We provide comprehensive clinical data indicating that the glutamine-proline regulatory axis plays an important role in the aggressive subclass of luminal BC and is therefore a potential therapeutic target.

Background Glutamine (Gln) is an abundant nutrient used by cancer cells. Breast cancers cells and particularly triple-receptor negative breast cancer (TNBC) are reported to be dependent on Gln to produce the energy required for survival and proliferation. Despite intense research on the role of the intracellular Gln pathway, few reports have focussed on Gln transporters in breast cancer and TNBC. Methods The role and localisation of the Gln transporter SLC38A2/SNAT2 in response to Gln deprivation or pharmacological stresses was examined in a panel of breast cancer cell lines. Subsequently, the effect of SLC38A2 knockdown in Gln-sensitive cell lines was analysed. The prognostic value of SLC38A2 in a cohort of breast cancer was determined by immunohistochemistry. Results SLC38A2 was identified as a strongly expressed amino acid transporter in six breast cancer cell lines. We confirmed an autophagic route of degradation for SLC38A2. SLC38A2 knockdown decreased Gln consumption, inhibited cell growth, induced autophagy and led to ROS production in a subgroup of Gln-sensitive cell lines. High expression of SLC38A2 protein was associated with poor breast cancer specific survival in a large cohort of patients (p = 0.004), particularly in TNBC (p = 0.02). Conclusions These results position SLC38A2 as a selective target for inhibiting growth of Gln-dependent breast cancer cell lines.

CHIP/STUB1 ubiquitin ligase is a negative co-chaperone for HSP90/HSC70, and its expression is reduced or lost in several cancers, including breast cancer. Using an extensive and well-annotated breast cancer tissue collection, we identified the loss of nuclear but not cytoplasmic CHIP to predict more aggressive tumorigenesis and shorter patient survival, with loss of CHIP in two thirds of ErbB2 and triple-negative breast cancers (TNBC) and in one third of ER breast cancers. Reduced CHIP expression was seen in breast cancer patient-derived xenograft tumors and in ErbB2 and TNBC cell lines. Ectopic CHIP expression in ErbB2 lines suppressed oncogenic traits and xenograft tumor growth. An unbiased screen for CHIP-regulated nuclear transcription factors identified many candidates whose DNA-binding activity was up- or downregulated by CHIP. We characterized myeloid zinc finger 1 (MZF1) as a CHIP target, given its recently identified role as a positive regulator of cathepsin B/L (CTSB/L)-mediated tumor cell invasion downstream of ErbB2. We show that CHIP negatively regulates CTSB/L expression in ErbB2 and other breast cancer cell lines. CTSB inhibition abrogates invasion and matrix degradation and halts ErbB2 breast cancer cell line xenograft growth. We conclude that loss of CHIP remodels the cellular transcriptome to unleash critical pro-oncogenic pathways, such as the matrix-degrading enzymes of the cathepsin family, whose components can provide new therapeutic opportunities in breast and other cancers with loss of CHIP expression. These findings reveal a novel targetable pathway of breast oncogenesis unleashed by the loss of tumor suppressor ubiquitin ligase CHIP/STUB1. .

The version in the Kent Academic Repository may differ from the final published version. Users are advised to check http://kar.kent.ac.uk for the status of the paper. Users should always cite the published version of record.

The majority of breast cancers are oestrogen-receptor-positive (ER+) and are subject to endocrine therapy; however, an unpredictable subgroup of patients will develop resistance to endocrine therapy. The SLC7A5/SLC3A2 complex is a major route for the transport of large neutral essential amino acids through the plasma membrane. Alterations in the expression and function of those amino-acid transporters lead to metabolic reprogramming, which contributes to the tumorigenesis and drug resistance. This study aims to assess the effects and roles of SLC7A5/SLC3A2 co-expression in predicting responses to endocrine therapy in patients with ER+ breast cancer. The biological and clinical impact of SLC7A5/SLC3A2 co-expression was assessed in large annotated cohorts of ER+/HER2− breast cancer with long-term follow-up at the mRNA and protein levels. In vitro experiments were conducted to investigate the effect of SLC7A5/SLC3A2 knockdown in the proliferation of cancer cells and to the sensitivity to tamoxifen. We found that proliferation-related genes are highly expressed in a subgroup of patients with high SLC7A5/SLC3A2, and knockdown of SLC7A5/SLC3A2 decreased proliferation of ER+ breast cancer cells. In patients treated with endocrine therapy, high SLC7A5/SLC3A2 co-expression was associated with poor patient outcome, and depletion of SLC7A5/SLC3A2 using siRNA increased the sensitivity of breast cancer cells to tamoxifen. On the basis of our findings, SLC7A5/SLC3A2 co-expression has the potential of identifying a subgroup of ER+/HER2− breast cancer patients who fail to benefit from endocrine therapy and could guide the choice of other alternative therapies.

Dysregulated cellular metabolism is regarded as one of the hallmarks of cancer with some tumours utilising the glutamine metabolism pathway for their sustained proliferation and survival. Glutamate dehydrogenase (GLUD1) is a key enzyme in glutaminolysis converting glutamate to α-Ketoglutarate for entry into the TCA cycle.Breast cancer (BC) comprises a heterogeneous group of tumours in terms of molecular biology and clinical behaviour, and we have previously shown that altered glutamine metabolism varies substantially among the different molecular subtypes.We hypothesise that the prognostic value of GLUD1 expression will differ between the BC molecular subtypes and may act as a potential therapeutic target for BC tumours.Methods: GLUD1 was assessed at the DNA, mRNA (n=1,980) and protein (n=1,300) levels in large and well-characterised cohorts and correlated with clinicopathological parameters, molecular subtypes, patient outcome and treatments.Results: There was a correlation between GLUD1 mRNA and GLUD1 protein expression which were highly expressed in low grade Luminal/ER+ BC (p<0.01). GLUD1 mRNA and protein was associated with good patient outcome but not in any specific molecular subtypes. However, high GLUD1 protein expression was associated with a better outcome in triple negative (TN) patients treated with chemotherapy (p=0.03).High GLUD1 mRNA was associated with the glutamine transporter, SLC1A5, and leucine transporter, SLC7A8 as well as mTOR (p<0.0001). Conclusion:We provide comprehensive data indicating GLUD1 plays an important role in Luminal/ ER+ BC. GLUD1 expression predicts a better patient outcome and we show that it has the potential for predicting response to chemotherapy in TNBC patients.

Identification of effective and reliable biomarkers that could be used to predict the efficacy of endocrine therapy are of crucial importance to the management of oestrogen receptor positive (ER+) breast cancer (BC). KIF18A, a key regulator of cell cycle, is overexpressed in many human cancers, including BC. In this study, we investigated the role of KIF18A as a biomarker to predict the benefit from endocrine treatment in early ER+ BC patients.Methods: KIF18A expression was assessed at the genomic level using the METABRIC dataset to explore its prognostic and predictive value in ER+ BC patients (n=1506). Predictive significance of KIF18A mRNA was validated using KM-Plot datasets (n=2061). KIF18A protein expression was assessed using immunohistochemistry in a large annotated series of early-stage ER+ BC (n=1592) with long-term follow-up.Results: High mRNA and protein expression of KIF18A were associated with short recurrence free survival (RFS), distant metastasis free survival (DMFS) and BC specific survival (BCSS) (all P<0.05) in ER+ BC in patients who received no adjuvant treatment or adjuvant endocrine therapy. In multivariate analysis, high KIF18A expression was an independent prognostic biomarker for poor RFS (P=0.027) and DMFS (P=0.028) in patients treated with adjuvant endocrine therapy.Conclusion: KIF18A appears to be a candidate biomarker of a subgroup of ER+ BC characterised by poor clinical outcome. High KIF18A expression has prognostic significance to predict poor benefit from endocrine treatment for patients with ER+ BC. Therefore, measurement of KIF18A on ER+ BC patients prior to treatment could guide clinician decision on benefit from endocrine therapy.

<p>Tables and Figures</p>