Targeting Tumor Hypoxia: Suppression of Breast Tumor Growth and Metastasis by Novel Carbonic Anhydrase IX Inhibitors

Yuanmei Lou,Andrea Scozzafava,Fabrizio Carta,Claudiu T Supuran,Christopher M Overall,Arusha Oloumi,Dawn Waterhouse,Ulrich Auf Dem Keller,Ardalan Ahmadi,Shoukat Dedhar,Brent W Sutherland,Christina Ostlund,Nadia Touisni,Samuel Leung,Fabio Pacchiano,Stephen Chia,Paul C Mcdonald,Calvin D Roskelley ,Marcel B Bally ,Blaise A Clarke ,Andrew I Minchinton ,David G Huntsman ,Alastair H Kyle ,Jean‐Yves Winum

doi:10.1158/0008-5472.can-10-4261

Abstract

Carbonic anhydrase IX (CAIX) is a hypoxia and HIF-1-inducible protein that regulates intra- and extracellular pH under hypoxic conditions and promotes tumor cell survival and invasion in hypoxic microenvironments. Interrogation of 3,630 human breast cancers provided definitive evidence of CAIX as an independent poor prognostic biomarker for distant metastases and survival. shRNA-mediated depletion of CAIX expression in 4T1 mouse metastatic breast cancer cells capable of inducing CAIX in hypoxia resulted in regression of orthotopic mammary tumors and inhibition of spontaneous lung metastasis formation. Stable depletion of CAIX in MDA-MB-231 human breast cancer xenografts also resulted in attenuation of primary tumor growth. CAIX depletion in the 4T1 cells led to caspase-independent cell death and reversal of extracellular acidosis under hypoxic conditions in vitro. Treatment of mice harboring CAIX-positive 4T1 mammary tumors with novel CAIX-specific small molecule inhibitors that mimicked the effects of CAIX depletion in vitro resulted in significant inhibition of tumor growth and metastasis formation in both spontaneous and experimental models of metastasis, without inhibitory effects on CAIX-negative tumors. Similar inhibitory effects on primary tumor growth were observed in mice harboring orthotopic tumors comprised of lung metatstatic MDA-MB-231 LM2-4(Luc+) cells. Our findings show that CAIX is vital for growth and metastasis of hypoxic breast tumors and is a specific, targetable biomarker for breast cancer metastasis.

Highlights

Cancer metastasis is a complex process that results in establishment of secondary tumors in distant organs [1]
Using a combination of gene depletion strategies and pharmacologic inhibition with novel small molecule inhibitors, we show a functional requirement of Carbonic anhydrase IX (CAIX) in the growth and metastasis of mouse and human breast tumors in several preclinical models
CAIX is a poor prognostic marker in a large cohort of breast cancer patients previous studies have reported that CAIX expression in several types of cancer, including breast cancer, correlates with poor patient prognosis [11, 34, 36, 37], the sample sizes have been relatively small and adjuvant treatments not uniform

Summary

Introduction

Cancer metastasis is a complex process that results in establishment of secondary tumors in distant organs [1]. Targeting CAIX for the treatment of cancer has garnered much scientific and clinical interest, appropriate carbonic anhydrase-relevant cell and animal models of tumor hypoxia for testing novel, CAIX-active compounds have only recently become available [23]. Neither the functional requirement of CAIX in breast tumor growth and metastasis in vivo, nor the benefit of therapeutic targeting this enzyme in aggressive breast cancer has been addressed. We provide definitive evidence, using a large (>3,600) cohort of human breast cancer samples, that CAIX is a poor prognostic marker for distant metastasis and survival. Using a combination of gene depletion strategies and pharmacologic inhibition with novel small molecule inhibitors, we show a functional requirement of CAIX in the growth and metastasis of mouse and human breast tumors in several preclinical models. Our findings establish CAIX as a therapeutic target for the treatment of CAIXpositive breast cancer

Materials and Methods

Results and Discussion

Disclosure of Potential Conflicts of Interest