Store-independent activation of Orai1 by SPCA2 in mammary tumors.

Ca(2+) is an essential and ubiquitous second messenger. Changes in cytosolic Ca(2+) trigger events critical for tumorigenesis, such as cellular motility, proliferation, and apoptosis. We show that an isoform of Secretory Pathway Ca(2+)-ATPase, SPCA2, is upregulated in breast cancer-derived cells and human breast tumors, and suppression of SPCA2 attenuates basal Ca(2+) levels and tumorigenicity. Contrary to its conventional role in Golgi Ca(2+) sequestration, expression of SPCA2 increased Ca(2+) influx by a mechanism dependent on the store-operated Ca(2+) channel Orai1. Unexpectedly, SPCA2-Orai1 signaling was independent of ER Ca(2+) stores or STIM1 and STIM2 sensors and uncoupled from Ca(2+)-ATPase activity of SPCA2. Binding of the SPCA2 amino terminus to Orai1 enabled access of its carboxyl terminus to Orai1 and activation of Ca(2+) influx. Our findings reveal a signaling pathway in which the Orai1-SPCA2 complex elicits constitutive store-independent Ca(2+) signaling that promotes tumorigenesis.

ORAI1 regulates sustained cytosolic free calcium fluctuations during breast cancer cell apoptosis and apoptotic resistance via a STIM1 independent pathway.

Excessive rapid increases in cytosolic free Ca2+ have a clear association with the induction of cancer cell death. Whereas, characterizing the Ca2+ signaling events that occur during the progression of the apoptotic cascade over a period of hours or days, has not yet been possible. Now using genetically encoded Ca2+ indicators complemented with automated epifluorescence microscopy we have shown that staurosporine-induced apoptosis in MDA-MB-231 breast cancer cells was associated with delayed development of cytosolic free Ca2+ fluctuations, which were then maintained for 24 h. These cytosolic free Ca2+ fluctuations were dependent on the Ca2+ channel ORAI1. Silencing of ORAI1, but not its canonical activators STIM1 and STIM2, promoted apoptosis in this model. The pathway for this regulation implicates a mechanism previously associated with the migration of cancer cells involving ORAI1, the chaperone protein SigmaR1, and Ca2+ -activated K+ channels.

ORAI1-Regulated Gene Expression in Breast Cancer Cells: Roles for STIM1 Binding, Calcium Influx and Transcription Factor Translocation.

A remodeling of calcium homeostasis, including calcium influx via store-operated calcium entry (SOCE), is a feature of breast cancers. SOCE is critical to maintain calcium balance in the endoplasmic reticulum calcium store and is an important mechanism for calcium signaling in a variety of cell types, including breast cancer cells. The canonical mechanism of SOCE is stromal interacting molecule 1 (STIM1)-mediated activation of ORAI. Elevated ORAI1 expression is a feature of basal breast cancer cells. However, the role of ORAI1 in the regulation of transcription in breast cancer cells of the basal molecular subtype is still unclear. Using CRISPR-Cas9 gene editing, ORAI1 protein expression was disrupted in MDA-MB-231 and MDA-MB-468 basal breast cancer cells. The ORAI1 wild-type and mutants were reintroduced into ORAI1 knockout cells to study the role of ORAI1 in gene transcriptional regulation. In the absence of calcium store depletion, ORAI1 regulated PTGS2 in MDA-MB-231 cells, and this was dependent on ORAI1 pore function and STIM1 binding. The activation of SOCE by thapsigargin resulted in ORAI1-dependent increases in IL6 transcription in MDA-MB-468 cells; this was also dependent on ORAI1 pore function and STIM1 binding and was associated with the translocation of NFAT1. Given the upregulation of ORAI1 in basal breast cancer cells, our results provide further evidence that ORAI1 may contribute to cancer progression through regulation of gene expression.

Calcium signalling and breast cancer.

The past two decades have seen the identification of important roles for calcium signalling in many of the hallmarks of cancer. One of the cancer types that has been a particular focus of such studies is breast cancer. The breast is intrinsically linked to the calcium ion due to the importance of milk calcium in neonatal growth and development. Indeed, some of the calcium channels and pumps involved in transporting calcium ions into milk also have altered expression in some breast cancers. However, altered expression is not confined to channels and pumps important in lactation, other calcium channels and pumps may also be modulated and may even be specific to breast cancer molecular subtypes. This review considers calcium signalling in the context of breast cancer and provides an overview of the roles that have been attributed to specific regulators of cellular calcium levels in processes relevant to breast cancer progression. Emerging areas in the study of calcium signalling in breast cancer are considered, such as the intersection between calcium signalling, the tumour microenvironment and breast cancer.

An Emerging Role for Calcium Signaling in Cancer-Associated Fibroblasts.

Tumors exist in a complex milieu where interaction with their associated microenvironment significantly contributes to disease progression. Cancer-associated fibroblasts (CAFs) are the primary component of the tumor microenvironment and participate in complex bidirectional communication with tumor cells. CAFs support the development of various hallmarks of cancer through diverse processes, including direct cell-cell contact, paracrine signaling, and remodeling and deposition of the extracellular matrix. Calcium signaling is a key second messenger in intra- and inter-cellular signaling pathways that contributes to cancer progression; however, the links between calcium signaling and CAFs are less well-explored. In this review, we put into context the role of calcium signaling in interactions between cancer cells and CAFs, with a focus on migration, proliferation, chemoresistance, and genetic instability.

Transient receptor potential cation channel subfamily V and breast cancer.

Transient receptor potential cation channel subfamily V (TRPV) channels play important roles in a variety of cellular processes. One example includes the sensory role of TRPV1 that is sensitive to elevated temperatures and acidic environments and is activated by the hot pepper component capsaicin. Another example is the importance of the highly Ca2+ selective channels TRPV5 and TRPV6 in Ca2+ absorption/reabsorption in the intestine and kidney. However, in some cases such as TRPV4 and TRPV6, breast cancer cells appear to overexpress TRPV channels. Moreover, TRPV mediated Ca2+ influx may contribute to enhanced breast cancer cell proliferation and other processes important in tumor progression such as angiogenesis. It appears that the overexpression of some TRPV channels in breast cancer and/or their involvement in breast cancer cell processes, processes important in the tumor microenvironment or pain may make some TRPV channels potential targets for breast cancer therapy. In this review, we provide an overview of TRPV expression in breast cancer subtypes, the roles of TRPV channels in various aspects of breast cancer progression and consider implications for future therapeutic approaches.

Differential engagement of ORAI1 and TRPC1 in the induction of vimentin expression by different stimuli.

The Ca2+ signal is essential in both hypoxia- and epidermal growth factor (EGF)-mediated epithelial to mesenchymal transition (EMT) in MDA-MB-468 breast cancer cells. This finding suggests that Ca2+-permeable ion channels participate in the induction of expression of some mesenchymal markers such as vimentin. However, the ion channels involved in vimentin expression induction have not been fully characterized. This work sought to define how differential modulation of the calcium signal effects the induction of vimentin and the Ca2+ influx pathways involved. We identified that the intracellular Ca2+ chelator EGTA-AM, cytochalasin D (a modulator of cytoskeletal dynamics and cell morphology), and the sarco/endoplasmic reticulum ATPase inhibitor thapsigargin are all inducers of vimentin in MDA-MB-468 breast cancer cells. EGTA-AM- and thapsigargin-mediated induction of vimentin expression in MDA-MB-468 cells involves store-operated Ca2+ entry, as evidenced by sensitivity to silencing of the molecular components of this pathway, STIM1 and ORAI1. In stark contrast, cytochalasin D-mediated vimentin induction was insensitive to silencing of ORAI1, despite sensitivity to silencing of its canonical activator the endoplasmic reticulum Ca2+ sensor STIM1. Cytochalasin D-mediated vimentin induction was, however, sensitive to silencing of another reported STIM1 target, TRPC1. Subsequent studies identified that EGTA-AM-induced vimentin expression also partially involved a TRPC1-dependent pathway. These studies define a complex interplay between vimentin expression in this model and the specific Ca2+-permeable ion channels involved. The complexity in the engagement of different Ca2+ influx pathways that regulate vimentin induction are opportunities but also potential challenges in targeting Ca2+ signaling to block EMT in cancer cells. Our findings further highlight the need to identify potential indispensable ion channels that can regulate induction of specific mesenchymal markers via different stimuli.

Assessment of doxorubicin-induced remodeling of Ca2+ signaling and associated Ca2+ regulating proteins in MDA-MB-231 breast cancer cells.

Triple-negative breast cancers (TNBC) are often associated with high relapse rates, despite treatment with chemotherapy agents such as doxorubicin. A better understanding of the signaling and molecular changes associated with doxorubicin may provide novel insights into strategies to enhance treatment efficacy. Calcium signaling is involved in many pathways influencing the efficacy of chemotherapy agents such as proliferation and cell death. However, there are a limited number of studies exploring the effect of doxorubicin on calcium signaling in TNBC. In this study, MDA-MB-231 triple-negative, basal breast cancer cells stably expressing the genetically-encoded calcium indicator GCaMP6m (GCaMP6m-MDA-MB-231) were used to define alterations in calcium signaling. The effects of doxorubicin in GCaMP6m-MDA-MB-231 cells were determined using live cell imaging and fluorescence microscopy. Changes in mRNA levels of specific calcium regulating proteins as a result of doxorubicin treatment were also assessed using real time qPCR. Doxorubicin (1 µM) produced alterations in intracellular calcium signaling, including enhancing the sensitivity of MDA-MB-231 cells to ATP stimulation and prolonging the recovery time after store-operated calcium entry. Upregulation in mRNA levels of ORAI1, TRPC1, SERCA1, IP3R2 and PMCA2 with doxorubicin 1 µM treatment was also observed. Doxorubicin treatment is associated with specific remodeling in calcium signaling in MDA-MB-231 cells, with associated changes in mRNA levels of specific calcium-regulating proteins.

Activation of the Ion Channel TRPV4 Induces Epithelial to Mesenchymal Transition in Breast Cancer Cells.

Epithelial to mesenchymal transition (EMT) in cancer is important in therapeutic resistance and invasiveness. Calcium signaling is key to the induction of EMT in breast cancer cells. Although inhibition of specific calcium-permeable ion channels regulates the induction of a sub-set of EMT markers in breast cancer cells, it is still unclear if activation of a specific calcium channel can be a driver for the induction of EMT events. In this study, we exploited the availability of a selective pharmacological activator of the calcium-permeable ion channel TRPV4 to assess the direct role of calcium influx in EMT marker induction. Gene association studies revealed a link between TRPV4 and gene-ontologies associated with EMT and poorer relapse-free survival in lymph node-positive basal breast cancers. TRPV4 was an important component of the calcium influx phase induced in MDA-MB-468 breast cancer cells by the EMT inducer epidermal growth factor (EGF). Pharmacological activation of TRPV4 then drove the induction of a variety of EMT markers in breast cancer cells. These studies demonstrate that calcium influx through specific pathways appears to be sufficient to trigger EMT events.

TRPC1 is a differential regulator of hypoxia-mediated events and Akt signalling in PTEN-deficient breast cancer cells.

Hypoxia is a feature of the tumour microenvironment that promotes invasiveness, resistance to chemotherapeutics and cell survival. Our studies identify the transient receptor potential canonical-1 (TRPC1) ion channel as a key component of responses to hypoxia in breast cancer cells. This regulation includes control of specific epithelial to mesenchymal transition (EMT) events and hypoxia-mediated activation of signalling pathways such as activation of the EGFR, STAT3 and the autophagy marker LC3B, through hypoxia-inducible factor-1α (HIF1α)-dependent and -independent mechanisms. TRPC1 regulated HIF1α levels in PTEN-deficient MDA-MB-468 and HCC1569 breast cancer cell lines. This regulation arises from effects on the constitutive translation of HIF1α under normoxic conditions via an Akt-dependent pathway. In further support of the role of TRPC1 in EMT, its expression is closely associated with EMT- and metastasis-related genes in breast tumours, and is enhanced in basal B breast cancer cell lines. TRPC1 expression is also significantly prognostic for basal breast cancers, particularly those classified as lymph node positive. The defined roles of TRPC1 identified here could be therapeutically exploited for the control of oncogenic pathways in breast cancer cells.

Pharmacological inhibition of store-operated calcium entry in MDA-MB-468 basal A breast cancer cells: consequences on calcium signalling, cell migration and proliferation.

Store-operated Ca2+ entry is a pathway that is remodelled in a variety of cancers, and altered expression of the components of store-operated Ca2+ entry is a feature of breast cancer cells of the basal molecular subtype. Studies of store-operated Ca2+ entry in breast cancer cells have used non-specific pharmacological inhibitors, complete depletion of intracellular Ca2+ stores and have mostly focused on MDA-MB-231 cells (a basal B breast cancer cell line). These studies compared the effects of the selective store-operated Ca2+ entry inhibitors Synta66 and YM58483 (also known as BTP2) on global cytosolic free Ca2+ ([Ca2+]CYT) changes induced by physiological stimuli in a different breast cancer basal cell line model, MDA-MB-468. The effects of these agents on proliferation as well as serum and epidermal growth factor (EGF) induced migration were also assessed. Activation with the purinergic receptor activator adenosine triphosphate, produced a sustained increase in [Ca2+]CYT that was entirely dependent on store-operated Ca2+ entry. The protease activated receptor 2 activator, trypsin, and EGF also produced Ca2+ influx that was sensitive to both Synta66 and YM58483. Serum-activated migration of MDA-MB-468 breast cancer cells was sensitive to both store-operated Ca2+ inhibitors. However, proliferation and EGF-activated migration was differentially affected by Synta66 and YM58483. These studies highlight the need to define the exact mechanisms of action of different store-operated calcium entry inhibitors and the impact of such differences in the control of tumour progression pathways.

An SAR study of hydroxy-trifluoromethylpyrazolines as inhibitors of Orai1-mediated store operated Ca2+ entry in MDA-MB-231 breast cancer cells using a convenient Fluorescence Imaging Plate Reader assay.

The proteins Orai1 and STIM1 control store-operated Ca2+ entry (SOCE) into cells. SOCE is important for migration, invasion and metastasis of MDA-MB-231 human triple negative breast cancer (TNBC) cells and has been proposed as a target for cancer drug discovery. Two hit compounds from a medium throughput screen, displayed encouraging inhibition of SOCE in MDA-MB-231 cells, as measured by a Fluorescence Imaging Plate Reader (FLIPR) Ca2+ assay. Following NMR spectroscopic analysis of these hits and reassignment of their structures as 5-hydroxy-5-trifluoromethylpyrazolines, a series of analogues was prepared via thermal condensation reactions between substituted acylhydrazones and trifluoromethyl 1,3-dicarbonyl arenes. Structure-activity relationship (SAR) studies showed that small lipophilic substituents at the 2- and 3-positions of the RHS and 2-, 3- and 4-postions of the LHS terminal benzene rings improved activity, resulting in a novel class of potent and selective inhibitors of SOCE.

Essential role of Orai1 store-operated calcium channels in lactation.

The nourishment of neonates by nursing is the defining characteristic of mammals. However, despite considerable research into the neural control of lactation, an understanding of the signaling mechanisms underlying the production and expulsion of milk by mammary epithelial cells during lactation remains largely unknown. Here we demonstrate that a store-operated Ca(2+) channel subunit, Orai1, is required for both optimal Ca(2+) transport into milk and for milk ejection. Using a novel, 3D imaging strategy, we visualized live oxytocin-induced alveolar unit contractions in the mammary gland, and we demonstrated that in this model milk is ejected by way of pulsatile contractions of these alveolar units. In mammary glands of Orai1 knockout mice, these contractions are infrequent and poorly coordinated. We reveal that oxytocin also induces a large transient release of stored Ca(2+) in mammary myoepithelial cells followed by slow, irregular Ca(2+) oscillations. These oscillations, and not the initial Ca(2+) transient, are mediated exclusively by Orai1 and are absolutely required for milk ejection and pup survival, an observation that redefines the signaling processes responsible for milk ejection. These findings clearly demonstrate that Ca(2+) is not just a substrate for nutritional enrichment in mammals but is also a master regulator of the spatiotemporal signaling events underpinning mammary alveolar unit contraction. Orai1-dependent Ca(2+) oscillations may represent a conserved language in myoepithelial cells of other secretory epithelia, such as sweat glands, potentially shedding light on other Orai1 channelopathies, including anhidrosis (an inability to sweat).

Evaluation of known and novel inhibitors of Orai1-mediated store operated Ca2+ entry in MDA-MB-231 breast cancer cells using a Fluorescence Imaging Plate Reader assay.

The Orai1 Ca2+ permeable ion channel is an important component of store operated Ca2+ entry (SOCE) in cells. It's over-expression in basal molecular subtype breast cancers has been linked with poor prognosis, making it a potential target for drug development. We pharmacologically characterised a number of reported inhibitors of SOCE in MDA-MB-231 breast cancer cells using a convenient Fluorescence Imaging Plate Reader (FLIPR) assay, and show that the rank order of their potencies in this assay is the same as those reported in a wide range of published assays. The assay was also used in a screening project seeking novel inhibitors. Following a broad literature survey of classes of calcium channel inhibitors we used simplified ligand structures to query the ZINC on-line database, and following two iterations of refinement selected a novel Orai1-selective dichlorophenyltriazole hit compound. Analogues of this were synthesized and evaluated in the FLIPR assay to develop structure-activity relationships (SAR) for the three domains of the hit; triazole (head), dichlorophenyl (body) and substituted phenyl (tail). For this series, the results suggested the need for a lipophilic tail domain and an out-of-plane twist between the body and tail domains.

Altered calcium signaling in cancer cells.

It is the nature of the calcium signal, as determined by the coordinated activity of a suite of calcium channels, pumps, exchangers and binding proteins that ultimately guides a cell's fate. Deregulation of the calcium signal is often deleterious and has been linked to each of the 'cancer hallmarks'. Despite this, we do not yet have a full understanding of the remodeling of the calcium signal associated with cancer. Such an understanding could aid in guiding the development of therapies specifically targeting altered calcium signaling in cancer cells during tumorigenic progression. Findings from some of the studies that have assessed the remodeling of the calcium signal associated with tumorigenesis and/or processes important in invasion and metastasis are presented in this review. The potential of new methodologies is also discussed. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

PMCA2 silencing potentiates MDA-MB-231 breast cancer cell death initiated with the Bcl-2 inhibitor ABT-263.

PMCA2 overexpression in some breast cancers suggests that this calcium pump isoform may play a role in breast pathophysiology. To investigate PMCA2 as a potential drug target for breast cancer therapy, we assessed the functional consequence of PMCA2 silencing on cell death pathways and calcium signals in the basal-like MDA-MB-231 breast cancer cell line. Silencing PMCA2 expression alone has no effect on MDA-MB-231 cell viability, however, PMCA2 silencing promotes calcium-induced cell death initiated with the calcium ionophore ionomycin. Assessment of cytoplasmic calcium responses generated with various agents including ionomycin demonstrates that in MDA-MB-231 cells, PMCA2 does not play a major role in shaping global calcium signals. We also examined the ability of PMCA2 silencing to modulate caspase-dependent cell death triggered by a Bcl-2 inhibitor that is in clinical development for the treatment of various cancers, ABT-263 (Navitoclax). Despite the lack of effect on global calcium responses, PMCA2 silencing augmented Bcl-2 inhibitor (ABT-263)-mediated MDA-MB-231 breast cancer cell death. These studies provide evidence that PMCA2 inhibitors could sensitize PMCA2-positive breast cancers to cell death initiators that work through mechanisms involving the Bcl-2 survival pathway.

Differential effects of two-pore channel protein 1 and 2 silencing in MDA-MB-468 breast cancer cells.

Two-pore channel proteins, TPC1 and TPC2, are calcium permeable ion channels found localized to the membranes of endolysosomal calcium stores. There is increasing interest in the role of TPC-mediated intracellular signaling in various pathologies; however their role in breast cancer has not been extensively evaluated. TPC1 and TPC2 mRNA was present in all non-tumorigenic and tumorigenic breast cell lines assessed. Silencing of TPC2 but not TPC1 attenuated epidermal growth factor-induced vimentin expression in MDA-MB-468 breast cancer cells. This effect was not due to a general inhibition of epithelial to mesenchymal transition (EMT) as TPC2 silencing had no effect on epidermal growth factor (EGF)-induced changes on E-cadherin expression. TPC1 and TPC2 were also shown to differentially regulate cyclopiazonic acid (CPA)-mediated changes in cytosolic free Ca(2+). These findings indicate potential differential regulation of signaling processes by TPC1 and TPC2 in breast cancer cells.

The voltage gated Ca(2+)-channel Cav3.2 and therapeutic responses in breast cancer.

BACKGROUND: Understanding the cause of therapeutic resistance and identifying new biomarkers in breast cancer to predict therapeutic responses will help optimise patient care. Calcium (Ca(2+))-signalling is important in a variety of processes associated with tumour progression, including breast cancer cell migration and proliferation. Ca(2+)-signalling is also linked to the acquisition of multidrug resistance. This study aimed to assess the expression level of proteins involved in Ca(2+)-signalling in an in vitro model of trastuzumab-resistance and to assess the ability of identified targets to reverse resistance and/or act as potential biomarkers for prognosis or therapy outcome. METHODS: Expression levels of a panel of Ca(2+)-pumps, channels and channel regulators were assessed using RT-qPCR in resistant and sensitive age-matched SKBR3 breast cancer cells, established through continuous culture in the absence or presence of trastuzumab. The role of Cav3.2 in the acquisition of trastuzumab-resistance was assessed through pharmacological inhibition and induced overexpression. Levels of Cav3.2 were assessed in a panel of non-malignant and malignant breast cell lines using RT-qPCR and in patient samples representing different molecular subtypes (PAM50 cohort). Patient survival was also assessed in samples stratified by Cav3.2 expression (METABRIC and KM-Plotter cohort). RESULTS: Increased mRNA of Cav3.2 was a feature of both acquired and intrinsic trastuzumab-resistant SKBR3 cells. However, pharmacological inhibition of Cav3.2 did not restore trastuzumab-sensitivity nor did Cav3.2 overexpression induce the expression of markers associated with resistance, suggesting that Cav3.2 is not a driver of trastuzumab-resistance. Cav3.2 levels were significantly higher in luminal A, luminal B and HER2-enriched subtypes compared to the basal subtype. High levels of Cav3.2 were associated with poor outcome in patients with oestrogen receptor positive (ER+) breast cancers, whereas Cav3.2 levels were correlated positively with patient survival after chemotherapy in patients with HER2-positive breast cancers. CONCLUSION: Our study identified elevated levels of Cav3.2 in trastuzumab-resistant SKBR3 cell lines. Although not a regulator of trastuzumab-resistance in HER2-positive breast cancer cells, Cav3.2 may be a potential differential biomarker for survival and treatment response in specific breast cancer subtypes. These studies add to the complex and diverse role of Ca(2+)-signalling in breast cancer progression and treatment.

Calcium and cancer: targeting Ca2+ transport.

Ca2+ is a ubiquitous cellular signal. Altered expression of specific Ca2+ channels and pumps are characterizing features of some cancers. The ability of Ca2+ to regulate both cell death and proliferation, combined with the potential for pharmacological modulation, offers the opportunity for a set of new drug targets in cancer. However, the ubiquity of the Ca2+ signal is often mistakenly presumed to thwart the specific therapeutic targeting of proteins that transport Ca2+. This Review presents evidence to the contrary and addresses the question: which Ca2+ channels and pumps should be targeted?

Sequence diversity and differential expression of major phenylpropanoid-flavonoid biosynthetic genes among three mango varieties.

BACKGROUND: Mango fruits contain a broad spectrum of phenolic compounds which impart potential health benefits; their biosynthesis is catalysed by enzymes in the phenylpropanoid-flavonoid (PF) pathway. The aim of this study was to reveal the variability in genes involved in the PF pathway in three different mango varieties Mangifera indica L., a member of the family Anacardiaceae: Kensington Pride (KP), Irwin (IW) and Nam Doc Mai (NDM) and to determine associations with gene expression and mango flavonoid profiles. RESULTS: A close evolutionary relationship between mango genes and those from the woody species poplar of the Salicaceae family (Populus trichocarpa) and grape of the Vitaceae family (Vitis vinifera), was revealed through phylogenetic analysis of PF pathway genes. We discovered 145 SNPs in total within coding sequences with an average frequency of one SNP every 316 bp. Variety IW had the highest SNP frequency (one SNP every 258 bp) while KP and NDM had similar frequencies (one SNP every 369 bp and 360 bp, respectively). The position in the PF pathway appeared to influence the extent of genetic diversity of the encoded enzymes. The entry point enzymes phenylalanine lyase (PAL), cinnamate 4-mono-oxygenase (C4H) and chalcone synthase (CHS) had low levels of SNP diversity in their coding sequences, whereas anthocyanidin reductase (ANR) showed the highest SNP frequency followed by flavonoid 3'-hydroxylase (F3'H). Quantitative PCR revealed characteristic patterns of gene expression that differed between mango peel and flesh, and between varieties. CONCLUSIONS: The combination of mango expressed sequence tags and availability of well-established reference PF biosynthetic genes from other plant species allowed the identification of coding sequences of genes that may lead to the formation of important flavonoid compounds in mango fruits and facilitated characterisation of single nucleotide polymorphisms between varieties. We discovered an association between the extent of sequence variation and position in the pathway for up-stream genes. The high expression of PAL, C4H and CHS genes in mango peel compared to flesh is associated with high amounts of total phenolic contents in peels, which suggest that these genes have an influence on total flavonoid levels in mango fruit peel and flesh. In addition, the particularly high expression levels of ANR in KP and NDM peels compared to IW peel and the significant accumulation of its product epicatechin gallate (ECG) in those extracts reflects the rate-limiting role of ANR on ECG biosynthesis in mango.