ADAM10 mediates trastuzumab resistance and is correlated with survival in HER2 positive breast cancer.

Trastuzumab prolongs survival in HER2 positive breast cancer patients. However, resistance remains a challenge. We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment. Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding. Therefore, we studied the role of ADAM10 in relation to trastuzumab treatment and resistance in HER2 positive breast cancer. ADAM10 expression was assessed in HER2 positive breast cancer cell lines and xenograft mice treated with trastuzumab. Trastuzumab treatment increased ADAM10 levels in HER2 positive breast cancer cells (p ≤ 0.001 in BT474; p ≤ 0.01 in SKBR3) and in vivo (p ≤ 0.0001) compared to control, correlating with a decrease in PKB phosphorylation. ADAM10 inhibition or knockdown enhanced trastuzumab response in naïve and trastuzumab resistant breast cancer cells. Trastuzumab monotherapy upregulated ADAM10 (p ≤ 0.05); and higher pre-treatment ADAM10 levels correlated with decreased clinical response (p ≤ 0.05) at day 21 in HER2 positive breast cancer patients undergoing a trastuzumab treatment window study. Higher ADAM10 levels correlated with poorer relapse-free survival (p ≤ 0.01) in a cohort of HER2 positive breast cancer patients. Our studies implicate a role of ADAM10 in acquired resistance to trastuzumab and establish ADAM10 as a therapeutic target and a potential biomarker for HER2 positive breast cancer patients

Mechanisms and effects of the heterogeneous response of CAIX to hypoxia

Breast cancer is the most common cancer in females and several molecular subtypes have been described. However, intra-tumour heterogeneity exists and is associated with evolution of cancer metastasis and therapy resistance. The presence of regions of low oxygen (hypoxia) is associated with poor prognosis and the hypoxic microenvironment is also associated with resistance to current therapeutic strategies. Moreover, hypoxia and metabolite deficiency can alter the tumour microenvironment and drive heterogeneity. The pH regulating enzyme carbonic anhydrase IX (CAIX) is strongly induced under hypoxic conditions and its overexpression is likewise associated with poor therapeutic outcome. In a previous study, taking advantage of the membranous localization of CAIX, it was shown that under hypoxia a CAIX positive and CAIX negative population is present in MCF7 cells. RNA sequencing suggested an enrichment of stem cell markers in the CAIX positive population as well as a potential symbiotic relationship between these two subpopulations. In this study MCF7CAIX positive and negative populations were further investigated. Additionally, CAIX positive and negative populations were also found in HCT116 cells and subsequently established as stable cell populations. Both population show similar levels of induction of the hypoxia induced transcription factor HIF-1. The underlying mechanism of CAIX regulation was confirmed to rely on epigenetic modifications, as it could be shown that HIF-1 binding to the CAIX promoter is decreased in the CAIX negative population. To investigate the interaction of CAIX positive and negative cells in 3D models, stably labelled cells using either mCherry of GFP were generated. Upon mixing the two populations in a 1:1 ratio an initially even mix of the cells was observed in the spheroid. However, at a 72 h time point CAIX positive cells moved more towards the centre of the spheroid. This was inhibited by knockdown or pharmacological inhibition of CAIX. A key factor in migration and invasion are metalloproteinases/peptidases (MMPs). We found that MMP14 was upregulated in CAIX positive cells in 2D and 3D models, indicating an underlying mechanism for the re-localisation seen. Analysis of co-culture experiments using 2D a trans-well system revealed that under hypoxia the number of the CAIX positive population was increased in the presence of CAIX negative cells but not vice versa. A similar effect was seen using conditioned media, pointing to a secreted factor. Using a FRET based intracellular sensor to measure lactate levels it could be shown that lactate was higher in the CAIX negative cells. After spiking of the media during live imaging the CAIX positive cells quickly absorbed and retained lactate. In comparison, the CAIX negative cells initially showed an uptake of the metabolite, which was followed by a much quicker release. During co-culture, lactate levels in the conditioned media of CAIX negative cells were higher. Incubation of CAIX positive cells with this conditioned media decreased lactate levels, indicating lactate uptake by the cells. Moreover, knockdown of LDHA and/or B decreased the survival benefit CAIX positive cells had from CAIX negative cells. Another metabolic pathway showing marked differences between the two populations was lipid droplet formation. Under hypoxia CAIX positive cells showed an increased production of lipid droplet in comparison to CAIX negative cells and this was partly due to fatty acid uptake. In line with this, mRNA levels of the fatty acid transporter CD36 as well as some members of the FAB proteins were higher in the CAIX positive cells. Following a previous report showing that lipid droplets formed under hypoxia provide a survival benefit upon re-oxygenation, it was confirmed that CAIX positive cells grew indeed better in this set-up. Therefore, the results suggest a metabolic symbiosis between the CAIX positive and negative populations based on a lactate shuttle and differential uptake of fatty acids. Of wider significance, these findings show that tumours are heterogeneous in the response to hypoxia and that cell populations interact with each other in a symbiotic manner. This highlights the need for a better utilisation and combination of biomarkers to assess metabolic pathways and a better stratification of tumours. Moreover, this work highlights a potential effect of therapeutics addressing metabolic symbiosis.</p

Profile of neratinib and its potential in the treatment of breast cancer

Katharina Feldinger,1 Anthony Kong,2 1Department of Oncology, The Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, 2The Robert Aitkin Institute, School of Cancer Sciences, University of Birmingham, Birmingham, UK Abstract: The HER (ErbB) receptor tyrosine kinase receptors are implicated in many cancers and several anti-HER treatments are now approved. In recent years, a new group of compounds that bind irreversibly to the adenosine triphosphate binding pocket of HER receptors have been developed. One of these compounds, neratinib, has passed preclinical phases and is currently undergoing various clinical trials. This manuscript reviews the preclinical as well as clinical data on neratinib. As a pan-HER inhibitor, this irreversible tyrosine kinase inhibitor binds and inhibits the tyrosine kinase activity of epidermal growth factor receptors, EGFR (or HER1), HER2 and HER4, which leads to reduced phosphorylation and activation of downstream signaling pathways. Neratinib has been shown to be effective against HER2-overexpressing or mutant tumors in vitro and in vivo. Neratinib is currently being investigated in various clinical trials in breast cancers and other solid tumors, including those with HER2 mutation. Earlier studies have already shown promising clinical activity for neratinib. However, more translational research is required to investigate biomarkers that could help to predict response and resistance for selection of appropriate patients for treatment with neratinib, either as monotherapy or in combination with other drug(s). Keywords: neratinib, HKI 272, pan-HER inhibitor, irreversible tyrosine kinase inhibitor, HER (ErbB), breast cance

Adaptation to HIF1α deletion in hypoxic cancer cells by upregulation of GLUT14 and creatine metabolism

Hypoxia-inducible factor 1α is a key regulator of the hypoxia response in normal and cancer tissues. It is well recognized to regulate glycolysis and is a target for therapy. However, how tumor cells adapt to grow in the absence of HIF1α is poorly understood and an important concept to understand for developing targeted therapies is the flexibility of the metabolic response to hypoxia via alternative pathways. We analyzed pathways that allow cells to survive hypoxic stress in the absence of HIF1α, using the HCT116 colon cancer cell line with deleted HIF1α versus control. Spheroids were used to provide a 3D model of metabolic gradients. We conducted a metabolomic, transcriptomic, and proteomic analysis and integrated the results. These showed surprisingly that in three-dimensional growth, a key regulatory step of glycolysis is Aldolase A rather than phosphofructokinase. Furthermore, glucose uptake could be maintained in hypoxia through upregulation of GLUT14, not previously recognized in this role. Finally, there was a marked adaptation and change of phosphocreatine energy pathways, which made the cells susceptible to inhibition of creatine metabolism in hypoxic conditions. Overall, our studies show a complex adaptation to hypoxia that can bypass HIF1α, but it is targetable and it provides new insight into the key metabolic pathways involved in cancer growth