Low dose HSP90 inhibition with AUY922 blunts rapid evolution of metastatic and drug resistant phenotypes induced by TGF-ß and paclitaxel in A549 cells.

OBJECTIVES: Despite advances in cancer treatment, drug resistance and metastasis continue to contribute to treatment failure. Since drug resistance and metastasis in cancer are features that often occur toward the late stages in the disease after withstanding numerous selective pressures, they may rely on a shared adaptive mechanism in order to persist. The heat shock response is one of the most well conserved adaptive responses to cellular stress found in nature. A major player in the heat shock response is HSP90, with some studies suggesting that it can facilitate the molecular evolution of drug resistance and metastasis in cancer. Non-small cell lung cancers (NSCLCs) are strongly associated with drug resistance and metastasis either at the time of diagnosis or early in the treatment process. MATERIALS AND METHODS: We explored the role of HSP90 in the evolution of metastatic and drug resistant features in NSCLC by treating A549 cells with AUY922, a clinically relevant HSP90 inhibitor, and inducing metastatic and drug resistant phenotypes via treatment with TGF-ß and paclitaxel, respectively. We measured phenotypic plasticity in E-Cadherin, a marker for epithelial to mesenchymal transition and two ABC transporters associated with drug resistant lung cancers. RESULTS: We found that metastatic and efflux dependent drug resistant features negatively correlated with AUY922 treatment. We followed our results with functional assays relevant to metastasis and ABC transporters to confirm our results. Specifically we found the expression of E-cadherin was significantly increased in A549 cultures pretreated with AUY922 prior to exposure to paclitaxel, while expression of the drug transporters ABCB1 and ABCC1 was significantly reduced under similar conditions. CONCLUSION: Together our data indicates that HSP90 inhibition with AUY922 can limit the acquisition of metastatic and drug resistant phenotypes in A549 cells at low, clinically appropriate doses.

Cdk1 Controls Global Epigenetic Landscape in Embryonic Stem Cells.

The cyclin-dependent kinase 1 (Cdk1) drives cell division. To uncover additional functions of Cdk1, we generated knockin mice expressing an analog-sensitive version of Cdk1 in place of wild-type Cdk1. In our study, we focused on embryonic stem cells (ESCs), because this cell type displays particularly high Cdk1 activity. We found that in ESCs, a large fraction of Cdk1 substrates is localized on chromatin. Cdk1 phosphorylates many proteins involved in epigenetic regulation, including writers and erasers of all major histone marks. Consistent with these findings, inhibition of Cdk1 altered histone-modification status of ESCs. High levels of Cdk1 in ESCs phosphorylate and partially inactivate Dot1l, the H3K79 methyltransferase responsible for placing activating marks on gene bodies. Decrease of Cdk1 activity during ESC differentiation de-represses Dot1l, thereby allowing coordinated expression of differentiation genes. These analyses indicate that Cdk1 functions to maintain the epigenetic identity of ESCs.

HSP90 Inhibition and Cellular Stress Elicits Phenotypic Plasticity in Hematopoietic Differentiation.

Cancer cells exist in a state of Darwinian selection using mechanisms that produce changes in gene expression through genetic and epigenetic alteration to facilitate their survival. Cellular plasticity, or the ability to alter cellular phenotype, can assist in survival of premalignant cells as they progress to full malignancy by providing another mechanism of adaptation. The connection between cellular stress and the progression of cancer has been established, although the details of the mechanisms have yet to be fully elucidated. The molecular chaperone HSP90 is often upregulated in cancers as they progress, presumably to allow cancer cells to deal with misfolded proteins and cellular stress associated with transformation. The objective of this work is to test the hypothesis that inhibition of HSP90 results in increased cell plasticity in mammalian systems that can confer a greater adaptability to selective pressures. The approach used is a murine in vitro model system of hematopoietic differentiation that utilizes a murine hematopoietic stem cell line, erythroid myeloid lymphoid (EML) clone 1, during their maturation from stem cells to granulocytic progenitors. During the differentiation protocol, 80%-90% of the cells die when placed in medium where the major growth factor is granulocyte-macrophage-colony stimulating factor. Using this selection point model, EML cells exhibit increases in cellular plasticity when they are better able to adapt to this medium and survive. Increases in cellular plasticity were found to occur upon exposure to geldanamycin to inhibit HSP90, when subjected to various forms of cellular stress, or inhibition of histone acetylation. Furthermore, we provide evidence that the cellular plasticity associated with inhibition of HSP90 in this model involves epigenetic mechanisms and is dependent upon high levels of stem cell factor signaling. This work provides evidence for a role of HSP90 and cellular stress in inducing phenotypic plasticity in mammalian systems that has new implications for cellular stress in progression and evolution of cancer.

D-cyclins repress apoptosis in hematopoietic cells by controlling death receptor Fas and its ligand FasL.

D-type cyclins (D1, D2, and D3) are components of the mammalian core cell-cycle machinery and function to drive cell proliferation. Here, we report that D-cyclins perform a rate-limiting antiapoptotic function in vivo. We found that acute shutdown of all three D-cyclins in bone marrow of adult mice resulted in massive apoptosis of all hematopoietic cell types. We demonstrate that adult hematopoietic stem cells are particularly dependent on D-cyclins for survival and that they are especially sensitive to cyclin D loss. Surprisingly, we found that the antiapoptotic function of D-cyclins also operates in quiescent hematopoietic stem and progenitor cells. Our analyses revealed that D-cyclins repress the expression of the death receptor Fas and its ligand, FasL. Acute ablation of D-cyclins upregulated these proapoptotic genes and led to Fas- and caspase 8-dependent apoptosis. These results reveal an unexpected function of cell-cycle proteins in controlling apoptosis in normal cell homeostasis.