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.

Regulation of Intracellular pH is Altered in Cardiac Myocytes of Ovariectomized Rats.

Background It is well known that after menopause women are exposed to a greater cardiovascular risk, but the intracellular modifications are not properly described. The sodium/proton exchanger (NHE) and the sodium/bicarbonate cotransporter (NBC) regulate the intracellular pH and, indirectly, the intracellular sodium concentration ([Na+]). There are 2 isoforms of NBC in the heart: the electrogenic (1Na+/2[Formula: see text]; NBCe1) and the electroneutral (1Na+/1[Formula: see text]; NBCn1). Because NHE and NBCn1 hyperactivity as well as the NBCe1 decreased activity have been associated with several cardiovascular pathologies, the aim of this study was to investigate the potential alterations of the alkalinizing transporters during the postmenopausal period. Methods and Results Three-month ovariectomized rats (OVX) were used. The NHE activity and protein expression are significantly increased in OVX. The NBCe1 activity is diminished, and the NBCn1 activity becomes predominant in OVX rats. p-Akt levels showed a significant diminution in OVX. Finally, NHE activity in platelets from OVX rats is also higher in comparison to sham rats, resulting in a potential biomarker of cardiovascular diseases. Conclusions Our results demonstrated for the first time that in the cardiac ventricular myocytes of OVX rats NHE and NBC isoforms are altered, probably because of the decreased level of p-Akt, compromising the ionic intracellular homeostasis.

The reduced myofilament responsiveness to calcium contributes to the negative force-frequency relationship in rat cardiomyocytes: role of reactive oxygen species and p-38 map kinase.

The force-frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. However, a decrease (negative FFR) or no effect (flat FFR) on contractile force in response to an elevation of heart rate is present in the normal rat or in human heart failure. Reactive oxygen species (ROS) can act as intracellular signaling molecules activating diverse kinases as calcium-calmodulin-dependent protein kinase II (CaMKII) and p-38 MAP kinase (p-38K). Our aim was to elucidate the intracellular molecules implicated in the FFR of isolated rat ventricular myocytes. The myocytes were field-stimulated via two-platinum electrodes. Sarcomere length was recorded with a video camera. Ca2+ transients and intracellular pHi were recorded by epifluorescence. Increasing frequency from 0.5 to 3 Hz decreased cell shortening without changes in pHi. This negative FFR was changed to positive FFR when the myocytes were pre-incubated with the ROS scavenger MPG, the NADPH oxidase blocker apocynin, or by inhibiting mitochondrial ROS production with 5-HD. Similar results were obtained when the cells were pre-incubated with the CaMKII blocker, KN-93, or the p-38K inhibitor, SB-202190. Consistently, the levels of phosphorylation of p-38K and the oxidation of CaMKII were significantly higher at 2 Hz than at 0.5 Hz. Despite the presence of positive inotropic effect during stimulation frequency enhancement, Ca2+ transient amplitudes were reduced in MPG- and SB-202190-treated myocytes. In conclusion, our results indicate that the activation of the intracellular pathway involving ROS-CaMKII-p-38K contributes to the negative FFR of rat cardiomyocytes, likely by desensitizing the response of contractile myofilaments to Ca2+.