Epithelial to mesenchymal transition (EMT), invasion, and motility are essential steps in colorectal cancer (CRC) metastasis regulated by HIF-1α and NF-κB. Since HSP90 activates HIF-1α and NF-κB, we hypothesized that inhibition of HSP90 leads to inhibition of HIF-1α and NF-κB resulting in inhibition of EMT, invasion, and motility. Treatment of colorectal cancer cell lines HT-29 and HCT-116 with ganetespib at 50 nM for 24 h inhibited EMT (downregulated vimentin and upregulated E-cadherin), matrigel invasion, and spheroid migration. Ganetespib treatment or HSP90 knockdown downregulated molecular pathways associated with EMT, invasion, and motility. The overexpression of HIF-1α or NF-κB resulted in increased EMT, invasion, and motility in both cell lines and these effects were inhibited by ganetespib. Similar effects were observed in animal xenografts treated with ganetespib. Taken together, our data demonstrate for the first time that inhibition of HSP90 downregulates both HIF-1α and NF-κB leading to inhibition of EMT, motility, and invasiveness in colorectal cancer.

The results confirm decreased mortality from advanced colorectal cancer in the era of modern combination chemotherapy in younger and older patients. Younger age, non-right-sided tumors, and absence of signet ring histology significantly associate with better survival.

Hypoxia enhances MMP2 expression and the invasion and metastatic potential of melanoma cells. CD147 has been shown to induce MMP2 in multiple cancers. To investigate the role of CD147 in hypoxiainduced MMP2 activation, we performed immunohistochemistry (IHC) staining in 206 normal and melanoma tissue samples, and analyzed the correlation between HIF1α and CD147. ChIP (chromosome Immunoprecipitation) in melanoma cell lines supports that HIF1α directly binds to CD147 promoter. Moreover, we made a series of deletion mutants of CD147 promoter, and identified a conserved HIF1α binding site. Point mutation in this site significantly decreased CD147 response to hypoxia. Importantly, knocking down CD147 attenuates MMP2 response to hypoxia in melanoma cell lines. MMP2 could not be efficiently activated by hypoxia in CD147 depletion cells. ELISA data showed that MMP2 secretion was reduced in CD147 depletion cells than control under hypoxia condition. To verify the data from cell culture model, we performed in vivo mouse xenograft experiment. IHC staining showed reduced MMP2 level in CD147 depleted xenografts compared to the control group, with the HIF1α level being comparable. Our study demonstrates a novel pathway mediated by CD147 to promote the MMP2 activation induced by hypoxia, and helps to understand the interplay between hypoxia and melanoma progression.

Purpose/Objectives: Epithelial to mesenchymal transformation (EMT), invasiveness and motility are essential steps in cancer metastasis. HIF-1α and NF-κB have a central role in cancer metastasis. These transcriptional factors (HIF-1α and NF-κB) required HSP90 for stability, folding and trafficking. Ganetespib is a potent functional inhibitor of HSP90 currently under evaluation in clinical trials. We tested the hypothesis that HSP90 functional inhibition by ganetespib can inhibit metastasis in colorectal cell lines. Methods: Two colorectal cancer (CRC) cell lines were used, HCT-116 and HT-29. Cells were either untreated (control) or treated with ganetespib at 50 nM for 24 hours. Western blot and RT-PCR analyses were carried out to determine the effect of ganetespib on different signal molecules involved in metastasis. Electrophoretic mobility shift assay (EMSA) was performed in CRC cells to evaluate the effects of ganetespib on NF-kB activity. Immunocytochemistry assay was also performed. Results: the treatment of colorectal cell lines with ganetespib can inhibit NF-κB and HIF-1α at transcriptional and translational level resulting in inhibition of EMT, invasion and motility. Our investigation also showed that ganetespib treatment significantly (p<0.001) inhibited vimentin and upregulated E-cadherin protein and mRNA levels in both cell lines. Treatment with ganetespib significantly (p<0.001) inhibited matrigel invasion and spheroid migration. In addition, ganetespib treatment significantly (p<0.001) downregulated molecular signaling pathways associated with invasion and motility including uPAR, p70S6K, FAK, MMP2, and MMP9. The overexpression of HIF-1α or NF-κB resulted in increased invasion and motility and its related signaling molecules in both cell lines. Functional inhibition of HSP90 resulted in inhibition of EMT due to NF-κB or HIF-1α downregulation. Similar effects were observed in tumors from animals treated with ganetespib. We also demonstrated the inhibition of HIF-1α, NF-κB and EMT in rectal tumors obtained from patients receiving ganetespib. Conclusion: In this study, we present in vitro, animal, and human data to confirm the role of targeting HSP90 as a method to prevent metastasis of colorectal cancer. We demonstrate for the first time that inhibition of HSP90 downregulates both HIF-1α and NF-κB leading to inhibition of EMT, motility and invasiveness in colorectal cancer. Accordingly, combining HSP90 inhibitors with chemo- or radiotherapy is a rational approach for future drug development in colorectal cancer. Citation Format: Purnachandra Ganji, Tua-Elisabeth Long, Phani Madhuri Mummareddi, Wungki Park, Field F. Willingham, Jerome C. Landry, Patrick Sullivan, LaTonia Taliaferro-Smith, Roberto Diaz, Bassel El-Rayes. Heat shock protein 90 functional inhibition regulates epithelial to mesenchymal transformation, invasion and migration via NF-kB and HIF-1α signaling in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2707. doi:10.1158/1538-7445.AM2013-2707