Antibodies toward Na+,HCO3--cotransporter NBCn1/SLC4A7 block net acid extrusion and cause pH-dependent growth inhibition and apoptosis in breast cancer.

BACKGROUND: Na+,HCO3--cotransporter NBCn1/Slc4a7 accelerates murine breast carcinogenesis. Lack of specific pharmacological tools previously restricted therapeutic targeting of NBCn1 and identification of NBCn1-dependent functions in human breast cancer. METHODS: We develop extracellularly-targeted anti-NBCn1 antibodies, screen for functional activity on cells, and evaluate (a) mechanisms of intracellular pH regulation in human primary breast carcinomas, (b) proliferation, cell death, and tumor growth consequences of NBCn1 in triple-negative breast cancer, and (c) association of NBCn1-mediated Na+,HCO3--cotransport with human breast cancer metastasis. RESULTS: We identify high-affinity (KD ≈ 0.14 nM) anti-NBCn1 antibodies that block human NBCn1-mediated Na+,HCO3--cotransport in cells, without cross-reactivity towards human NBCe1 or murine NBCn1. These anti-NBCn1 antibodies abolish Na+,HCO3--cotransport activity in freshly isolated primary organoids from human breast carcinomas and lower net acid extrusion effectively in primary breast cancer tissue from patients with macrometastases in axillary lymph nodes. Inhibitory anti-NBCn1 antibodies decelerate tumor growth in vivo by ~50% in a patient-derived xenograft model of triple-negative breast cancer and pH-dependently reduce colony formation, cause G2/M-phase cell cycle accumulation, and increase apoptosis of metastatic triple-negative breast cancer cells in vitro. CONCLUSIONS: Inhibitory anti-NBCn1 antibodies block net acid extrusion in human breast cancer tissue, particularly from patients with disseminated disease, and pH-dependently limit triple-negative breast cancer growth.

Carbonic anhydrases reduce the acidity of the tumor microenvironment, promote immune infiltration, decelerate tumor growth, and improve survival in ErbB2/HER2-enriched breast cancer.

BACKGROUND: Carbonic anhydrases catalyze CO2/HCO3- buffer reactions with implications for effective H+ mobility, pH dynamics, and cellular acid-base sensing. Yet, the integrated consequences of carbonic anhydrases for cancer and stromal cell functions, their interactions, and patient prognosis are not yet clear. METHODS: We combine (a) bioinformatic analyses of human proteomic data and bulk and single-cell transcriptomic data coupled to clinicopathologic and prognostic information; (b) ex vivo experimental studies of gene expression in breast tissue based on quantitative reverse transcription and polymerase chain reactions, intracellular and extracellular pH recordings based on fluorescence confocal microscopy, and immunohistochemical protein identification in human and murine breast cancer biopsies; and (c) in vivo tumor size measurements, pH-sensitive microelectrode recordings, and microdialysis-based metabolite analyses in mice with experimentally induced breast carcinomas. RESULTS: Carbonic anhydrases-particularly the extracellular isoforms CA4, CA6, CA9, CA12, and CA14-undergo potent expression changes during human and murine breast carcinogenesis. In patients with basal-like/triple-negative breast cancer, elevated expression of the extracellular carbonic anhydrases negatively predicts survival, whereas, surprisingly, the extracellular carbonic anhydrases positively predict patient survival in HER2/ErbB2-enriched breast cancer. Carbonic anhydrase inhibition attenuates cellular net acid extrusion and extracellular H+ elimination from diffusion-restricted to peripheral and well-perfused regions of human and murine breast cancer tissue. Supplied in vivo, the carbonic anhydrase inhibitor acetazolamide acidifies the microenvironment of ErbB2-induced murine breast carcinomas, limits tumor immune infiltration (CD3+ T cells, CD19+ B cells, F4/80+ macrophages), lowers inflammatory cytokine (Il1a, Il1b, Il6) and transcription factor (Nfkb1) expression, and accelerates tumor growth. Supporting the immunomodulatory influences of carbonic anhydrases, patient survival benefits associated with high extracellular carbonic anhydrase expression in HER2-enriched breast carcinomas depend on the tumor inflammatory profile. Acetazolamide lowers lactate levels in breast tissue and blood without influencing breast tumor perfusion, suggesting that carbonic anhydrase inhibition lowers fermentative glycolysis. CONCLUSIONS: We conclude that carbonic anhydrases (a) elevate pH in breast carcinomas by accelerating net H+ elimination from cancer cells and across the interstitial space and (b) raise immune infiltration and inflammation in ErbB2/HER2-driven breast carcinomas, restricting tumor growth and improving patient survival.

Loss of RPTPγ primes breast tissue for acid extrusion, promotes malignant transformation and results in early tumour recurrence and shortened survival.

BACKGROUND: While cellular metabolism and acidic waste handling accelerate during breast carcinogenesis, temporal patterns of acid-base regulation and underlying molecular mechanisms responding to the tumour microenvironment remain unclear. METHODS: We explore data from human cohorts and experimentally investigate transgenic mice to evaluate the putative extracellular HCO3--sensor Receptor Protein Tyrosine Phosphatase (RPTP)γ during breast carcinogenesis. RESULTS: RPTPγ expression declines during human breast carcinogenesis and particularly in high-malignancy grade breast cancer. Low RPTPγ expression associates with poor prognosis in women with Luminal A or Basal-like breast cancer. RPTPγ knockout in mice favours premalignant changes in macroscopically normal breast tissue, accelerates primary breast cancer development, promotes malignant breast cancer histopathologies, and shortens recurrence-free survival. In RPTPγ knockout mice, expression of Na+,HCO3--cotransporter NBCn1-a breast cancer susceptibility protein-is upregulated in normal breast tissue but, contrary to wild-type mice, shows no further increase during breast carcinogenesis. Associated augmentation of Na+,HCO3--cotransport in normal breast tissue from RPTPγ knockout mice elevates steady-state intracellular pH, which has known pro-proliferative effects. CONCLUSIONS: Loss of RPTPγ accelerates cellular net acid extrusion and elevates NBCn1 expression in breast tissue. As these effects precede neoplastic manifestations in histopathology, we propose that RPTPγ-dependent enhancement of Na+,HCO3--cotransport primes breast tissue for cancer development.

Amplified Ca2+ dynamics and accelerated cell proliferation in breast cancer tissue during purinergic stimulation.

Intracellular Ca2+ dynamics shape malignant behaviors of cancer cells. Whereas previous studies focused on cultured cancer cells, we here used breast organoids and colonic crypts freshly isolated from human and murine surgical biopsies. We performed fluorescence microscopy to evaluate intracellular Ca2+ concentrations in breast and colon cancer tissue with preferential focus on intracellular Ca2+ release in response to purinergic and cholinergic stimuli. Inhibition of the sarco-/endoplasmic reticulum Ca2+ ATPase with cyclopiazonic acid elicited larger Ca2+ responses in breast cancer tissue, but not in colon cancer tissue, relative to respective normal tissue. The resting intracellular Ca2+ concentration was elevated, and ATP, UTP and acetylcholine induced strongly augmented intracellular Ca2+ responses in breast cancer tissue compared with normal breast tissue. In contrast, resting intracellular Ca2+ levels and acetylcholine-induced increases in intracellular Ca2+ concentrations were unaffected and ATP- and UTP-induced Ca2+ responses were smaller in colon cancer tissue compared with normal colon tissue. In accordance with the amplified Ca2+ responses, ATP and UTP substantially increased proliferative activity-evaluated by bromodeoxyuridine incorporation-in breast cancer tissue, whereas the effect was minimal in normal breast tissue. ATP caused cell death-identified with ethidium homodimer-1 staining-in breast cancer tissue only at concentrations above the expected pathophysiological range. We conclude that intracellular Ca2+ responses are amplified in breast cancer tissue, but not in colon cancer tissue, and that nucleotide signaling stimulates breast cancer cell proliferation within the extracellular concentration range typical for solid cancer tissue.

Tumor-infiltrating lymphocytes predict improved overall survival after post-mastectomy radiotherapy: a study of the randomized DBCG82bc cohort.

BACKGROUND: The predictive value of tumor-infiltrating lymphocytes (TILs) on the benefit from radiotherapy (RT) remains unclear. Our aim was to investigate the association between TILs and post-mastectomy RT (PMRT) regarding the risk of recurrence and survival in a randomized cohort. MATERIAL AND METHODS: Stromal TILs were histologically estimated in 1011 tumors from high-risk breast cancer (BC) patients from the DBCG82bc trial. Patients were diagnosed between 1982 and 90, treated with total mastectomy and partial axillary lymph node dissection, randomized to ± PMRT followed by adjuvant systemic treatment. A competing risk model, Kaplan-Meier analysis and multivariate Cox regression analysis were used for correlating TILs and clinical outcome. RESULTS: 106 of 1011 patients (10.5%) showed high TILs using a 30% cut-off. In multivariate regression analysis, a high level of TILs was an independent factor associated with lower risk of distant metastasis (DM) and improved overall survival (OS), but without association with loco-regional control. High TILs were associated with a significantly greater OS after PMRT at 20 years compared to low TILs (8% improvement for low TILs (23% to 31%) vs. 22% for high TILs (26% to 48%), interaction-test: p = 0.028). The association between TILs and PMRT was more pronounced among estrogen-receptor negative (ER-) tumors, and patients having ER-/low TILs tumors showed no OS benefit from PMRT at 20 years (-4% improvement for low TILs (28% to 24%) vs. 23% for high TILs (20% to 43%). A similar trend in the association between high TILs and reduced risk of DM after PMRT was seen. CONCLUSION: High TILs predict improved OS from PMRT in BC patients, and the association appeared especially strong for ER- tumors. A trend in the association between high TILs and reduced risk of DM after PMRT was seen. These findings may indicate that RT triggers an immune response inducing a systemic effect outside the treatment field.

Targeting the Acidic Tumor Microenvironment: Unexpected Pro-Neoplastic Effects of Oral NaHCO3 Therapy in Murine Breast Tissue.

The acidic tumor microenvironment modifies malignant cell behavior. Here, we study consequences of the microenvironment in breast carcinomas. Beginning at carcinogen-based breast cancer induction, we supply either regular or NaHCO3-containing drinking water to female C57BL/6j mice. We evaluate urine and blood acid-base status, tumor metabolism (microdialysis sampling), and tumor pH (pH-sensitive microelectrodes) in vivo. Based on freshly isolated epithelial organoids from breast carcinomas and normal breast tissue, we assess protein expression (immunoblotting, mass spectrometry), intracellular pH (fluorescence microscopy), and cell proliferation (bromodeoxyuridine incorporation). Oral NaHCO3 therapy increases breast tumor pH in vivo from 6.68 ± 0.04 to 7.04 ± 0.09 and intracellular pH in breast epithelial organoids by ~0.15. Breast tumors develop with median latency of 85.5 ± 8.2 days in NaHCO3-treated mice vs. 82 ± 7.5 days in control mice. Oral NaHCO3 therapy does not affect tumor growth, histopathology or glycolytic metabolism. The capacity for cellular net acid extrusion is increased in NaHCO3-treated mice and correlates negatively with breast tumor latency. Oral NaHCO3 therapy elevates proliferative activity in organoids from breast carcinomas. Changes in protein expression patterns-observed by high-throughput proteomics analyses-between cancer and normal breast tissue and in response to oral NaHCO3 therapy reveal complex influences on metabolism, cytoskeleton, cell-cell and cell-matrix interaction, and cell signaling pathways. We conclude that oral NaHCO3 therapy neutralizes the microenvironment of breast carcinomas, elevates the cellular net acid extrusion capacity, and accelerates proliferation without net effect on breast cancer development or tumor growth. We demonstrate unexpected pro-neoplastic consequences of oral NaHCO3 therapy that in breast tissue cancel out previously reported anti-neoplastic effects.

Metformin is distributed to tumor tissue in breast cancer patients in vivo: A 11C-metformin PET/CT study.

PURPOSE: Epidemiological studies and randomized clinical trials suggest that the antidiabetic drug, metformin, may have anti-neoplastic effects. The mechanism that mediates these beneficial effects has been suggested to involve direct action on cancer cells, but this will require distribution of metformin in tumor tissue. The present study was designed to investigate metformin distribution in vivo in breast and liver tissue in breast cancer patients. METHODS: Seven patients recently diagnosed with ductal carcinoma were recruited. Using PET/CT, tissue distribution of metformin was determined in vivo for 90 min after injection of a carbon-11-labeled metformin tracer. After surgery, tumor tissue was investigated for gene expression levels of metformin transporter proteins. RESULTS: Tumor tissue displayed a distinct uptake of metformin compared to normal breast tissue AUC0-90 min (75.4 ± 5.5 vs 42.3 ± 6.3) g/ml*min (p = 0.01). Maximal concentration in tumor was at 1 min where it reached approximately 30% of the activity in the liver. The metformin transporter protein with the highest gene expression in tumor tissue was multidrug and toxin extrusion 1 (MATE 1) followed by plasma membrane monoamine transporter (PMAT). CONCLUSION: This study confirms that metformin is transported into tumor tissue in women with breast cancer. This finding support that metformin may have direct anti-neoplastic effects on tumor cells in breast cancer patients. However, distribution of metformin in tumor tissue is markedly lower than in liver, an established metformin target tissue.

Na+,HCO3--cotransporter NBCn1 (Slc4a7) accelerates ErbB2-induced breast cancer development and tumor growth in mice.

Metabolic acid production challenges cellular pH homeostasis in solid cancer tissue, and mechanisms of net acid extrusion represent promising new targets for breast cancer therapy. Here, we used genetically engineered mice to investigate the contribution of the Na+,HCO3--cotransporter NBCn1 (Slc4a7) to intracellular acid-base regulation in ErbB2-induced breast cancer tissue and the consequences of NBCn1 knockout for breast tumor development and growth. We demonstrate an approximately 2-fold increase of NBCn1 protein abundance in ErbB2-induced breast cancer tissue compared to normal breast tissue despite a 4-fold decrease in the NBCn1 mRNA level. In congruence, we show that NBCn1 facilitates net acid extrusion and elevates steady-state intracellular pH in breast cancer tissue. Disruption of NBCn1 expression delayed ErbB2-induced breast carcinogenesis from a median tumor-free survival of 9.5 months in wild-type mice to 12 months in NBCn1-knockout mice and decelerated the tumor growth rate by approximately 1/3. Glycolytic metabolism-evaluated based on the interstitial concentrations of lactate and glucose measured in microdialysates-was increased in breast cancer tissue compared to normal breast tissue, but was unaffected by NBCn1 knockout. Disruption of NBCn1 expression inhibited cell proliferation-evaluated by staining for the proliferative marker Ki67-particularly in central tumor areas with predicted increase in acid loading from glycolytic metabolism. In conclusion, NBCn1 regulates intracellular pH in ErbB2-induced breast cancer tissue by providing a pathway for cellular uptake of HCO3-, which can neutralize metabolic acidic waste products. Disrupting NBCn1 expression delays ErbB2-induced breast cancer development, inhibits cancer cell proliferation, and decelerates tumor growth.

Diabetes and Breast Cancer Subtypes.

BACKGROUND: Women with diabetes have a worse survival after breast cancer diagnosis compared to women without diabetes. This may be due to a different etiological profile, leading to the development of more aggressive breast cancer subtypes. Our aim was to investigate whether insulin and non-insulin treated women with diabetes develop specific clinicopathological breast cancer subtypes compared to women without diabetes. METHODS AND FINDINGS: This cross-sectional study included randomly selected patients with invasive breast cancer diagnosed in 2000-2010. Stratified by age at breast cancer diagnosis (≤50 and >50 years), women with diabetes were 2:1 frequency-matched on year of birth and age at breast cancer diagnosis (both in 10-year categories) to women without diabetes, to select ~300 patients with tumor tissue available. Tumor MicroArrays were stained by immunohistochemistry for estrogen and progesterone receptor (ER, PR), HER2, Ki67, CK5/6, CK14, and p63. A pathologist scored all stains and revised morphology and grade. Associations between diabetes/insulin treatment and clinicopathological subtypes were analyzed using multivariable logistic regression. Morphology and grade were not significantly different between women with diabetes (n = 211) and women without diabetes (n = 101), irrespective of menopausal status. Premenopausal women with diabetes tended to have more often PR-negative (OR = 2.44(95%CI:1.07-5.55)), HER2-negative (OR = 2.84(95%CI:1.11-7.22)), and basal-like (OR = 3.14(95%CI:1.03-9.60) tumors than the women without diabetes, with non-significantly increased frequencies of ER-negative (OR = 2.48(95%CI:0.95-6.45)) and triple negative (OR = 2.60(95%CI:0.88-7.67) tumors. After adjustment for age and BMI, the associations remained similar in size but less significant. We observed no evidence for associations of clinicopathological subtypes with diabetes in postmenopausal women, or with insulin treatment in general. CONCLUSIONS: We found no compelling evidence that women with diabetes, treated with or without insulin, develop different breast cancer subtypes than women without diabetes. However, premenopausal women with diabetes tended to develop breast tumors that do not express hormonal receptors, which are typically associated with poor prognosis.

Importance of margin width in breast-conserving treatment of early breast cancer.

BACKGROUND AND METHOD: The association between margin width and ipsilateral breast tumour recurrence (IBTR, defined as invasive recurrence) was investigated in a population-based nationwide cohort of 11,900 patients undergoing breast-conserving therapy for invasive cancer. RESULTS: The median follow-up was 4.9 years. The cumulative incidence of IBTR at 5 and 9 years was 2.4% and 5.9%, respectively. A final positive margin increased the risk of IBTR (HR 2.51; 95% CI 1.02-6.23). No decrease in IBTR with a wider negative margin compared to a narrow but negative margin was observed in the adjusted analysis of margin width (>0 to <2 mm vs. ≥2 to <5 mm vs. ≥5 mm (reference): HR 1.54 (CI 95% 0.81-2.93) vs. 0.95 (CI 95% 0.56-1.62) vs. 1). However, few patients had narrow margins. The factors associated with increased IBTR were young age (P < 0.001), >4 positive lymph nodes (P = 0.008) and re-excision (P = 0.003). A reduced risk of IBTR was observed with chemotherapy (P < 0.001), boost radiation (P = 0.023) and ER positivity (P < 0.001). CONCLUSION: An overall low rate of IBTR was observed. A final positive margin was associated with a more than twofold risk of IBTR. There was no evidence for better local control with wider margins, but the data were insufficient to show whether narrow margins were as good as wider negative margins in terms of local control. J. Surg. Oncol. 2016;113:609-615. © 2016 Wiley Periodicals, Inc.

The Influence of Repeat Surgery and Residual Disease on Recurrence After Breast-Conserving Surgery: A Danish Breast Cancer Cooperative Group Study.

BACKGROUND: A significant proportion of women who have breast-conserving surgery (BCS) subsequently undergo re-excision or proceed to mastectomy. This study aimed to identify factors associated with residual disease after repeat surgery and to determine their effect on ipsilateral breast tumor recurrence (IBTR) and survival. METHODS: The study cohort was identified within the national population-based registry of the Danish Breast Cancer Cooperative Group, including women who underwent BCS for unilateral invasive breast cancer between 2000 and 2009. RESULTS: The study investigated 12,656 women. Within 2 months after initial BCS, 1342 (11 %) of these women had a re-excision, and 756 (6 %) of the women had a mastectomy. Residual disease was found in 20 % of re-excisions and 59 % of mastectomies. In adjusted analysis, ductal carcinoma in situ (DCIS) outside the invasive tumor, positive initial margin, and age younger than 50 years were associated with increased risk of residual disease. In the adjusted analysis, patients with residual disease after re-excision had an increased risk of IBTR regardless whether residual findings were invasive carcinoma [hazard ratio (HR), 2.97; 95 % confidence interval (CI) 1.57-5.62] or DCIS (HR, 2.58; 95 % CI 1.50-4.45). However, no difference was seen in overall survival comparing patients receiving one excision with those having repeat surgery with or without residual disease (p = 0.96). CONCLUSION: A higher risk of IBTR seen after re-excision was associated with residual disease. Overall survival was similar regardless of repeat surgery and residual findings.

Breast carcinosarcoma: clinical and pathological features.

Carcinosarcoma of the breast is an extremely rare and highly aggressive breast tumor.It has two distinct malignant cell lines involving epithelial (carcinomatous) and mesenchymal (sarcomatous) components. The literature on the topic is sparse. We report a rare case of carcinosarcoma of the breast containing a small fraction of a pancytokeratin positive sarcomatous-appearing cell population i.e. a metaplastic cell population. The patient was treated with a multidisciplinary approach.

Na+,HCO3- -cotransport is functionally upregulated during human breast carcinogenesis and required for the inverted pH gradient across the plasma membrane.

Metabolic and biochemical changes during breast carcinogenesis enhance cellular acid production. Extrusion of the acid load from the cancer cells raises intracellular pH, while it decreases extracellular pH creating an inverted pH gradient across the plasma membrane compared to normal cells and promoting cancer cell metabolism, proliferation, migration, and invasion. We investigated the effects of breast carcinogenesis on the mechanisms of cellular pH control using multicellular epithelial organoids freshly isolated from human primary breast carcinomas and matched normal breast tissue. Intracellular pH was measured by fluorescence microscopy, while protein expression was investigated by immunofluorescence imaging and immunoblotting. We found that cellular net acid extrusion increased during human breast carcinogenesis due to enhanced Na(+),HCO3 (-)-cotransport, which created an alkaline shift (~0.3 units of magnitude) in steady-state intracellular pH of human primary breast carcinomas compared to normal breast tissue. Na(+)/H(+)-exchange activity and steady-state intracellular pH in the absence of CO2/HCO3 (-) were practically unaffected by breast carcinogenesis. These effects were evident under both acidic (pH 6.8, representative of the tumor microenvironment) and physiological (pH 7.4) extracellular conditions. Protein expression of the Na(+),HCO3 (-)-cotransporter NBCn1 (SLC4A7), which has been linked to breast cancer susceptibility in multiple genome-wide association studies, was twofold higher in human breast carcinomas compared to matched normal breast tissue. Protein expression of the Na(+)/H(+)-exchanger NHE1 (SLC9A1) was markedly less affected. We propose that upregulated NBCn1 during human breast carcinogenesis contributes to the characteristic acid distribution within human breast carcinomas and thereby plays a pathophysiological role for breast cancer development and progression.

Apocrine carcinoma arising in a complex fibroadenoma: a case report.

A carcinoma arising in a fibroadenoma is a rare event, which often entails a diagnostic challenge. The most common type is the lobular carcinoma and secondary a ductal carcinoma. We present an extremely rare case of malignant development of an invasive apocrine carcinoma in a complex fibroadenoma and underline the importance for clinicians to recognize the possibility of benign and malignant co-existence especially in older women.

Contribution of Na+,HCO3(-)-cotransport to cellular pH control in human breast cancer: a role for the breast cancer susceptibility locus NBCn1 (SLC4A7).

Genome-wide association studies recently linked the locus for Na(+),HCO(3)(-)-cotransporter NBCn1 (SLC4A7) to breast cancer susceptibility, yet functional insights have been lacking. To determine whether NBCn1, by transporting HCO(3)(-) into cells, may dispose of acid produced during high metabolic activity, we studied the expression of NBCn1 and the functional impact of Na(+),HCO(3)(-)-cotransport in human breast cancer. We found that the plasmalemmal density of NBCn1 was 20-30% higher in primary breast carcinomas and metastases compared to matched normal breast tissue. The increase in NBCn1 density was similar in magnitude to that observed for Na(+)/H(+)-exchanger NHE1 (SLC9A1), a transporter previously implicated in cell migration, proliferation and malignancy. In primary breast carcinomas, the apparent molecular weight for NBCn1 was increased compared to normal tissue. Using pH-sensitive fluorophores, we showed that Na(+),HCO(3)(-)-cotransport is the predominant mechanism of acid extrusion and is inhibited 34 ± 9% by 200 µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in human primary breast carcinomas. At intracellular pH (pH(i) ) levels >6.6, CO(2)/HCO(3)(-)-dependent mechanisms accounted for >90% of total net acid extrusion. Na(+)/H(+)-exchange activity was prominent only at lower pH(i) -values. Furthermore, steady-state pH(i) was 0.35 ± 0.06 units lower in the absence than in the presence of CO(2)/HCO(3)(-). In conclusion, expression of NBCn1 is upregulated in human primary breast carcinomas and metastases compared to normal breast tissue. Na(+),HCO(3)(-)-cotransport is a major determinant of pH(i) in breast cancer and the modest DIDS-sensitivity is consistent with NBCn1 being predominantly responsible. Hence, our results suggest a major pathophysiological role for NBCn1 that may be clinically relevant.