quiescent leukaemic cells account for minimal residual disease in childhood lymphoblastic leukaemia. Leukemia 2013; 27: 1204–1207.

29.

Wookey

McLean

Hwang

, et al. The expression of calcitonin receptor detected in malignant cells of the brain tumour glioblastoma multiforme and functional properties in the cell line A172. Histopathology 2012; 60: 895–910.

30.

Ostrovskaya

Hick

Hutchinson

, et al. Expression and activity of the calcitonin receptor family in a sample of primary human high-grade gliomas. BMC Cancer 2019; 19: 157–168.

31.

Wookey

Zulli

, et al. Calcitonin receptor (CTR) expression in embryonic, foetal and adult tissues: developmental and pathophysiological implications. In: Hay

Dickerson

(eds) The calcitonin gene-related peptide family; form, function and future perspectives. Netherlands: Springer, 2010, pp.199–233.

32.

Wang

Rout

Bagchi

, et al. Expression of calcitonin receptors in mouse preimplantation embryos and their function in the regulation of blastocyst differentiation by calcitonins. Development 1998; 125: 4293–4302.

33.

Burgess

. The effect of calcitonin on the prechordal mesoderm, neural plate and neural crest of Xenopus embryos. J Anat 1985; 140: 49–55.

34.

Jagger

Chambers

Pondel

. Transgenic mice reveal novel sites of calcitonin receptor gene expression during development. Biochem Biophys Res Commun 2000; 274: 124–129.

35.

Jagger

Gallagher

Chambers

, et al. The porcine calcitonin receptor promoter directs expression of a linked reporter gene in a tissue and developmental specific manner in transgenic mice. Endocrinology 1999; 140: 492–499.

36.

Tolcos

Tikellis

Rees

, et al. Ontogeny of calcitonin receptor mRNA and protein in the developing central nervous system of the rat. J Comp Neurol 2003; 456: 29–38.

37.

Wookey

Turner

Furness

. Transient expression of the calcitonin receptor by enteric neurons of the embryonic and early post-natal mouse. Cell Tis Res 2012; 347: 311–317.

38.

Tikellis

Xuereb

Casley

, et al. Calcitonin receptor isoforms expressed in the developing rat kidney. Kidney Int 2003; 63: 416–426.

39.

Fischer

Tobler

Kaufmann

, et al. Calcitonin: regional distribution of the hormone and its binding sites in the human brain and pituitary. Proc Natl Acad Sci 1981; 78: 7801–7805.

40.

Sexton

McKenzie

Mendelsohn

FAO

. Evidence for a new subclass of calcitonin/calcitonin gene-related peptide binding sites in rat brain. Neurochem Int 1988; 12: 323–335.

41.

Becskei

Riediger

Zund

, et al. Immunohistochemical mapping of calcitonin receptors in the adult rat brain. Brain Res 2004; 1030: 221–233.

42.

Paxinos

Chai

Christopoulos

, et al. In vitro autoradiographic localization of calcitonin and amylin binding sites in monkey brain. J Chem Neuroanat 2004; 27: 217–236.

43.

Goda

Doi

Umezaki

, et al. Calcitonin receptors are ancient modulators for rhythms of preferential temperature in insects and body temperature in mammals. Genes Dev 2018; 32: 140–155.

44.

Walker

Eftekhari

Bower

, et al. A second trigeminal CGRP receptor: function and expression of the AMY1 receptor. Ann Clin Transl Neurol 2015; 2: 595–608.

45.

Marx

Woodward

Aurbach

. Calcitonin receptors of kidney and bone. Science 1972; 178: 999–1001.

46.

Nicholson

Moseley

Sexton

, et al. Abundant calcitonin receptors in isolated rat osteoclasts. Biochemical and autoradiographic characterization. J Clin Invest 1986; 78: 355–360.

47.

Hattersley

Chalmers

. Calcitonin receptors as markers for osteoclast differentiation: correlation between generation of bone-resorptive cells and cells that express calcitonin receptors in mouse bone marrow cultures. Endocrinology 1989; 125: 1606–1612.

48.

Zaidi

Pazianas

Shankar

, et al. Osteoclast function and its control. Exp Physiol 1993; 78: 721–739.

49.

Gooi

Pompolo

Karsdal

, et al. Calcitonin impairs the anabolic effect of PTH in young rats and stimulates expression of sclerostin by osteocytes. Bone 2010; 46: 1486–1497.

50.

Sexton

Adam

Moseley

, et al. Localization and characterization of renal calcitonin receptors by in vitro autoradiography. Kidney Int 1987; 32: 862–868.

51.

Hanna

Smith

Johnston

, et al. Expression of a novel receptor for the calcitonin peptide family and a salmon calcitonin-like peptide in the alpha-thyrotropin thyrotroph cell line. Endocrinology 1995; 136: 2377–2382.

52.

Fukada

Uezumi

Ikemoto

, et al. Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells 2007; 25: 2448–2459.

53.

Gnocchi

White

Ono

, et al. Further characterisation of the molecular signature of quiescent and activated mouse muscle satellite cells. PLoS One 2009; 4: e5205.

54.

Yamaguchi

Watanabe

Ohtani

, et al. Calcitonin receptor signaling inhibits muscle stem cells from escaping the Quiescent State and the Niche. Cell Rep 2015; 13: 302–314.

55.

Baghdadi

Castel

Machado

, et al .Reciprocal signalling by Notch-Collagen V- CALCR retains muscle stem cells in their niche. Nature 2018; 557: 714–718.

56.

Silvestroni

Menditto

Frajese

, et al. Identification of calcitonin receptors in human spermatozoa. J Clin Endocrinol Metab 1987; 65: 742–746.

57.

Adeoya-Osiguwa

Fraser

. Calcitonin acts as a first messenger to regulate adenylyl cyclase/cAMP and mammalian sperm function. Mol Reprod Dev 2003; 65: 228–236.

58.

Nicholson

D’Santos

Evans

, et al. Human placental calcitonin receptors. Biochem J 1988; 250: 877–882.

59.

Kovacs

Chafe

Woodland

, et al. Calcitropic gene expression in the murine placenta suggests a role for the intraplacental yolk sac in maternal-fetal calcium exchange. Am J Physiol 2002; 282: E721–E732.

60.

Burzon

di Sant’Agnese

, et al. Calcitonin receptor mRNA expression in human prostate. Urology 1996; 47: 376–381.

61.

Furness

SGB

Hare

Kourakis

, et al. A novel ligand of calcitonin receptor reveals a potential new sensor that modulates programmed cell death. Cell Death Disc 2016; 2: 16062.

62.

Meeuwsen

Persoon-Deen

Bsibsi

, et al. Cytokine, chemokine and growth factor gene profiling of cultured human astrocytes after exposure to proinflammatory stimuli. Glia 2003; 43: 243–253.

63.

Wookey

Zulli

Buxton

, et al. Calcitonin receptor immunoreactivity associated with specific cell types in diseased radial and internal mammary arteries. Histopathology 2008; 52: 605–612.

64.

Wookey

Zulli

Hare

. The elevated expression of calcitonin receptor by cells recruited into the endothelial layer and neo-intima of atherosclerotic plaque. Histochem Cell Biol 2009; 132: 181–189.

65.

Gilabert-Oriol

Furness

SGB

Stringer

, et al. Dianthin-30 or gelonin versus monomethyl auristatin E, each configured with an anti-calcitonin receptor antibody, are differentially potent in vitro in high grade glioma cell lines derived from glioblastoma. Cancer Immunol Immunother 2017; 66: 1217–1228.

66.

Pal

Patil

Kumar

, et al. Loss-of-function mutations in Calcitonin Receptor (CALCR) identify highly aggressive glioblastoma with poor outcome. Clin Cancer Res 2018; 24: 1448–1458.

67.

Ritchie

Thomas

Andrews

, et al. Effects of the calciotrophic peptides calcitonin and parathyroid hormone on prostate cancer growth and chemotaxis. Prostate 1997; 30: 183–187.

68.

Thomas

Chigurupati

Anbalagan

, et al. Calcitonin increases tumorigenicity of prostate cancer cells: evidence for the role of protein kinase A and urokinase-type plasminogen receptor. Mol Endocrinol 2006; 20: 1894–1911.

69.

Frendo

Delage-Mourroux

Cohen

, et al. Calcitonin receptor mRNA is expressed in human medullary thyroid carcinoma. Thyroid 1998; 8: 141–147.

70.

Frendo

Pichaud

Mourroux

, et al. An isoform of the human calcitonin receptor is expressed in TT cells and in medullary carcinoma of the thyroid. FEBS Lett 1994; 342: 214–216.

71.

Cappagli

Potes

Ferreira

, et al. Calcitonin receptor expression in medullary thyroid carcinoma. PeerJ 2017; 5: e3778.

72.

Boot

Oosting

de Miranda

, et al. Imprinted survival genes preclude loss of heterozygosity of chromosome 7 in cancer cells. J Pathol 2016; 240: 72–83.

73.

Nicholson

Horton

Sexton

, et al. Calcitonin receptors of human osteoclastoma. Horm Metab Res 1987; 19: 585–589.

74.

Gorn

Rudolph

Flannery

, et al. Expression of two human skeletal calcitonin receptor isoforms cloned from a giant cell tumor of bone. The first intracellular domain modulates ligand binding and signal transduction. J Clin Invest 1995; 95: 2680–2691.

75.

Silvestris

Cafforio

De Matteo

, et al. Expression and function of the calcitonin receptor by myeloma cells in their osteoclast-like activity in vitro. Leuk Res 2008; 32: 611–623.

76.

Mould

Pondel

. Calcitonin receptor gene expression in K562 chronic myelogenous leukemic cells. Cancer Cell Int 2003; 3: 6–12.

77.

Gorn

Lin

Yamin

, et al. Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line. J Clin Invest 1992; 90: 1726–1735.

78.

Findlay

Michelangeli

Moseley

, et al. Calcitonin binding and degradation by two cultured human breast cancer cell lines (MCF 7 and T 47D). Biochem J 1981; 196: 513–520.

79.

Gillespie

Thomas

, et al. Calcitonin receptors, bone sialoprotein and osteopontin are expressed in primary breast cancers. Intern J Cancer 1997; 73: 812–815.

80.

Kuestner

Elrod

Grant

, et al. Cloning and characterization of an abundant subtype of the human calcitonin receptor. Mol Pharmacol 1994; 46: 246–255.

81.

Furness

SGB

Gupta

Zhong

, et al. Strategy to develop and validate antibodies against linear epitopes of human calcitonin receptor: identification of insert-positive isoforms and ubiquity across mammalian species suggests new physiological functions. Unpublished data.

82.

Dal Maso

Just

Hick

, et al. Characterization of signalling and regulation of common calcitonin receptor splice variants and polymorphisms. Biochem Pharmacol 2018; 148: 111–129.

83.

Beaudreuil

Balasubramanian

Chenais

, et al. Molecular characterization of two novel isoforms of the human calcitonin receptor. Gene 2004; 343: 143–151.

84.

Liang

Khoshouei

Radjainia

, et al. Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature 2017; 546: 118–123.

85.

Cohen

Thaw

Varma

, et al. Human calcitonin receptors exhibit agonist-independent (constitutive) signaling activity. Endocrinology 1997; 138: 1400–1405.

86.

Raggatt

Evdokiou

Findlay

. Sustained activation of Erk1/2 MAPK and cell growth suppression by the insert-negative, but not the insert-positive isoform of the human calcitonin receptor. J Endocrinol 2000; 167: 93–105.

87.

Furness

SGB

Liang

Nowell

, et al. Ligand-dependent modulation of G protein conformation alters drug efficacy. Cell 2016; 167: 739–749.e711.

88.

Dermer

Cohen

Thaw

, et al. Intracellular retention and rapid degradation of human calcitonin receptors overexpressed in COS cells. Endocrinology 1996; 137: 5502–5508.

89.

Seck

Baron

Horne

. Binding of filamin to the C-terminal tail of the calcitonin receptor controls recycling. J Biol Chem 2003; 278: 10408–10416.

90.

Safaei

Larson

Cheng

, et al. Abnormal lysosomal trafficking and enhanced exosomal export of cisplatin in drug-resistant human ovarian carcinoma cells. Mol Cancer Ther 2005; 4: 1595–1604.

91.

Livesey

Larkins

, et al. Calcitonin effects on growth and on selective activation of type II isoenzyme of cyclic adenosine 3’:5’-monophosphate-dependent protein kinase in T 47D human breast cancer cells. Cancer Res 1983; 43: 794–800.

92.

Dayer

Vassalli

Bobbitt

, et al. Calcitonin stimulates plasminogen activator in porcine renal tubular cells: LLC-PK1. J Cell Biol 1981; 91: 195–200.

93.

Evdokiou

Raggatt

Atkins

, et al. Calcitonin receptor-mediated growth suppression of HEK-293 cells is accompanied by induction of p21WAF1/CIP1 and G2/M arrest. Mol Endocrinol 1999; 13: 1738–1750.

94.

LaBaer

Garrett

Stevenson

, et al. New functional activities for the p21 family of CDK inhibitors. Genes Dev 1997; 11: 847–862.

95.

Santra

Mann

Mercer

, et al. Ectopic expression of decorin protein core causes a generalized growth suppression in neoplastic cells of various histogenetic origin and requires endogenous p21, an inhibitor of cyclin-dependent kinases. J Clin Invest 1997; 100: 149–157.

96.

Findlay

Raggatt

Bouralexis

, et al. Calcitonin decreases the adherence and survival of HEK-293 cells by a caspase-independent mechanism. J Endocrinol 2002; 175: 715–725.

97.

Venkatanarayan

Raulji

Norton

, et al. IAPP-driven metabolic reprogramming induces regression of p53-deficient tumours in vivo. Nature 2015; 517: 626–630.

98.

Shah

Thomas

Muralidharan

, et al. Calcitonin promotes in vivo metastasis of prostate cancer cells by altering cell signaling, adhesion, and inflammatory pathways. Endocr Relat Cancer 2008; 15: 953–964.

99.

Thomas

Muralidharan

Shah

. Knock-down of calcitonin receptor expression induces apoptosis and growth arrest of prostate cancer cells. Int J Oncol 2007; 31: 1425–1437.

100.

Plotkin

Weinstein

Parfitt

, et al. Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J Clin Invest 1999; 104: 1363–1374.

101.

Selander

Harkonen

Valve

, et al. Calcitonin promotes osteoclast survival in vitro. Mol Cell Endocrinol 1996; 122: 119–129.

102.

Shih

Holland

. Notch signaling enhances nestin expression in gliomas. Neoplasia 2006; 8: 1072–1082.

103.

Morton

Barnes

. Collagen polymorphism in the normal and diseased blood vessel wall. Investigation of collagens types I, III and V. Atherosclerosis 1982; 42: 41–51.

104.

Ooshima

. Collagen alpha B chain: increased proportion in human atherosclerosis. Science 1981; 213: 666–668.

105.

Mak

Png

Lee

. Type V collagen in health, disease, and fibrosis. Anat Rec (Hoboken) 2016; 299: 613–629.

106.

Inkinen

Wolff

Von Boguslawski

, et al. Type V collagen in experimental granulation tissue. Connect Tiss Res 1998; 39: 281–294.

107.

Golan-Gerstl

Cohen

Shilo

, et al. Splicing factor hnRNP A2/B1 regulates tumor suppressor gene splicing and is an oncogenic driver in glioblastoma. Cancer Res 2011; 71: 4464–4472.

108.

Goncalves

Pereira

JFS

Jordan

. Signaling pathways driving aberrant splicing in cancer cells. Genes (Basel) 2017; 9: 9.

109.

Nishikawa

Ishikawa

Yamamoto

, et al. A novel calcitonin receptor gene in human osteoclasts from normal bone marrow. FEBS Lett 1999; 458: 409–414.

110.

Hebden

Smalt

Chambers

, et al. Multiple promoters regulate human calcitonin receptor gene expression. Biochem Biophys Res Commun 2000; 272: 738–743.

111.

Pondel

Jagger

Hebden

, et al. Transcriptional regulation of the calcitonin receptor gene. Biochem Soc Trans 2002; 30: 423–427.

112.

Shen

Crotti

Flannery

, et al. A novel promoter regulates calcitonin receptor gene expression in human osteoclasts. Biochim Biophys Acta 2007; 1769: 659–667.

113.

Pondel

Partington

Mould

. Tissue-specific activity of the proximal human calcitonin receptor promoter is mediated by Sp1 and an epigenetic phenomenon. FEBS Lett 2003; 554: 433–438.

114.

Koizume

Miyagi

. Diverse mechanisms of Sp1-dependent transcriptional regulation potentially involved in the adaptive response of cancer cells to oxygen-deficient conditions. Cancers 2015; 8: 2.

115.

Yamin

Gorn

Flannery

, et al. Cloning and characterization of a mouse brain calcitonin receptor complementary deoxyribonucleic acid and mapping of the calcitonin receptor gene. Endocrinology 1994; 135: 2635–2643.

116.

Anusaksathien

Laplace

, et al. Tissue-specific and ubiquitous promoters direct the expression of alternatively spliced transcripts from the calcitonin receptor gene. J Biol Chem 2001; 276: 22663–22674.

117.

Lin

Harris

Flannery

, et al. Expression cloning of an adenylate cyclase-coupled calcitonin receptor. Science 1991; 254: 1022–1024.

118.

Zolnierowicz

Cron

Solinas-Toldo

, et al. Isolation, characterization, and chromosomal localization of the porcine calcitonin receptor gene. Identification of two variants of the receptor generated by alternative splicing. J Biol Chem 1994; 269: 19530–19538.

119.

Malter

. Identification of an AUUUA-specific messenger RNA binding protein. Science 1989; 246: 664–666.

120.

Shaw

Kamen

. A conserved AU sequence from the 3’ untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 1986; 46: 659–667.

121.

Fuchs

. Dianthin and its potential in targeted tumor therapies. Toxins (Basel) 2019; 11: 592.

122.

von Mallinckrodt

Thakur

Weng

, et al. Dianthin-EGF is an effective tumor targeted toxin in combination with saponins in a xenograft model for colon carcinoma. Future Oncol 2014; 10: 2161–2175.

123.

Sherr

. Principles of tumor suppression. Cell 2004; 116: 235–246.

124.

Allen

Bjerke

Edlund

, et al. Origin of the U87MG glioma cell line: good news and bad news. Sci Transl Med 2016; 8: 354re353.

Building the case for the calcitonin receptor as a viable target for the treatment of glioblastoma

Abstract

Keywords

Introduction

The widespread expression of the calcitonin receptor (CT Receptor)

Pharmacology of CT Receptora and CT Receptorb

Hypothesis on the mechanism of the CT Receptorb isoform

Calcitonin/CT Receptor, cell survival/apoptosis and the cell cycle

Stem cell quiescence and a role for a surrogate ligand of CT Receptor

Genotyped profiles of GBM

Regulation of CALCR transcription and stability of mRNA

Targeting inactivated CT Receptor in GBM

Evidence for CTR as a tumour suppressor and/or an oncogene

Conclusion

Footnotes

Acknowledgements

Conflict of interest statement

Funding

ORCID iD

References

Pharmacology of CT Receptor_a and CT Receptor_b

Hypothesis on the mechanism of the CT Receptor_b isoform