Multiomics analysis identifies oxidative phosphorylation as a cancer vulnerability arising from myristoylation inhibition.

BACKGROUND: In humans, two ubiquitously expressed N-myristoyltransferases, NMT1 and NMT2, catalyze myristate transfer to proteins to facilitate membrane targeting and signaling. We investigated the expression of NMTs in numerous cancers and found that NMT2 levels are dysregulated by epigenetic suppression, particularly so in hematologic malignancies. This suggests that pharmacological inhibition of the remaining NMT1 could allow for the selective killing of these cells, sparing normal cells with both NMTs. METHODS AND RESULTS: Transcriptomic analysis of 1200 NMT inhibitor (NMTI)-treated cancer cell lines revealed that NMTI sensitivity relates not only to NMT2 loss or NMT1 dependency, but also correlates with a myristoylation inhibition sensitivity signature comprising 54 genes (MISS-54) enriched in hematologic cancers as well as testis, brain, lung, ovary, and colon cancers. Because non-myristoylated proteins are degraded by a glycine-specific N-degron, differential proteomics revealed the major impact of abrogating NMT1 genetically using CRISPR/Cas9 in cancer cells was surprisingly to reduce mitochondrial respiratory complex I proteins rather than cell signaling proteins, some of which were also reduced, albeit to a lesser extent. Cancer cell treatments with the first-in-class NMTI PCLX-001 (zelenirstat), which is undergoing human phase 1/2a trials in advanced lymphoma and solid tumors, recapitulated these effects. The most downregulated myristoylated mitochondrial protein was NDUFAF4, a complex I assembly factor. Knockout of NDUFAF4 or in vitro cell treatment with zelenirstat resulted in loss of complex I, oxidative phosphorylation and respiration, which impacted metabolomes. CONCLUSIONS: Targeting of both, oxidative phosphorylation and cell signaling partly explains the lethal effects of zelenirstat in select cancer types. While the prognostic value of the sensitivity score MISS-54 remains to be validated in patients, our findings continue to warrant the clinical development of zelenirstat as cancer treatment.

Transcription factor ZIC2 regulates the tumorigenic phenotypes associated with both bulk and cancer stem cells in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy in North America. Current therapeutic regimens are ineffective against advanced EOC. A better understanding of the molecular mechanisms that regulate the biology of EOC will be a critical step toward developing more efficacious therapies against EOC. Herein, we demonstrate that elevated expression of transcription factor ZIC2 was associated with lower survival of EOC patients. Knockout of endogenous ZIC2 in EOC cells attenuated the tumorigenic phenotypes associated with both bulk and cancer stem cells in vitro and in vivo, indicating a pro-tumorigenic role of ZIC2 in EOC. On the other hand, however, overexpression of ZIC2 in EOC cells that do not express endogenous ZIC2 promoted cell migration and sphere formation, but inhibited cell growth and colony formation in vitro and tumor growth in vivo, indicating that the role for ZIC2 in EOC is context dependent. Our transcriptomic analysis showed that ZIC2-regulated genes were involved in multiple biological processes and signaling pathways associated with tumor progression. In conclusion, our findings reveal a context-dependent role for ZIC2 in regulating tumorigenic phenotypes in EOC, providing evidence that ZIC2 can be a potential therapeutic target for EOCs that express a high level of ZIC2.

Cancer-associated Fibroblast-specific Expression of the Matricellular Protein CCN1 Coordinates Neovascularization and Stroma Deposition in Melanoma Metastasis.

Melanoma is the leading cause of skin cancer-related death. As prognosis of patients with melanoma remains problematic, identification of new therapeutic targets remains essential. Matricellular proteins are nonstructural extracellular matrix proteins. They are secreted into the tumor microenvironment to coordinate behavior among different cell types, yet their contribution to melanoma is underinvestigated. Examples of matricellular proteins include those comprising the CCN family. The CCN family member, CCN1, is highly proangiogenic. Herein, we show that, in human patients with melanoma, although found in several tumor cell types, CCN1 is highly expressed by a subset of cancer-associated fibroblasts (CAF) in patients with melanoma and this expression correlates positively with expression of proangiogenic genes and progressive disease/resistance to anti-PD1 checkpoint inhibitors. Consistent with these observations, in a syngeneic C57BL6 mouse model of melanoma, loss of CCN1 expression from Col1A2-Cre-, herein identified as "universal," fibroblasts, impaired metastasis of subcutaneously injected B16F10 tumor cells to lung, concomitant with disrupted neovascularization and collagen organization. Disruption of the extracellular matrix in the loss of CCN1 was validated using a novel artificial intelligence-based image analysis platform that revealed significantly decreased phenotypic fibrosis and composite morphometric collagen scores. As drug resistance is linked to matrix deposition and neoangiogenesis, these data suggest that CCN1, due to its multifaceted role, may represent a novel therapeutic target for drug-resistant melanoma. Our data further emphasize the essential role that cancer-associated, (universal) Col1A2-Cre-fibroblasts and extracellular matrix remodeling play in coordinating behavior among different cell types within the tumor microenvironment. SIGNIFICANCE: In human patients, the expression of proangiogenic matricellular protein CCN1 in CAFs correlates positively with expression of stroma and angiogenic markers and progressive disease/resistance to checkpoint inhibitor therapy. In an animal model, loss of CCN1 from CAFs impaired metastasis of melanoma cells, neovascularization, and collagen deposition, emphasizing that CAFs coordinate cellular behavior in a tumor microenvironment and that CCN1 may be a novel target.

RNA cytosine methyltransferase NSUN5 promotes protein synthesis and tumorigenic phenotypes in glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. The standard treatment achieves a median overall survival for GBM patients of only 15 months. Hence, novel therapies based on an increased understanding of the mechanistic underpinnings of GBM are desperately needed. In this study, we show that elevated expression of 28S rRNA (cytosine-C(5))-methyltransferase NSUN5, which methylates cytosine 3782 of 28S rRNA in GBM cells, is strongly associated with the poor survival of GBM patients. Moreover, we demonstrate that overexpression of NSUN5 increases protein synthesis in GBM cells. NSUN5 knockdown decreased protein synthesis, cell proliferation, sphere formation, migration, and resistance to temozolomide in GBM cell lines. NSUN5 knockdown also decreased the number and size of GBM neurospheres in vitro. As a corollary, mice harboring U251 tumors wherein NSUN5 was knocked down survived longer than mice harboring control tumors. Taken together, our results suggest that NSUN5 plays a protumorigenic role in GBM by enabling the enhanced protein synthesis requisite for tumor progression. Accordingly, NSUN5 may be a hitherto unappreciated target for the treatment of GBM.

A novel splice site CUL3 variant in a patient with neurodevelopmental delay.

BACKGROUND: CUL3-related neurodevelopmental disorder is a recently described rare genetic condition characterized by global developmental delay and intellectual disability. Five affected individuals have been reported worldwide. The molecular and phenotypic spectrum of the disorder has yet to be fully elucidated. Splice variants in CUL3 are a well-described cause of pseudohypoaldosteronism type IIE; however, splice variants have not been associated with the neurodevelopmental disorder. We report the first individual with a neurodevelopmental disorder attributed to a CUL3 splice site variant. CASE REPORT: The patient presented with congenital developmental dysplasia of the hip and global developmental delay. A de novo splice site variant (c.379-2A > G) was identified in CUL3 and is predicted to abolish the acceptor splice site. CONCLUSION: This is the first report of an individual with a splice site variant causing CUL3-related neurodevelopmental disorder and expands our understanding of this rare condition.

A 79-kb paternally inherited 7q32.2 microdeletion involving MEST in a patient with a Silver-Russell syndrome-like phenotype.

Maternal uniparental disomy of human chromosome 7 [upd(7)mat] is well-characterized as a cause of the growth disorder Silver-Russell syndrome (SRS). However, the causative gene is not currently known. There is growing evidence that molecular changes at the imprinted MEST region in 7q32.2 are associated with a phenotype evocative of SRS. This report details a patient with a SRS-like phenotype and a paternally inherited microdeletion of 79 kilobases (35-fold smaller than the previously reported smallest deletion) in the 7q32.2 region. This microdeletion encompasses only five genes, including MEST, which corroborates the hypothesis that MEST plays a central role in the 7q32.2 microdeletion growth disorder, as well as further implicating MEST in upd(7)mat SRS itself.

Expanding the clinical spectrum of autosomal-recessive renal tubular dysgenesis: Two siblings with neonatal survival and review of the literature.

BACKGROUND: Autosomal-recessive renal tubular dysgenesis (AR-RTD) is a rare genetic disorder caused by defects in the renin-angiotensin system that manifests as fetal anuria leading to oligohydramnios and Potter sequence. Although the most common outcome is neonatal death from renal failure, pulmonary hypoplasia, and/or refractory arterial hypotension; several cases have been reported that describe survival past the neonatal period. METHODS: Herein, we report the first family with biallelic ACE variants and more than one affected child surviving past the neonatal period, as well as provide a review of the previously reported 18 cases with better outcomes. RESULTS: While both siblings with identical compound heterozygous ACE variants have received different treatments, neither required renal replacement therapy. We show that both vasopressin and fludrocortisone in the neonatal period may provide survival advantages, though outcomes may also be dependent on the type of gene variant, as well as other factors. CONCLUSION: While AR-RTD is most often a lethal disease in the neonatal period, it is not universally so. A better understanding of the factors affecting survival will help to guide prognostication and medical decision-making.

Delineating the expanding phenotype of HERC2-related disorders: The impact of biallelic loss of function versus missense variation.

HECT And RLD Domain-Containing E3 Ubiquitin Protein Ligase 2, or HERC2, codes an ubiquitin ligase that has an important role in key cellular processes including cell cycle regulation, DNA repair, mitochondrial functions, and spindle formation during mitosis. While HERC2 Neurodevelopmental Disorder in Old Order Amish is a well characterized human disorder involving HERC2, bi-allelic HERC2 loss of function has only been described in three families and results in a more severe neurodevelopmental disorder. Herein, we delineate the HERC2 loss of function phenotype by describing three previously unreported patients, and by summarizing the molecular and phenotypic information of all known HERC2 missense variants and biallelic loss of function patients. Collectively, these twelve individuals present with recurring features that define a syndrome with varying combinations of severe neurodevelopmental delay, structural brain anomalies, seizures, hypotonia, feeding difficulties, hearing and vision issues, and renal anomalies. This study describes a distinct neurodevelopmental disorder, emphasizing the importance of further characterization of HERC2-related disorders, as well as highlighting the importance of ongoing work into understanding these critical neurodevelopmental pathways.

The solubility of calcium oxalates explains some aspects of their underrepresentation in the oral cavity.

OBJECTIVE: Clarifying the discrepancy between frequently high oxalate concentrations present in saliva, but negligible amounts of calcium oxalate deposits found on oral surfaces. METHODS: Studying the calcium oxalate concentration range that can lead to heterogeneous crystallization in the oral cavity. a) Minimum: calcium oxalate monohydrate (COM) seed crystals were pre-grown ([Ca2+] = [C2O42-] = 1 mM, 30 min, 37 °C), and then re-immersed for ≥6 h to find the solubility equilibrium concentration (no growth, no dissolution). The concentrations tested were [Ca2+]/[C2O42-] : 0.055/0.050, 0.060/0.055, 0.070/0.065 and 0.080/0.075 mM. Supersaturations were calculated via the Debye-Hückel-theory and COM morphologies examined by scanning electron microscopy (SEM). b) Maximum (at the heterogeneous/homogeneous crystallization equilibrium): hydroxyapatite (HA) seed crystals were used to heterogeneously crystallize COM (37 °C, 24 h), using oxalate concentrations between 0.2 and 0.5 mM and calcium concentrations of 0.5 mM. COM-forming oxalate consumption was spectroscopically examined; COM precipitates were investigated by SEM; and HA identity was confirmed by X-ray analysis. RESULTS: Within the concentration range of [Ca2+]/[C2O42-]:0.060/0.055 mM (minimum) and [Ca2+]/[C2O42-]:0.50/0.25 mM (maximum) COM precipitates heterogeneously. In terms of mass, this corresponds to a range of 8.04-36.53 mg/l (daily) or an average of 14.32 mg COM (mimicking e.g. plaque mineralization). Higher concentrations react homogeneously (mimicking precipitation within saliva). CONCLUSION: In vivo, only ∼0.05 % oxalate present in saliva reacts with oral surfaces daily, corresponding to ∼0.0665 µmol/l or ∼9.72 µg COM per day. Calcium-consuming calcium phosphate formation and phosphoproteins such as statherin obviously hinder intraoral COM formation.

Targeting N-myristoylation for therapy of B-cell lymphomas.

Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, is critical for membrane targeting and cell signaling. Because cancer cells often have increased N-myristoyltransferase (NMT) expression, NMTs were proposed as anti-cancer targets. To systematically investigate this, we performed robotic cancer cell line screens and discovered a marked sensitivity of hematological cancer cell lines, including B-cell lymphomas, to the potent pan-NMT inhibitor PCLX-001. PCLX-001 treatment impacts the global myristoylation of lymphoma cell proteins and inhibits early B-cell receptor (BCR) signaling events critical for survival. In addition to abrogating myristoylation of Src family kinases, PCLX-001 also promotes their degradation and, unexpectedly, that of numerous non-myristoylated BCR effectors including c-Myc, NFκB and P-ERK, leading to cancer cell death in vitro and in xenograft models. Because some treated lymphoma patients experience relapse and die, targeting B-cell lymphomas with a NMT inhibitor potentially provides an additional much needed treatment option for lymphoma.

Translational control of breast cancer plasticity.

Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5' Untranslated Regions (5'UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. The accumulation of the corresponding proteins induces plasticity and "fate-switching" toward stem cell-like phenotypes. Mechanistically, we observe that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem-cell-like phenotypes. These effects are overcome with drugs that antagonize translational reprogramming caused by eIF2α phosphorylation (e.g. ISRIB), suggesting that the Integrated Stress Response drives breast cancer plasticity. Collectively, our findings reveal a mechanism of induction of plasticity of breast cancer cells and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.

Insights into Fibroblast Plasticity: Cellular Communication Network 2 Is Required for Activation of Cancer-Associated Fibroblasts in a Murine Model of Melanoma.

Tumor stroma resembles a fibrotic microenvironment, being characterized by the presence of myofibroblast-like cancer-associated fibroblasts (CAFs). In wild-type mice injected with melanoma cells, we show that the stem cell transcription factor Sox2 is expressed by tumor cells and induced in CAFs derived from synthetic fibroblasts. These fibroblasts were labeled postnatally with green fluorescent protein using mice expressing a tamoxifen-dependent Cre recombinase under the control of a fibroblast-specific promoter/enhancer. Conversely, fibroblast activation was impaired in mice with a fibroblast-specific deletion of cellular communication network 2 (Ccn2), associated with reduced expression of α-smooth muscle actin and Sox2. Multipotent Sox2-expressing skin-derived precursor (SKP) spheroids were cultured from murine back skin. Using lineage tracing and flow cytometry, approximately 40% of SKPs were found to be derived from type I collagen-lineage cells and acquired multipotency in culture. Inhibition of mechanotransduction pathways prevented myofibroblast differentiation of SKPs and expression of Ccn2. In SKPs deleted for Ccn2, differentiation into a myofibroblast, but not an adipocyte or neuronal phenotype, was also impaired. In human melanoma, CCN2 expression was associated with a profibrotic integrin alpha (ITGA) 11-expressing subset of CAFs that negatively associated with survival. These results suggest that synthetic dermal fibroblasts are plastic, and that CCN2 is required for the differentiation of dermal progenitor cells into a myofibroblast/CAF phenotype and is, therefore, a therapeutic target in melanoma.

Activation of cancer-associated fibroblasts is required for tumor neovascularization in a murine model of melanoma.

Metastatic melanoma is highly fatal. Within the tumor microenvironment, the role of cancer-associated fibroblasts (CAFs) in melanoma metastasis and progression is relatively understudied. The matricellular protein CCN2 (formerly termed connective tissue growth factor, CTGF) is overexpressed, in a fashion independent of BRAF mutational status, by CAFs in melanoma. Herein, we find, in human melanoma patients, that CCN2 expression negatively correlates with survival and positively correlates with expression of neovascularization markers. To assess the role of CAFs in melanoma progression, we used C57BL/6 mice expressing a tamoxifen-dependent cre recombinase expressed under the control of a fibroblast-specific promoter/enhancer (COL1A2) to delete CCN2 postnatally in fibroblasts. Mice deleted or not for CCN2 in fibroblasts were injected subcutaneously with B16-F10 melanoma cells. Loss of CCN2 in CAFs resulted in reduced CAF activation, as detected by staining with anti-α-smooth muscle actin antibodies, and reduced tumor-induced neovascularization, as detected by micro-computed tomography (micro-CT) and staining with anti-CD31 antibodies. CCN2-deficient B16(F10) cells were defective in a tubule formation/vasculogenic mimicry assay in vitro. Mice deleted for CCN2 in CAFs also showed impaired vasculogenic mimicry of subcutaneously-injected B16-F10 cells in vivo. Our results provide new insights into the cross-talk among different cell types in the tumor microenvironment and suggest CAFs play a heretofore unappreciated role by being essential for tumor neovascularization via the production of CCN2. Our data are consistent with the hypothesis that activated CAFs are essential for melanoma metastasis and that, due to its role in this process, CCN2 is a therapeutic target for melanoma.

COX-2 induces oncogenic micro RNA miR655 in human breast cancer.

We show that Cyclooxygenase-2 over-expression induces an oncogenic microRNA miR655 in human breast cancer cells by activation of EP4. MiR655 expression positively correlated with COX-2 in genetically disparate breast cancer cell lines and increased in all cell lines when grown as spheroids, implicating its link with stem-like cells (SLCs). Ectopic miR655 over-expression in MCF7 and SKBR3 cells resulted in increased proliferation, migration, invasion, spheroid formation and Epithelial to Masenchymal transition (EMT). Conversely, knocking down miR655 in aggressive MCF7-COX2 and SKBR3-COX2 cells reverted these phenotypes. MCF7-miR655 cells displayed upregulated NOTCH/WNT genes; both pathway inhibitors abrogated miR655-induced spheroid formation, linking miR655 with SLC-related pathways. MiR655 expression was dependent on EP4 activity and EP4 downstream signaling pathways PI3K/AKT, ERK and NF-kB and led to TGFß resistance for Smad3 phosphorylation. Tail vein injection of MCF7-miR655 and SKBR3-miR655 cells in NOD/SCID/GUSB-null mice revealed increased lung colony growth and micrometastases to liver and spleen. MiR655 expression was significantly high in human breast tumors (n = 105) compared to non-tumor tissues (n = 20) and associated with reduced patient survival. Thus miR655 could serve as a prognostic breast cancer biomarker.

Matricellular proteins in cancer: a focus on secreted Frizzled-related proteins.

Tumours are complex entities, wherein cancer cells interact with myriad soluble, insoluble and cell associated factors. These microenvironmental mediators regulate tumour growth, progression and metastasis, and are produced by cancer cells and by stromal components such as fibroblast, adipocytes and immune cells. Through their ability to bind to extracellular matrix proteins, cell surface receptors and growth factors, matricellular proteins enable a dynamic reciprocity between cancer cells and their microenvironment. Hence, matricellular proteins play a critical role in tumour progression by regulating where and when cancer cells are exposed to key growth factors and regulatory proteins. Recent studies suggest that, in addition to altering Wingless (Wnt) signalling, certain members of the Secreted Frizzled Related Protein (sFRP) family are matricellular in nature. In this review, we outline the importance of matricellular proteins in cancer, and discuss how sFRPs may function to both inhibit and promote cancer progression in a context-dependent manner. By considering the matricellular functionality of sFRPs, we may better understand their apparently paradoxical roles in cancers.

Deletion of F4L (ribonucleotide reductase) in vaccinia virus produces a selective oncolytic virus and promotes anti-tumor immunity with superior safety in bladder cancer models.

Bladder cancer has a recurrence rate of up to 80% and many patients require multiple treatments that often fail, eventually leading to disease progression. In particular, standard of care for high-grade disease, Bacillus Calmette-Guérin (BCG), fails in 30% of patients. We have generated a novel oncolytic vaccinia virus (VACV) by mutating the F4L gene that encodes the virus homolog of the cell-cycle-regulated small subunit of ribonucleotide reductase (RRM2). The F4L-deleted VACVs are highly attenuated in normal tissues, and since cancer cells commonly express elevated RRM2 levels, have tumor-selective replication and cell killing. These F4L-deleted VACVs replicated selectively in immune-competent rat AY-27 and xenografted human RT112-luc orthotopic bladder cancer models, causing significant tumor regression or complete ablation with no toxicity. It was also observed that rats cured of AY-27 tumors by VACV treatment developed anti-tumor immunity as evidenced by tumor rejection upon challenge and by ex vivo cytotoxic T-lymphocyte assays. Finally, F4L-deleted VACVs replicated in primary human bladder cancer explants. Our findings demonstrate the enhanced safety and selectivity of F4L-deleted VACVs, with application as a promising therapy for patients with BCG-refractory cancers and immune dysregulation.

A pan-cancer analysis of secreted Frizzled-related proteins: re-examining their proposed tumour suppressive function.

Secreted frizzled-related proteins (SFRPs), containing five family members (SFRPs 1-5) are putative extracellular Wnt inhibitors. Given their abilities to inhibit Wnt signalling, as well as the loss of SFRP1 in many cancers, this family is generally considered to be tumour suppressive. In this study we analyzed gene expression, promoter methylation and survival data from over 8000 tumour and normal samples from 29 cancers in order to map the context-specific associations of SFRPs 1-5 with patient survival, gene silencing and gene expression signatures. We show that only SFRP1 associates consistently with tumour suppressive functions, and that SFRP2 and SFRP4 typically associate with a poor prognosis concomitant with the expression of genes associated with epithelial-to-mesenchymal transition. Moreover, our results indicate that while SFRP1 is lost in cancer cells via the process of DNA methylation, SFRP2 and 4 are likely derived from the tumour stroma, and thus tend to increase in tumours as compared to normal tissues. This in-depth analysis highlights the need to study each SFRP as a separate entity and suggests that SFRP2 and SFRP4 should be approached as complex matricellular proteins with functions that extend far beyond their putative Wnt antagonistic ability.

Investigating the utility of human melanoma cell lines as tumour models.

Melanoma researchers utilize cell lines to model many tumour phenomena. It is thus important to understand similarities and differences between cell lines and the tumours that they represent, so that the optimal models can be chosen to answer specific research questions. Herein, we compared the transcriptomes of 42 melanoma cell lines to hundreds of tumours from The Cancer Genome Atlas and thousands of single melanoma cells. Tumour purity was accounted for using the ESTIMATE algorithm, so that differences likely resulting from non-tumour cells could be accounted for. In addition, UV mutational signatures and the expression of skin-associated genes were analyzed in order to identify the putative origin of various cell lines. We found the transcriptional and mutational characteristics of melanoma cell lines to mirror those of the tumours, with the exception of immune-associated transcripts, which were absent in cell culture. We also determined cell lines that highly or poorly recapitulate melanomas and have identified colon (COLO 741) and lung (COLO 699) cancer cell lines that may actually be melanoma. In summary, this study represents a comprehensive comparison of melanoma cell lines and tumours that can be used as a guide for researchers when selecting melanoma cell line models.

Defining the Na+/H+ exchanger NHE1 interactome in triple-negative breast cancer cells.

Mounting evidence supports a major role for the Na+/H+ exchanger NHE1 in cancer progression and metastasis. NHE1 is hyperactive at the onset of oncogenic transformation, resulting in intracellular alkalinization and extracellular microenvironmental acidification. These conditions promote invasion and facilitate metastasis. However, the signal pathways governing the regulation of exchanger activity are still unclear. This is especially important in the aggressively metastatic, triple-negative basal breast cancer subtype. We used affinity chromatography followed by mass spectrometry to identify novel and putative interaction partners of NHE1 in MDA-MB-231 triple-negative breast cancer cells. NHE1 associated with several types of proteins including cytoskeletal proteins and chaperones. We validated protein interactions by co-immunoprecipitation for: 14-3-3, AKT, α-enolase, CHP1, HSP70 and HSP90. Additionally, we used The Cancer Genome Atlas (TCGA) to study NHE1 gene expression in primary patient breast tumours versus adjacent normal tissue. NHE1 expression was elevated in breast tumour samples and, when broken down by breast cancer subtype, NHE1 gene expression was significantly lower in tumours of the basal subtype compared to luminal and HER2+ subtypes. Reverse phase protein array (RPPA) analysis showed that NHE1 expression positively correlated with p90RSK expression in basal, but not luminal, primary tumours. Other proteins were negatively correlated with NHE1 expression in basal breast cancer tumours. Taken together, our data provides the first insight into the signalling molecules that form the NHE1 interactome in triple-negative breast cancer cells. These results will focus our search for novel targeted therapies.