Differential expression and function of CAIX and CAXII in breast cancer: A comparison between tumorgraft models and cells.

Zhijuan Chen,Robert Mckenna,Kevin D Brown,Mam Y Mboge,Susan C Frost,Lingbao Ai,Chingkuang Tu,Coy D Heldermon,Aamir Ahmad

doi:10.1371/journal.pone.0199476

Abstract

Carbonic anhydrase IX (CAIX) and XII (CAXII) are transmembrane proteins that are associated with cancer progression. We have previously described the catalytic properties of CAIX in MDA-MB-231 breast cancer cells, a line of cells that were derived from a patient with triple negative breast cancer. We chose this line because CAIX expression in breast cancer is a marker of hypoxia and a prognosticator for reduced survival. However, CAXII expression is associated with better survival statistics than those patients with low CAXII expression. Yet CAIX and CAXII have similar catalytic activities. Here we compare the potential roles of CAIX and CAXII in the context of TNBC and estrogen receptor (ER)-positive breast cancer. In tumor graft models, we show that CAIX and CAXII exhibit distinct expression patterns and non-overlapping. We find the same pattern across a panel of TNBC and luminal breast cancer cell lines. This affords an opportunity to compare directly CAIX and CAXII function. Our data suggest that CAIX expression is associated with growth potentiation in the tumor graft model and in a TNBC line using knockdown strategies and blocking activity with an impermeant sulfonamide inhibitor, N-3500. CAXII was not associated with growth potentiation. The catalytic activities of both CAIX and CAXII were sensitive to inhibition by N-3500 and activated at low pH. However, pH titration of activity in membrane ghosts revealed significant differences in the catalytic efficiency and pKa values. These features provide evidence that CAIX is a more efficient enzyme than CAXII at low pH and that CAIX shifts the equilibrium between CO2 and bicarbonate in favor of CO2 production by consuming protons. This suggests that in the acidic microenvironment of tumors, CAIX plays a role in stabilizing pH at a value that favors cancer cell survival.

Highlights

There is a strong correlation between lactic acid production and metastatic incidence [1]
Breast cancer patients that show high expression of CAXII mRNA show higher RFS rates relative to those with low expression (p = 8.3e-13) (Fig 1C). This holds true for the estrogen receptor positive patients (p = 0.0056) (Fig 1D), surprisingly not as impressive as for all breast cancer patients. While these data suggest a fairly clear association between Carbonic anhydrase IX (CAIX) and CAXII mRNA with specific subtypes of breast cancer, the survival data vary significantly when other subtypes are evaluated for CAIX or CAXII mRNA expression (S1 Fig and S2 Fig)
We show that across all types of breast cancer, elevated expression of CAIX mRNA is associated with lower patient survival compared to breast cancers with low expression (Fig 1A) but especially those with the Triple negative breast cancer (TNBC) phenotype (Fig 1B)

Summary

Introduction

There is a strong correlation between lactic acid production and metastatic incidence [1]. All of the clinical breast cancer studies to date have shown a correlation between CAXII expression and better survival statistics for patients [18,19,20,21]. These later studies show a strong association between luminal cancers and CAXII expression. CAXII is a biomarker of favorable prognosis in lung [22] and brain [23] tumors, but is associated with poor prognosis in colorectal cancer [24] Despite these obvious differences, CAIX and CAXII have similar activities suggesting that their native environments may influence their activities and/or biochemical function. We present data that confirms that the activity profile of CAIX over CAXII is favored at pH values associated with acidic tumors

Materials and methods

Results

Discussion