Increased metabolism and insufficient blood supply cause acidic waste product accumulation in solid cancers. During carcinogenesis, cellular acid extrusion is upregulated but the underlying molecular mechanisms and their consequences for cancer growth and progression have not been established. Genome-wide association studies have indicated a possible link between the Na⁺, HCO₃⁻-cotransporter NBCn1 (SLC4A7) and breast cancer. We tested the functional consequences of NBCn1 knockout (KO) for breast cancer development. NBCn1 protein expression increased 2.5-fold during breast carcinogenesis and was responsible for the increased net acid extrusion and alkaline intracellular pH of breast cancer compared with normal breast tissue. Genetic disruption of NBCn1 delayed breast cancer development: tumor latency was ~50% increased while tumor growth rate was ~65% reduced in NBCn1 KO compared with wild-type (WT) mice. Breast cancer histopathology in NBCn1 KO mice differed from that in WT mice and included less aggressive tumor types. The extracellular tumor microenvironment in NBCn1 KO mice contained higher concentrations of glucose and lower concentrations of lactate than that in WT mice. Independently of NBCn1 genotype, the cleaved fraction of poly(ADP-ribose) polymerase (PARP)-1 and expression of monocarboxylate transporter (MCT)1 increased while phosphorylation of Akt and ERK1 decreased as functions of tumor volume. Cell proliferation, evaluated from Ki-67 and phospho-histone H₃staining, was ~60% lower in breast cancer of NBCn1 KO than that of WT mice when corrected for variations in tumor size. We conclude that NBCn1 facilitates acid extrusion from breast cancer tissue, maintains the alkaline intracellular environment and promotes aggressive cancer development and growth.

High metabolism and insufficient blood supply are characteristics of cancer tissue, which in combination with biochemical changes favor glycolytic metabolism and result in prominent intracellular acid production. Although extracellular pH at the core of malignant tumors is as low as one unit below normal, intracellular pH (pHi) in tumor cells is typically normal or even slightly alkaline. Thus, cancer cells must possess efficient mechanisms of acid extrusion to eliminate the excess acid load. We investigated the role of the Na+,HCO3–cotransporter NBCn1 (SLC4A7), which in recent genome-wide association studies has been linked to human breast cancer. Based on immunohistochemistry of tumor slices and immunoblotting of enzymatically isolated epithelial organoids, we found that NBCn1 expression is upregulated in human and murine primary breast carcinomas and metastases compared to normal breast tissue. The upregulation of NBCn1 was of similar or greater magnitude than that observed for the Na+/H+-exchanger NHE1, which has previously been implicated in cell migration, proliferation and malignancy. Measurements of pHi from slices of human and murine breast cancers and from malignant and normal breast epithelial organoids showed that Na+,HCO3–cotransport is the major mechanism of acid extrusion in the near-physiological pHi range. Na+/H+-exchange was important for acid extrusion only at very low pHi values. We furthermore found that Na+,HCO3–cotransport activity was substantially greater in malignant compared to normal breast epithelial organoids of both human and murine origin, while no apparent difference in Na+/H+-exchange activity was detected between cancer and normal breast tissue. Steady-state pHi was higher in the breast cancer tissue compared to normal breast epithelium in the presence of CO2/HCO3- but not in its nominal absence. We propose that NBCn1 plays a major role for cellular acid extrusion and pHi regulation in human and murine breast cancer. The upregulated expression of NBCn1 and the functional importance of Na+,HCO3–cotransport for pHi regulation support a causative role for NBCn1 in breast cancer development or progression. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-02.