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. Cancer Res; 71(9); 3364–76. ©2011 AACR.

Anticancer drug combinations can act synergistically or antagonistically against tumor cells in vitro depending on the ratios of the individual agents comprising the combination. The importance of drug ratios in vivo, however, has heretofore not been investigated, and combination chemotherapy treatment regimens continue to be developed based on the maximum tolerated dose of the individual agents. We systematically examined three different drug combinations representing a range of anticancer drug classes with distinct molecular mechanisms (irinotecan/floxuridine, cytarabine/daunorubicin, and cisplatin/daunorubicin) for drug ratio–dependent synergy. In each case, synergistic interactions were observed in vitro at certain drug/drug molar ratio ranges (1:1, 5:1, and 10:1, respectively), whereas other ratios were additive or antagonistic. We were able to maintain fixed drug ratios in plasma of mice for 24 hours after i.v. injection for all three combinations by controlling and overcoming the inherent dissimilar pharmacokinetics of individual drugs through encapsulation in liposomal carrier systems. The liposomes not only maintained drug ratios in the plasma after injection, but also delivered the formulated drug ratio directly to tumor tissue. In vivo maintenance of drug ratios shown to be synergistic in vitro provided increased efficacy in preclinical tumor models, whereas attenuated antitumor activity was observed when antagonistic drug ratios were maintained. Fixing synergistic drug ratios in pharmaceutical carriers provides an avenue by which anticancer drug combinations can be optimized prospectively for maximum therapeutic activity during preclinical development and differs from current practice in which dosing regimens are developed empirically in late-stage clinical trials based on tolerability. [Mol Cancer Ther 2006;5(7):1854–63]

An unbiased proteomic screen to identify integrin-linked kinase (ILK) interactors revealed rictor as an ILK-binding protein. This finding was interesting because rictor, originally identified as a regulator of cytoskeletal dynamics, is also a component of mammalian target of rapamycin complex 2 (mTORC2), a complex implicated in Akt phosphorylation. These functions overlap with known ILK functions. Coimmunoprecipitation analyses confirmed this interaction, and ILK and rictor colocalized in membrane ruffles and leading edges of cancer cells. Yeast two-hybrid assays showed a direct interaction between the NH₂- and COOH-terminal domains of rictor and the ILK kinase domain. Depletion of ILK and rictor in breast and prostate cancer cell lines resulted in inhibition of Akt Ser⁴⁷³ phosphorylation and induction of apoptosis, whereas, in several cell lines, depletion of mTOR increased Akt phosphorylation. Akt and Ser⁴⁷³P-Akt were detected in ILK immunoprecipitates and small interfering RNA–mediated depletion of rictor, but not mTOR, inhibited the amount of Ser⁴⁷³P-Akt in the ILK complex. Expression of the NH₂-terminal (1–398 amino acids) rictor domain also resulted in the inhibition of ILK-associated Akt Ser⁴⁷³ phosphorylation. These data show that rictor regulates the ability of ILK to promote Akt phosphorylation and cancer cell survival. [Cancer Res 2008;68(6):1618–24]

Copper complexes could define a promising new class of therapeutics, provided that there is a suitable way in which to formulate them for pharmaceutical use.