The SRC-family serves as a therapeutic target in triple negative breast cancer with acquired resistance to chemotherapy.

BACKGROUND: Resistance to chemotherapy, combined with heterogeneity among resistant tumors, represents a significant challenge in the clinical management of triple negative breast cancer (TNBC). By dissecting molecular pathways associated with treatment resistance, we sought to define patient sub-groups and actionable targets for next-line treatment. METHODS: Bulk RNA sequencing and reverse phase protein array profiling were performed on isogenic patient-derived xenografts (PDX) representing paclitaxel-sensitive and -resistant tumors. Pathways identified as upregulated in the resistant model were further explored as targets in PDX explants. Their clinical relevance was assessed in two distinct patient cohorts (NeoAva and MET500). RESULTS: Increased activity in signaling pathways involving SRC-family kinases (SFKs)- and MAPK/ERK was found in treatment resistant PDX, with targeted inhibitors being significantly more potent in resistant tumors. Up-regulation of SFKs- and MAPK/ERK-pathways was also detected in a sub-group of chemoresistant patients after neoadjuvant treatment. Furthermore, High SFK expression (of either SRC, FYN and/or YES1) was detected in metastatic lesions of TNBC patients with fast progressing disease (median disease-free interval 27 vs 105 months). CONCLUSIONS: Upregulation of SFK-signaling is found in a subset of chemoresistant tumors and is persistent in metastatic lesions. Based on pre-clinical results, these patients may respond favorably to treatment targeting SFKs.

Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells.

Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential.

Serum levels of inflammation-related markers and metabolites predict response to neoadjuvant chemotherapy with and without bevacizumab in breast cancers.

Angiogenesis is necessary for tumor growth and has been targeted in breast cancer; however, it is unclear which patients will respond and benefit from antiangiogenic therapy. We report noninvasive monitoring of patient response to neoadjuvant chemotherapy given alone or in combination with anti-vascular endothelial growth factor (bevacizumab) in a randomized clinical trial. At four time points during neoadjuvant chemotherapy ± bevacizumab of receptor tyrosine-protein kinase erbB-2-negative breast cancers, we measured metabolites and inflammation-related markers in patient's serum. We report significant changes in the levels of several molecules induced by bevacizumab, the most prominent being an increase in pentraxin 3 (PTX3) and von Willebrand factor (VWF). Serum levels of AXL, VWF and pulmonary and activation-regulated cytokine (PARC/CCL18) reflected response to chemotherapy alone or in combination with bevacizumab. We further analyzed serum cytokines in relation to tumor characteristics such as gene expression, tumor metabolites and tumor infiltrating leukocytes. We found that VWF and growth-differentiation factor 15 tumor mRNA levels correlated with their respective serum protein levels suggesting that these cytokines may be produced by tumors and outflow to the bloodstream while influencing the tumor microenvironment locally. Finally, we used binomial logistic regression which allowed to predict patient's response using only 10 noninvasive biomarkers. Our study highlights the potential of monitoring circulating levels of cytokines and metabolites during breast cancer therapy.

Immune phenotype of tumor microenvironment predicts response to bevacizumab in neoadjuvant treatment of ER-positive breast cancer.

Antiangiogenic drugs are potentially a useful supplement to neoadjuvant chemotherapy for a subgroup of patients with human epidermal growth factor receptor 2 (HER2) negative breast cancer, but reliable biomarkers for improved response are lacking. Here, we report on a randomized phase II clinical trial to study the added effect of bevacizumab in neoadjuvant chemotherapy with FEC100 (5-fluorouracil, epirubicin and cyclophosphamide) and taxanes (n = 132 patients). Gene expression from the tumors was obtained before neoadjuvant treatment, and treatment response was evaluated by residual cancer burden (RCB) at time of surgery. Bevacizumab increased the proportion of complete responders (RCB class 0) from 5% to 20% among patients with estrogen receptor (ER) positive tumors (P = .02). Treatment with bevacizumab was associated with improved 8-year disease-free survival (P = .03) among the good responders (RCB class 0 or I). Patients treated with paclitaxel (n = 45) responded better than those treated with docetaxel (n = 21; P = .03). Improved treatment response was associated with higher proliferation rate and an immune phenotype characterized by high presence of classically activated M1 macrophages, activated NK cells and memory activated CD4 T cells. Treatment with bevacizumab increased the number of adverse events, including hemorrhage, hypertension, infection and febrile neutropenia, but despite this, the ECOG status was not affected.

Immune stimulatory effect of anti-EpCAM immunotoxin - improved overall survival of metastatic colorectal cancer patients.

Introduction: In a recent phase I trial in a heterogeneous group of carcinoma patients with advanced disease, we did not observe objective responses by CT at 8 weeks in patients treated with either the anti-EpCAM immunotoxin MOC31PE alone or administered in combination with the immunosuppressor cyclosporin (CsA). We have now assessed overall survival (OS) data for the two groups to reveal potential differences, and to elucidate putative underlying mechanisms.Material and methods: The OS time of MOC31PE monotherapy (34 patients) and MOC31PE in combination with CsA (23 patients), was assessed. Pre- and post-treatment patient sera were analyzed in a multiplex immunoassay, and the immunogenic effects of MOC31PE were studied in vitro and in a dendritic cell maturation assay.Results: When the data were analyzed for all treated patients regardless of cancer type, the MOC31PE alone group had a median OS of 12.7 months (95% CI = 5.6-19.8 months) compared to 6.2 months (95% CI = 5.6-6.8 months) (p=.066) for the patients treated with MOC31PE + CsA group. For the subgroup of patients with colorectal cancer, the median OS survival was 16.3 months (95% CI = 5.6-27.0) for the MOC31PE only cohort (n = 15), compared to 6.0 months (CI = 5.8-6.2) (p < .001) for the combination group. The cytokine profile in patient sera and the in vitro immunological studies indicate that MOC31PE induced an immunogenic response leading to T-cell activation; a response that was suppressed in patients treated with MOC31PE + CsA.Conclusions: The results reveal a promising clinical benefit of anti-EpCAM immunotoxin treatment in patients with advanced disease, an effect apparently explained by a previously unknown immunogenic effect of MOC31PE.

Assessing Treatment Response and Prognosis by Serum and Tissue Metabolomics in Breast Cancer Patients.

Patients with locally advanced breast cancer have a worse prognosis compared to patients with localized tumors and require neoadjuvant treatment before surgery. The aim of this study was to characterize the systemic metabolic effect of neoadjuvant chemotherapy in patients with large primary breast cancers and to relate these changes to treatment response and long-term survival. This study included 132 patients with large primary breast tumors randomized to receive neoadjuvant chemotherapy with or without the addition of the antiangiogenic drug Bevacizumab. Tumor biopsies and serum were collected before and during treatment and, serum additionally 6 weeks after surgery. Samples were analyzed by nuclear magnetic resonance spectroscopy (NMR). Correlation analysis showed low correlations between metabolites measured in cancer tissue and serum. Multilevel partial least squares discriminant analysis (PLS-DA) showed clear changes in serum metabolite levels during treatment (p-values ≤ 0.001), including unfavorable changes in lipid levels. PLS-DA revealed metabolic differences between tissue samples from survivors and nonsurvivors collected 12 weeks into treatment with an accuracy of 72% (p-value = 0.005); however, this was not evident in serum samples. Our results demonstrate a potential clinical application for serum-metabolomics for patient monitoring during and after treatment, and indicate potential for tissue NMR spectroscopy for predicting patient survival.

Interplay of choline metabolites and genes in patient-derived breast cancer xenografts.

INTRODUCTION: Dysregulated choline metabolism is a well-known feature of breast cancer, but the underlying mechanisms are not fully understood. In this study, the metabolomic and transcriptomic characteristics of a large panel of human breast cancer xenograft models were mapped, with focus on choline metabolism. METHODS: Tumor specimens from 34 patient-derived xenograft models were collected and divided in two. One part was examined using high-resolution magic angle spinning (HR-MAS) MR spectroscopy while another part was analyzed using gene expression microarrays. Expression data of genes encoding proteins in the choline metabolism pathway were analyzed and correlated to the levels of choline (Cho), phosphocholine (PCho) and glycerophosphocholine (GPC) using Pearson's correlation analysis. For comparison purposes, metabolic and gene expression data were collected from human breast tumors belonging to corresponding molecular subgroups. RESULTS: Most of the xenograft models were classified as basal-like (N = 19) or luminal B (N = 7). These two subgroups showed significantly different choline metabolic and gene expression profiles. The luminal B xenografts were characterized by a high PCho/GPC ratio while the basal-like xenografts were characterized by highly variable PCho/GPC ratio. Also, Cho, PCho and GPC levels were correlated to expression of several genes encoding proteins in the choline metabolism pathway, including choline kinase alpha (CHKA) and glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5). These characteristics were similar to those found in human tumor samples. CONCLUSION: The higher PCho/GPC ratio found in luminal B compared with most basal-like breast cancer xenograft models and human tissue samples do not correspond to results observed from in vitro studies. It is likely that microenvironmental factors play a role in the in vivo regulation of choline metabolism. Cho, PCho and GPC were correlated to different choline pathway-encoding genes in luminal B compared with basal-like xenografts, suggesting that regulation of choline metabolism may vary between different breast cancer subgroups. The concordance between the metabolic and gene expression profiles from xenograft models with breast cancer tissue samples from patients indicates that these xenografts are representative models of human breast cancer and represent relevant models to study tumor metabolism in vivo.

Triple-negative breast cancer and the need for new therapeutic targets.

Triple-negative breast cancers (TNBCs) are a diverse and heterogeneous group of tumors that by definition lack estrogen and progesterone receptors and amplification of the HER2 gene. The majority of the tumors classified as TNBCs are highly malignant, and only a subgroup responds to conventional chemotherapy with a favorable prognosis. Results from decades of research have identified important molecular characteristics that can subdivide this group of breast cancers further. High-throughput molecular analyses including sequencing, pathway analyses, and integrated analyses of alterations at the genomic and transcriptomic levels have improved our understanding of the molecular alterations involved in tumor development and progression. How this knowledge should be used for rational selection of therapy is a challenging task and the subject of numerous ongoing research programs. This review summarizes the current knowledge on the clinical characteristics and molecular alterations of TNBCs. Currently used conventional therapeutic strategies and targeted therapy studies are discussed, with references to recently published results on the molecular characterization of TNBCs.

Associations between tumor vascularization assessed by in vivo DCE-MRI and the presence of disseminated tumor cells in bone marrow in breast cancer patients at the time of diagnosis.

PURPOSE: To explore possible associations between in vivo pharmacokinetic dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) parameters and the presence of disseminated tumor cells (DTCs) in bone marrow in breast cancer patients at the time of diagnosis. MATERIALS AND METHODS: Thirty-seven women with breast cancer (stage T2-4N0-1M0) were included. Patients were classified as DTC+ if one or more DTCs were detected by immunocytochemistry. DCE-MRI was acquired with a radial 3D T1 -weighted spoiled gradient echo sequence with k-space weighted image contrast. K(trans), kep, and ve were calculated using the extended Tofts model and a population-derived arterial input function. The nonparametric Mann-Whitney U-test was used to compare the histogram distributions of the pharmacokinetic parameters for the DTC+ and the DTC- patients. RESULTS: DTCs were detected in 7 of the 37 patients (19%). In DTC+ patients, the distribution of tumor K(trans) and kep were significantly (P < 0.01) more shifted towards lower values than in DTC- patients. CONCLUSION: An association between vascular dependent pharmacokinetic DCE-MRI parameters and the presence of DTCs were found. Compared to DTC- patients, DTC+ patients had poorer perfusion and permeability, indicative of hypoxia. Thus, pharmacokinetic parameters might be surrogate biomarkers of metastatic potential and future relapse.

Positron emission tomography and pharmacokinetics of 2-[18F]-fluoroethyl choline for metabolic studies in breast cancer xenografts.

BACKGROUND: Breast carcinomas (BC) can have abnormal choline (Cho) metabolism. Earlier studies indicated that Cho uptake can differ between different subtypes of BC. The purpose of this study was to investigate uptake of 2-[(18)F]-fluoroethyl-choline ([(18)F]FECh) in three different patient-derived breast cancer xenografts (BCXs) using dynamic positron emission tomography (dPET). MATERIAL AND METHODS: Nine athymic nude mice bearing bilateral MAS98.12 (basal-like), HBCx34 or MAS98.06 (both luminal B) BCXs were subjected to a 90-minute dPET scan following a bolus injection of 10 MBq of [(18)F]FECh. A Patlak Plot analysis and a well-established two-tissue compartment model were fitted to the uptake curves of the whole tumors, providing estimates of transfer rates between the vascular, non-metabolized and metabolized compartments. Patlak slope KP and intercept V, the rate constants k1, k2, k3, the intravascular fraction vb and MR[(18)F]FECh were estimated. Additionally, analyses of terminal blood samples and tumor cell suspension incubated with [(18)F]FECh were performed. RESULTS: [(18)F]FECh uptake in all BCXs was similar to surrounding normal tissue, thus creating no image contrast. The average liver uptake was 10 times higher than the tumor uptake. The uptake in MAS98.12 was higher than in the other two BCXs during the whole course of the acquisition, and was significantly higher than in HBCx34 at 10-30 minutes after injection. No significant differences were found for k1, MR[(18)F]FECh and intravascular fraction vb. Patlak slope KP, k2 and k3 were significantly lower for the MAS98.12 xenograft, in line with in vitro results. KP was correlated with both MR[(18)F]FECh and k3. CONCLUSIONS: dPET demonstrated that different subtypes of breast cancer have different uptake of [(18)F]FECh. Differences in rate constants and KP were in line with in vitro uptake in cell suspensions and earlier spectroscopy and gene expression analysis.

An integrated omics approach highlights how epigenetic events can explain and predict response to neoadjuvant chemotherapy and bevacizumab in breast cancer.

Treatment with the anti-angiogenic drug bevacizumab in addition to chemotherapy has shown efficacy for breast cancer in some clinical trials, but better biomarkers are needed to optimally select patients for treatment. Here, we present an omics approach where DNA methylation profiles are integrated with gene expression and results from proteomic data in breast cancer patients to predict response to therapy and pinpoint response-related epigenetic events. Fresh-frozen tumor biopsies taken before, during, and after treatment from human epidermal growth factor receptor 2 negative non-metastatic patients receiving neoadjuvant chemotherapy with or without bevacizumab were subjected to molecular profiling. Here, we report that DNA methylation at enhancer CpGs related to cell cycle regulation can predict response to chemotherapy and bevacizumab for the estrogen receptor positive subset of patients (AUC = 0.874), and we validated this observation in an independent patient cohort with a similar treatment regimen (AUC = 0.762). Combining the DNA methylation scores with the scores from a previously published protein signature resulted in a slight increase in the prediction performance (AUC = 0.784). We also show that tumors receiving the combination treatment underwent more extensive epigenetic alterations. Finally, we performed an integrative expression-methylation quantitative trait loci analysis on alterations in DNA methylation and gene expression levels, showing that the epigenetic alterations that occur during treatment are different between responders and non-responders and that these differences may be explained by the proliferation-epithelial-to-mesenchymal transition axis through the activity of grainyhead like transcription factor 2. Using tumor purity computed from copy number data, we developed a method for estimating cancer cell-specific methylation to confirm that the association to response reflects DNA methylation in cancer cells. Taken together, these results support the potential for clinical benefit of the addition of bevacizumab to chemotherapy when administered to the correct patients.

Low-molecular contrast agent dynamic contrast-enhanced (DCE)-MRI and diffusion-weighted (DW)-MRI in early assessment of bevacizumab treatment in breast cancer xenografts.

PURPOSE: To investigate the effect of bevacizumab treatment on vascular architecture and function in two xenograft models with different angiogenic properties using diffusion-weighted magnetic resonance imaging (DW-MRI) and dynamic contrast-enhanced MRI (DCE-MRI). MATERIALS AND METHODS: Mice carrying basal-like (MAS98.12) or luminal-like (MAS98.06) orthotopic breast cancer xenografts were treated with bevacizumab (5 mg/kg), doxorubicin (8 mg/kg), or both drugs in combination. DW-MRI and DCE-MRI were performed before and 3 days after treatment using a Bruker 7T preclinical scanner. Mean microvessel density (MVD) and proliferating microvessel density (pMVD) in the tumors were determined for evaluation of vascular response to bevacizumab treatment. RESULTS: No changes in DCE-MRI or DW-MRI parameters were observed in untreated controls during the experiment period. DW-MRI showed increased apparent diffusion coefficient (ADC) values in all treatment groups in both basal-like and luminal-like xenografts. DCE-MRI showed increased contrast agent uptake, particularly in central regions of the tumors, after bevacizumab/combination treatment in both xenograft models. This was accompanied by decreased MVD and pMVD in basal-like xenografts. Doxorubicin treatment had no effect on DCE-MRI parameters in any of the xenograft models. CONCLUSION: Both DW-MRI and DCE-MRI demonstrated an early response to bevacizumab treatment in the xenograft tumors. Increased contrast agent uptake and reduced MVD/pMVD is consistent with a normalization of vascular function.

High-throughput screen in vitro identifies dasatinib as a candidate for combinatorial treatment with HER2-targeting drugs in breast cancer.

Human epidermal growth factor receptor 2-positive (HER2+) breast cancer is an aggressive subtype of this disease. Targeted treatment has improved outcome, but there is still a need for new therapeutic strategies as some patients respond poorly to treatment. Our aim was to identify compounds that substantially affect viability in HER2+ breast cancer cells in response to combinatorial treatment. We performed a high-throughput drug screen of 278 compounds in combination with trastuzumab and lapatinib using two HER2+ breast cancer cell lines (KPL4 and SUM190PT). The most promising drugs were validated in vitro and in vivo, and downstream molecular changes of the treatments were analyzed. The screen revealed multiple drugs that could be used in combination with lapatinib and/or trastuzumab. The Src-inhibitor dasatinib showed the largest combinatorial effect together with lapatinib in the KPL4 cell line compared to treatment with dasatinib alone (p < 0.01). In vivo, only lapatinib significantly reduced tumor growth (p < 0.05), whereas dasatinib alone, or in combination with lapatinib, did not show significant effects. Protein analyses of the treated xenografts showed significant alterations in protein levels compared to untreated controls, suggesting that all drugs reached the tumor and exerted a measurable effect. In silico analyses suggested activation of apoptosis and reduced activity of survival pathways by all treatments, but the opposite pattern was observed for the combinatorial treatment compared to lapatinib alone.

Liver X receptors induce antiproliferative effects in basal-like breast cancer.

Liver X receptors (LXRs) are nuclear transcription factors important in the regulation of cholesterol transport, and glucose and fatty acid metabolism. The antiproliferative role of LXRs has been studied in a variety of malignancies and may represent a therapeutic opportunity in cancers lacking targeted therapies, such as triple-negative breast cancer. In this study, we investigated the impact of LXR agonists alone and in combination with carboplatin in preclinical models of breast cancer. In vitro experiments revealed a dose-dependent decrease in tumor cell proliferation in estrogen receptor-positive breast cancer cells, whereas LXR activation in vivo resulted in an increased growth inhibitory effect in a basal-like breast cancer model (in combination with carboplatin). Functional proteomic analysis identified differences in protein expression between responding and nonresponding models related to Akt activity, cell-cycle progression, and DNA repair. Furthermore, pathway analysis suggested that the LXR agonist in combination with carboplatin inhibits the activity of targets of E2F transcription factors and affects cholesterol homeostasis in basal-like breast cancer.

In vivo MRI and histopathological assessment of tumor microenvironment in luminal-like and basal-like breast cancer xenografts.

PURPOSE: To explore tumor pathophysiology with special attention to the microenvironment in two molecular subtypes of human breast cancer using in vivo magnetic resonance imaging (MRI) and histopathology. The impact of tumor growth, size, and the influence of estradiol were also investigated. MATERIALS AND METHODS: Two orthotopic and directly transplanted human breast cancer models representing luminal-like and basal-like molecular subtypes were characterized by dynamic contrast-enhanced MRI and diffusion-weighted MRI. Ex vivo measurements of vascularization, hypoxia, mitoses, and the level of VEGF activations were associated with the calculated in vivo MRI parameters of the tumors. RESULTS: The vascular permeability and perfusion (K(trans) ) was significantly higher in basal-like compared to luminal-like tumors. These findings were confirmed by a 4-fold higher proliferating microvessel density (pMVD) in basal-like tumors, reflecting the difference in aggressiveness between the subtypes. No effect of tumor growth was observed during 6 days of growth in any of the models; however, large tumors had lower K(trans) , higher extracellular extravascular volume fraction (v(e) ), and more hypoxia than medium-sized tumors. Estradiol withdrawal induced increased K(trans) , v(e) , and tumor water diffusion (ADC) in luminal-like tumors, corresponding to increased VEGFR2 activation, which is likely to cause increased tumor vessel permeability. CONCLUSION: These novel data confirm the potential of functional MRI methods to map histopathologically proven changes in breast tumor vasculature and microenvironment in vivo.

Quantification of Tumor Hypoxia through Unsupervised Modelling of Consumption and Supply Hypoxia MR Imaging in Breast Cancer.

The purpose of the present study is to investigate if consumption and supply hypoxia (CSH) MR-imaging can depict breast cancer hypoxia, using the CSH-method initially developed for prostate cancer. Furthermore, to develop a generalized pan-cancer application of the CSH-method that doesn't require a hypoxia reference standard for training the CSH-parameters. In a cohort of 69 breast cancer patients, we generated, based on the principles of intravoxel incoherent motion modelling, images reflecting cellular density (apparent diffusion coefficient; ADC) and vascular density (perfusion fraction; fp). Combinations of the information in these images were compared to a molecular hypoxia score made from gene expression data, aiming to identify a way to apply the CSH-methodology in breast cancer. Attempts to adapt previously proposed models for prostate cancer included direct transfers and model parameter rescaling. A novel approach, based on rescaling ADC and fp data to give more nuanced response in the relevant physiologic range, was also introduced. The new CSH-method was validated in a prostate cancer cohort with known hypoxia status. The proposed CSH-method gave estimates of hypoxia that was strongly correlated to the molecular hypoxia score in breast cancer, and hypoxia as measured in pathology slices stained with pimonidazole in prostate cancer. The generalized approach to CSH-imaging depicted hypoxia in both breast and prostate cancers and requires no model training. It is easy to implement using readily available technology and encourages further investigation of CSH-imaging in other cancer entities and in other settings, with the goal being to overcome hypoxia-induced resistance to treatment.

13C high-resolution-magic angle spinning MRS reveals differences in glucose metabolism between two breast cancer xenograft models with different gene expression patterns.

Tumor cells have increased glycolytic activity, and glucose is mainly used to form lactate and alanine, even when high concentrations of oxygen are present (Warburg effect). The purpose of the present study was to investigate glucose metabolism in two xenograft models representing basal-like and luminal-like breast cancer using (13) C high-resolution-magic angle spinning (HR-MAS) MRS and gene expression analysis. Tumor tissue was collected from two groups for each model: untreated mice (n=19) and a group of mice (n=16) that received an injection of [1-(13) C]-glucose 10 or 15 min before harvesting the tissue. (13) C HR-MAS MRS was performed on the tumor samples and differences in the glucose/alanine (Glc/Ala), glucose/lactate (Glc/Lac) and alanine/lactate (Ala/Lac) ratios between the models were studied. The expression of glycolytic genes was studied using tumor tissue from the same models. In the natural abundance MR spectra, a significantly lower Glc/Ala and Glc/Lac ratio (p<0.001) was observed in the luminal-like model compared with the basal-like model. In the labeled samples, the predominant glucose metabolites were lactate and alanine. Significantly lower Glc/Ala and Glc/Lac ratios were observed in the luminal-like model (p<0.05). Most genes contributing to glycolysis were expressed at higher levels in the luminal-like model (fdr<0.001). The lower Glc/Ala and Glc/Lac ratios and higher glycolytic gene expression observed in the luminal-like model indicates that the transformation of glucose to lactate and alanine occurred faster in this model than in the basal-like model, which has a growth rate several times faster than that of the luminal-like model. The results from the present study suggest that the tumor growth rate is not necessarily a determinant of glycolytic activity.

Detection of phenotype-specific therapeutic vulnerabilities in breast cells using a CRISPR loss-of-function screen.

Cellular phenotype plasticity between the epithelial and mesenchymal states has been linked to metastasis and heterogeneous responses to cancer therapy, and remains a challenge for the treatment of triple-negative breast cancer (TNBC). Here, we used isogenic human breast epithelial cell lines, D492 and D492M, representing the epithelial and mesenchymal phenotypes, respectively. We employed a CRISPR-Cas9 loss-of-function screen targeting a 2240-gene 'druggable genome' to identify phenotype-specific vulnerabilities. Cells with the epithelial phenotype were more vulnerable to the loss of genes related to EGFR-RAS-MAPK signaling, while the mesenchymal-like cells had increased sensitivity to knockout of G2 -M cell cycle regulators. Furthermore, we discovered knockouts that sensitize to the mTOR inhibitor everolimus and the chemotherapeutic drug fluorouracil in a phenotype-specific manner. Specifically, loss of EGFR and fatty acid synthase (FASN) increased the effectiveness of the drugs in the epithelial and mesenchymal phenotypes, respectively. These phenotype-associated genetic vulnerabilities were confirmed using targeted inhibitors of EGFR (gefitinib), G2 -M transition (STLC), and FASN (Fasnall). In conclusion, a CRISPR-Cas9 loss-of-function screen enables the identification of phenotype-specific genetic vulnerabilities that can pinpoint actionable targets and promising therapeutic combinations.

Protein Signature Predicts Response to Neoadjuvant Treatment With Chemotherapy and Bevacizumab in HER2-Negative Breast Cancers.

PURPOSE: Antiangiogenic therapy using bevacizumab has proven effective for a number of cancers; however, in breast cancer (BC), there is an unmet need to identify patients who benefit from such treatment. PATIENTS AND METHODS: In the NeoAva phase II clinical trial, patients (N = 132) with large (≥ 25 mm) human epidermal growth factor receptor 2 (HER2)-negative primary tumors were randomly assigned 1:1 to treatment with neoadjuvant chemotherapy (CTx) alone or in combination with bevacizumab (Bev plus CTx). The ratio of the tumor size after relative to before treatment was calculated into a continuous response scale. Tumor biopsies taken prior to neoadjuvant treatment were analyzed by reverse-phase protein arrays (RPPA) for expression levels of 210 BC-relevant (phospho-) proteins. Lasso regression was used to derive a predictor of tumor shrinkage from the expression of selected proteins prior to treatment. RESULTS: We identified a nine-protein signature score named vascular endothelial growth factor inhibition response predictor (ViRP) for use in the Bev plus CTx treatment arm able to predict with accuracy pathologic complete response (pCR) (area under the curve [AUC] = 0.85; 95% CI, 0.74 to 0.97) and low residual cancer burden (RCB 0/I) (AUC = 0.80; 95% CI, 0.68 to 0.93). The ViRP score was significantly lower in patients with pCR (P < .001) and in patients with low RCB (P < .001). The ViRP score was internally validated on mRNA data and the resultant surrogate mRNA ViRP score significantly separated the pCR patients (P = .016). Similarly, the mRNA ViRP score was validated (P < .001) in an independent phase II clinical trial (PROMIX). CONCLUSION: Our ViRP score, integrating the expression of nine proteins and validated on mRNA data both internally and in an independent clinical trial, may be used to increase the likelihood of benefit from treatment with bevacizumab combined with chemotherapy in patients with HER2-negative BC.

RAB5A expression is a predictive biomarker for trastuzumab emtansine in breast cancer.

HER2 is a predictive biomarker for HER2-targeted therapeutics. For antibody-drug conjugates (ADCs; e.g., trastuzumab emtansine (T-DM1)), HER2 is utilized as a transport gate for cytotoxic agents into the cell. ADC biomarkers may therefore be more complex, also reflecting the intracellular drug transport. Here we report on a positive correlation between the early endosome marker RAB5A and T-DM1 sensitivity in five HER2-positive cell lines. Correlation between RAB5A expression and T-DM1 sensitivity is confirmed in breast cancer patients treated with trastuzumab emtansine/pertuzumab in the I-SPY2 trial (NCT01042379), but not in the trastuzumab/paclitaxel control arm. The clinical correlation is further verified in patients from the KAMILLA trial (NCT01702571). In conclusion, our results suggest RAB5A as a predictive biomarker for T-DM1 response and outline proteins involved in endocytic trafficking as predictive biomarkers for ADCs.