Solid tumors are characterized by their ability to thrive in conditions that favor a high intracellular pH and low extracellular pH.1 Carbonic anhydrase IX (CAIX) is an enzyme that is induced in response to hypoxia (low oxygen) and plays a role in maintaining intra- and extracellular pH balance. Overexpressed in many types of tumors including breast cancer, CAIX is thought to provide a survival advantage to the cancer cells. Despite much research, there are contradictory findings about the role of CAIX and how expression levels relate to survival.

Expression and prognostic significance in breast cancer subtypes

Ivanova et al2 explored the prognostic significance of CAIX expression in different breast cancer subtypes. Using an online tool called Kaplan-Meier Plotter,3 they integrated gene expression and clinical data on 3455 breast cancer patients, using relapse-free survival and overall survival as endpoints. The survival data was also analyzed by the treatment received (chemotherapy, endocrine therapy, or no systemic treatment). They determined that CAIX mRNA expression is associated with a shorter relapse-free survival in the basal subtype and triple negative breast cancer, and a worse overall survival in the luminal B subtype. There was no association with the luminal A or HER2+ subtypes.

A likely role in tumorgenicity, but not proliferation or invasion

To explore the functional role of CAIX, the researchers used RNA silencing to inhibit CAIX expression in three different breast cancer cell lines. They stably transfected cell lines representing basal-like (MDA-MD-231), luminal A (MCF7) and HER2+ (SKBR-3) subtypes with short hairpin RNA to selectively silence CAIX gene expression. The transfected cell lines were grown under normal or hypoxic conditions, and subjected to assays that tested proliferation, invasion and tumor-forming ability. The team found that CAIX inhibition did not influence proliferation or survival in the breast cancer cell lines, suggesting that CAIX inhibition is unlikely to lead to tumor shrinkage. However, CAIX inhibition did reduce tumorgenicity under hypoxia, suggesting that CAIX inhibition may help to regulate hypoxic cancer stem cells.

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CAIX inhibition synergizes with doxorubicin

Assays were repeated in the presence of doxorubicin, a conventional chemotherapy drug. Doxorubicin synergized with CAIX inhibition to impede tumorgenicity, especially in the basal-like cell line. The team confirmed this result by treating the non-transformed (non-CAIX silenced) breast cancer cell lines with both doxorubicin and acetazolamide, a pharmacological inhibitor of CAIX. The researchers observed synergistic activity with both doxorubicin and acetazolamide, although they did not explore the molecular mechanisms by which this might take place.

This study was complex, testing many different combinations of cell lines, assays, growth conditions and inhibitory agents.  The final results (1) confirm CAIX as a worthy therapeutic target, (2) imply that CAIX inhibition may reduce the self-renewal capacity of breast cancer cells, and (3) suggest that combining CAIX inhibition and doxorubicin may be a promising therapeutic strategy for patients with basal-like and triple negative cancers with high CAIX expression.

  1. Parks SK1, Chiche J, and Pouyssegur J. pH control mechanisms of tumor survival and growth. J Cell Physiol. 2011 Feb;226(2):299-308. doi: 10.1002/jcp.22400.
  1. Ivanova L, Zandberga, E, Silina, K et al. Prognostic relevance of carbonic anhydrase IX expression is distinct in various subtypes of breast cancer and its silencing suppresses self-renewal capacity of breast cancer cells. Cancer Chemother Pharmacol.2015 Feb;75(2):235-46. doi: 10.1007/s00280-014-2635-1.
  1. Gyorffy B, Surowiak P, Budczies J, et al. Online survival analysis software to assess the prognostic value of biomarkers using transcriptomic data in non-small-cell lung cancer. PLoS One. 2013 Dec 18;8(12):e82241. doi: 10.1371/journal.pone.0082241. Tool available at: http://kmplot.com/analysis/