Ligand-induced activation of peroxisome proliferator-activated receptor γ (PPARγ) inhibits proliferation in cancer cells in vitro and in vivo; however, the downstream targets remain undefined. We report the identification of a peroxisome proliferator response element in the promoter region of the Na + / H + transporter gene NHE1, the overexpression of which has been associated with carcinogenesis. Exposure of breast cancer cells expressing high levels of PPARγ to its natural and synthetic agonists resulted in downregulation of NHE1 transcription as well as protein expression. Furthermore, the inhibitory effect of activated PPARγ on tumor colony-forming ability was abrogated on overexpression of NHE1, whereas small interfering RNA-mediated gene silencing of NHE1 significantly increased the sensitivity of cancer cells to growthinhibitory stimuli. Finally, histopathologic analysis of breast cancer biopsies obtained from patients with type II diabetes treated with the synthetic agonist rosiglitazone showed significant repression of NHE1 in the tumor tissue. These data provide evidence for tumor-selective downregulation of NHE1 by activated PPARγ in vitro and in pathologic specimens from breast cancer patients and could have potential implications for the judicious use of low doses of PPARγ ligands in combination chemotherapy regimens for an effective therapeutic response. [Cancer Res 2009;69(22):8636-44]

Host interactions with tumor cells contribute to tumor progression by several means. This study was done to determine whether mammary epithelium could interact with breast carcinoma by producing substances capable of inducing motility in the cancer cells. Conditioned medium of immortalized 184A1 mammary epithelium collected in serum-free conditions induced dose-dependent motility in the MCF-7 breast carcinoma cell line by both a semiquantitative scattering assay and a Boyden chamber assay. Purification of the motility factor revealed that it was laminin 332 (formerly laminin 5) by mass spectroscopy. A Western blot of the 184A1 conditioned medium using a polyclonal antibody confirmed the presence of laminin 332 in the conditioned medium. Blockage of the motility with antibodies to the laminin 332 and its receptor components, alpha(3) and beta(1) integrins, provided further evidence that tumor cell motility was caused by the laminin 332 in the conditioned medium. Invasion of MCF-7, BT-20, and MDA-MB-435 S was induced by purified laminin 332 and 184A1 conditioned medium and blocked by an anti-alpha(3) integrin antibody. Staining of carcinoma in situ from breast cancer specimens revealed that laminin 332 in the myoepithelium adjacent to the preinvasive cells provided a source of laminin 332 that could potentially encourage the earliest steps of stromal invasion. In metaplastic breast carcinomas, the presence of laminin 332-producing cells coexpressing alpha(3) integrin and the greater metastatic potential of tumors with higher laminin 332 levels suggest that laminin 332 expression is associated with aggressive features in these human breast cancers.

Among the many factors involved in the maintenance of homeostatic growth is the tight regulation of cellular pH. Intracellular pH of normal cells is maintained within a physiological range thanks to the activity of a number of pH regulators that respond to the acidbase shifts associated with normal cellular metabolic processes. Interestingly, there is a preponderance of evidence that dysregulation of intracellular pH is associated with processes that favor cell transformation such as cell cycle progression, enhanced proliferation, insensitivity to growth inhibitory stimuli, resistance to apoptosis, genomic instability and angiogenesis. Among the strategies employed by the cells to regulate intracellular pH, the Na⁺/H⁺ exchanger 1 (NHE1) protein from the Na⁺/H⁺ exchanger (NHE) family has been directly associated with cellular transformation, invasion and metastasis. These observations have heightened the interest in NHE1 as a promising novel drug target for more effective and selective anti-cancer therapeutics. Here we present a review of the basic biology of this remarkable protein and present evidence to support targeting NHE1 as a potential anti-cancer strategy.

The mechanism of Na þ /H þ exchanger 1 (NHE1) gene repression upon exposure of cells to non-apoptotic concentrations of hydrogen peroxide (H 2 O 2 ) was investigated. We show that continuous presence of H 2 O 2 was not required for inhibition of NHE1 promoter activity. However, the downregulation of NHE1 promoter activity and protein expression was abrogated by the presence of beta mercaptoethanol (bME) and dithiothreitol. The pan-caspase inhibitor zVAD-fmk also blocked the effect of H 2 O 2 on NHE1 promoter activity and expression, but unlike bME, caspase inhibition was ineffective in rescuing the early phase of NHE1 repression. Interestingly, the effect of caspase inhibition was observed only after 9 h of exposure to H 2 O 2 and completely restored NHE1 promoter activity by 18-24 h. Using tetrapeptide inhibitors of a variety of caspases and siRNA-mediated gene silencing, caspases 3 and 6 were identified as mediators of H 2 O 2 -induced NHE1 repression, independent of initiator/amplifier caspase activation. Furthermore, incubation of cells with the iron chelator, desferioxamine, not only blocked the activities of caspases 3 and 6, but also affected NHE1 promoter and protein expression in a manner similar to zVAD-fmk. These data show that a mild oxidative stress represses NHE1 promoter activity and expression via an early oxidation phase blocked by reducing agents, and a late phase requiring an iron-dependent increase in caspases 3 and 6 activities.

Supplementary Table 1 from Repression of NHE1 Expression by PPARγ Activation Is a Potential New Approach for Specific Inhibition of the Growth of Tumor Cells <i>In vitro</i> and <i>In vivo</i>

Supplementary Methods from Repression of NHE1 Expression by PPARγ Activation Is a Potential New Approach for Specific Inhibition of the Growth of Tumor Cells <i>In vitro</i> and <i>In vivo</i>

Supplementary Figure Legends 1-6 from Repression of NHE1 Expression by PPARγ Activation Is a Potential New Approach for Specific Inhibition of the Growth of Tumor Cells <i>In vitro</i> and <i>In vivo</i>

Supplementary Table 1 from Repression of NHE1 Expression by PPARγ Activation Is a Potential New Approach for Specific Inhibition of the Growth of Tumor Cells <i>In vitro</i> and <i>In vivo</i>