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

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 LM2-4Lucþ 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. Cancer Res; 71(9); 3364-76. Ó2011 AACR.

Glycosyl Coumarin Carbonic Anhydrase IX and XII Inhibitors Strongly Attenuate the Growth of Primary Breast Tumors

“…Furthermore, some of these compounds show significant primary tumor growth inhibitory effects in a mouse breast cancer model. Correlated with the previous demonstration 15 of their antimetastatic effects, we claim that members of this class of compounds possess important anticancer activity through a novel mechanism of action that takes advantage of the fact that CA IX and XII are overexpressed only in hypoxic tumors (which do not respond to classical chemo-and radiotherapy) and exhibit very limited expression in normal tissues. Thus, tumor CA IX/ XII inhibition may lead to significantly fewer side effects compared to classical anticancer agents in clinical use.…”

“…15 Furthermore, we have demonstrated that treatment of 4T1 tumors with certain novel ureido-substituted benzenesulfonamides 14 results in attenuation of primary tumor growth and metastasis in spontaneous 15 and experimental metastatic disease. 14,15 Novel CA IX-selective glycosyl coumarin inhibitors were also found to limit the metastatic burden in a model of experimental metastasis. 15 To evaluate the effect of pharmacologic inhibition of CA IX activity on primary tumor growth in vivo using the novel glycosyl coumarin compounds described here, we treated mice harboring established 4T1 tumors with the glycosyl coumarin 6, a compound that demonstrates excellent CA IX and XII inhibitory activities in vitro, and a very good selectivity ratio for inhibiting the tumor-associated CA isoforms compared to the cytosolic CA isoforms, as discussed above.…”

“…Furthermore, the antimetastatic effects (but not the effects on primary tumors) have also been observed with coumarin-based CAIs. 15 Here we report the discovery of 7-glycosyl coumarins as potent and CA IX/XII-selective inhibitors. Furthermore, some of these compounds show significant primary tumor growth inhibitory effects in a mouse breast cancer model.…”

“…2,9 CAIs of the sulfonamide type have been used clinically for decades, 3 for various classes of diuretics and systemically acting antiglaucoma agents, but their main drawback is their potent inhibition of CA I and II, ubiquitous enzymes playing important physiological roles. 3,4,10 Thus, critical barriers to the use of CAIs as therapeutic agents are related to the large number of isoforms in humans (i.e., 15 CAs, of which 12 have catalytic activity), their diffuse localization in many tissues and organs, and the lack of isozyme selectivity for many of the presently available inhibitors of the sulfonamide and/or sulfamate type. 3,4,10−13 Thus, there is a stringent need for CAIs with an inhibition profile more selective than those of the classical sulfonamides and their isosteres, and the coumarins represent an interesting such class because of the fact that several isoform-selective CAIs targeting CA isoforms IX, XII, and XIII have been reported recently by our group.…”

Therapeutic Mechanism and Efficacy of the Antibody–Drug Conjugate BAY 79-4620 Targeting Human Carbonic Anhydrase 9

“…Although some recently published small molecule inhibitors (27,28) successfully target the enzymatic activity of CAIX, the work described here uses a different therapeutic approach in exploiting CAIX as an anchor for the selective delivery of a toxic payload to tumors via an antibody-based therapeutic. A rigorous discovery cascade was implemented to generate and select fully human antibodies that are highly selective for binding to, and internalization by, CAIX-expressing cancer cell lines.…”