Carbonic Anhydrase IX Promotes Tumor Growth and Necrosis In Vivo and Inhibition Enhances Anti-VEGF Therapy

Abstract

Purpose Bevacizumab, an anti-VEGFA antibody, inhibits the developing vasculature of tumours, but resistance is common. Antiangiogenic therapy induces hypoxia and we observed increased expression of hypoxia-regulated genes, including carbonic anhydrase IX (CAIX), in response to Bevacizumab treatment in xenografts. CAIX expression correlates with poor prognosis in most tumour types and with worse outcome in Bevacizumab-treated metastatic colorectal cancer patients, malignant astrocytoma and recurrent malignant glioma. Experimental Design We knocked-down CAIX expression by shRNA in a colon cancer (HT29) and a glioblastoma (U87) cell line which have high hypoxic-induction of CAIX, and over-expressed CAIX in HCT116 cells which has low CAIX. We investigated the effect on growth rate in 3D culture and in vivo, and examined the effect of CAIX knockdown in combination with Bevacizumab. Results CAIX expression was associated with increased growth rate in spheroids and in vivo. Surprisingly, CAIX expression was associated with increased necrosis and apoptosis in vivo and in vitro. We found that acidity inhibits CAIX activity over the pH range found in tumours (pK=6.84), and this may be the mechanism whereby excess acid self-limits the build-up of extracellular acid. Expression of another hypoxia inducible CA isoform, CAXII, was upregulated in 3D but not 2D culture in response to CAIX knockdown. CAIX knockdown enhanced the effect of Bevacizumab treatment, reducing tumour growth rate in vivo. Conclusion This work provides evidence that inhibition of the hypoxic adaptation to anti-angiogenic therapy enhances Bevacizumab treatment and highlights the value of developing small molecules or antibodies which inhibit CAIX for combination therapy.

The chemistry, physiology and pathology of pH in cancer

“…Excessive extracellular acidification could become detrimental, but the extent of this may be curtailed by the inhibitory (i.e. negative feedback) effect of H þ ions on CA activity [33][34][35]. 2 and H þ -lactate diffusion away from the surface of cells is expected to produce a more alkaline pH i that better supports cell proliferation.…”

Carbamoylphosphonates inhibit autotaxin and metastasis formationin vivo

“…It has been proposed that CAIX could serve as a specific biomarker for this kind of tumor. The necessity of CAIX for tumor and metastasis formation has also been recently demonstrated by using the knock-down approach 25 . Further, inhibition of CAIX has been shown to result in regression or growth inhibition of mouse and human breast tumors as well as inhibition of cancer metastasis formation 25 .…”

Synthesis and inhibition potency of novel ureido benzenesulfonamides incorporating GABA as tumor-associated carbonic anhydrase IX and XII inhibitors

“…As mentioned above all hypoxic tumors overexpressed CA IX and CA XII isoforms after activation of the hypoxia inducible factor-1 (HIF-1) 6 with a pH imbalance between the tumor tissue and the normal tissue as main consequence 6,8,14 . The hCA IX/XII isoforms inhibition with sulfonamides was recently shown to reverse the effect of tumor acidification 6,8,14 , leading to inhibition of the primary tumor and metastases growth, and CA IX/XII have been proposed as novel therapeutic antitumor targets 1,13 .…”

“…The hCA IX/XII isoforms inhibition with sulfonamides was recently shown to reverse the effect of tumor acidification 6,8,14 , leading to inhibition of the primary tumor and metastases growth, and CA IX/XII have been proposed as novel therapeutic antitumor targets 1,13 . Reported here, there are novel ureido-benzenesulfonamide CAIs obtained considering compounds A-D as leads, some of which incorporate the same R-ureido linker, where R was phenyl, benzyl, cyclopentyl and 4-iodophenyl 21 together with a GABA portion connecting the 4-aminomethyl or 4-aminoethylbenzene sulfonamide and the ureido moieties.…”

“…Among the various biological functions, CAs are involved in pH buffering of the intra-and extra-cellular spaces [4][5][6][7] . Mainly two carbonic anhydrase isoforms hCA IX [8][9][10] and CA XII 11,12 , possessing extracellular active site, are over-expressed in most of hypoxic tumors, such as gliomas/ ependymomas, mesotheliomas, carcinomas of the bladder, cervix, kidneys, lungs, breast, and other dysplasia resistant to classic chemo-and radiotherapy 7,13,14 . Indeed, in response to hypoxia event, such as the one occuring in the tumor area, the activated hypoxia inducible factor-1 (HIF-1) causes an over-expression of hCA IX 6 , which increases the production of protons in the district involved.…”