Therapeutic drugs in cancer and resistance.

We investigated the mechanism of toxicity and resistance development of small molecule tyrosine kinase inhibitor lapatinib in HCT 116 colon cancer cells. Lapatinib mediated cell death in HCT 116 cells was caspase independent and involved cytosolic release of apoptosis inducing factor. Treatment of HCT 116 cells with 10µM Lapatinib lead to the outgrowth of lapatinib resistant HCT 116 cells. Our studies show that alterations in the expression and activation of Bcl-2 family proteins allow lapatinib resistant HCT 116 cells to resist cytotoxic effects of lapatinib as well as of other commonly used chemotherapeutic agents. In hepatoma and pancreatic cancer cells, the effects of combining multi-kinase inhibitor sorafenib with histone deacetylase inhibitors (HDACIs) namely, vorinostat and sodium valproate were investigated. It was found that sorafenib synergizes with HDACIs resulting in enhanced cell death compared to death induced by the drugs individually. The mechanism of action of sorafenib and vorinostat combination treatment as well as sorafenib and sodium valproate combined treatment was shown to involve activation of the CD95 death receptor pathway. Alterations in the CD95 pathway can render cancer cells resistant to chemotherapeutic agents. Hence, we combined sorafenib+sodium valproate with a BH-3 domain mimetic named obatoclax (GX-15-070) which resulted in enhanced toxicity to cancer cells. More importantly, knock-down of CD95 (to mimic non-functional CD95 pathway) reduced cell death induced by sorafenib+sodium valproate combined but failed to protect cells from cell death induced by sorafenib+sodium valproate+obatoclax combined. This suggests that combining sorafenib+HDACI with obatoclax may not only enhance toxicity to cancer cells but may also reduce chances of resistance development via alterations in the CD95 pathway. These studies enhance our knowledge of existing treatment strategies for cancer as well as throw light on how current approaches can be improved in order to better diagnose and treat cancer. Understanding mechanisms of drug action as well as resistance development will allow us to combine existing therapies effectively in order best target cancer cells as well as provide us with information that can help us design new cancer treatment strategies

BCL-2 Family Inhibitors Enhance Histone Deacetylase Inhibitor and Sorafenib Lethality via Autophagy and Overcome Blockade of the Extrinsic Pathway to Facilitate KillingS⃞

We examined whether the multikinase inhibitor sorafenib and histone deacetylase inhibitors (HDACI) interact to kill pancreatic carcinoma cells and determined the impact of inhibiting BCL-2 family function on sorafenib and HDACI lethality. The lethality of sorafenib was enhanced in pancreatic tumor cells in a synergistic fashion by pharmacologically achievable concentrations of the HDACIs vorinostat or sodium valproate. Overexpression of cellular FLICE-like inhibitory protein (c-FLIP-s) or knockdown of CD95 suppressed the lethality of the sorafenib/HDACI combination (sorafenib + HDACI). In immunohistochemical analyses or using expression of fluorescence-tagged proteins, treatment with sorafenib and vorinostat together (sorafenib + vorinostat) promoted colocalization of CD95 with caspase 8 and CD95 association with the endoplasmic reticulum markers calnexin, ATG5, and Grp78/BiP. In cells lacking CD95 expression or in cells expressing c-FLIP-s, the lethality of sorafenib + HDACI exposure was abolished and was restored when cells were coexposed to BCL-2 family inhibitors [ethyl [2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)]-4H-chromene-3-carboxylate (HA14-1), obatoclax (GX15-070)]. Knockdown of BCL-2, BCL-XL, and MCL-1 recapitulated the effects of GX15-070 treatment. Knockdown of BAX and BAK modestly reduced sorafenib + HDACI lethality but abolished the effects of GX15-070 treatment. Sorafenib + HDACI exposure generated a CD95- and Beclin1-dependent protective form of autophagy, whereas GX15-070 treatment generated a Beclin1-dependent toxic form of autophagy. The potentiation of sorafenib + HDACI killing by GX15-070 was suppressed by knockdown of Beclin1 or of BAX + BAK. Our data demonstrate that pancreatic tumor cells are susceptible to sorafenib + HDACI lethality and that in tumor cells unable to signal death from CD95, use of a BCL-2 family antagonist facilitates sorafenib + HDACI killing via autophagy and the intrinsic pathway

Inhibition of MCL-1 in breast cancer cells promotes cell death in vitro and in vivo

The present studies have examined approaches to suppress MCL-1 function in breast cancer cells, as a means to promote tumor cell death. Treatment of breast cancer cells with CDK inhibitors (flavopiridol; roscovitine) enhanced the lethality of the ERBB1 inhibitor lapatinib in a synergistic fashion. CDK inhibitors interacted with lapatinib to reduce MCL-1 expression and overexpression of MCL-1 or knock down of BAX and BAK suppressed drug combination lethality. Lapatinib-mediated inhibition of ERK1/2 and to a lesser extent AKT facilitated CDK inhibitor-induced suppression of MCL-1 levels. Treatment of cells with the BH3 domain/MCL-1 inhibitor obatoclax enhanced the lethality of lapatinib in a synergistic fashion. Knock out of MCL-1 and BCL-XL enhanced lapatinib toxicity to a similar extent as obatoclax and suppressed the ability of obatoclax to promote lapatinib lethality. Pre-treatment of cells with lapatinib or with obatoclax enhanced basal levels of BAX and BAK activity and further enhanced drug combination toxicity. In vivo tumor growth data in xenograft and syngeneic model systems confirmed our in vitro findings. Treatment of cells with CDK inhibitors enhanced the lethality of obatoclax in a synergistic fashion. Overexpression of MCL-1 or knock down of BAX and BAK suppressed the toxic interaction between CDK inhibitors and obatoclax. Obatoclax and lapatinib treatment or obatoclax and CDK inhibitor treatment or lapatinib and CDK inhibitor treatment radiosensitized breast cancer cells. Lapatinib and obatoclax interacted to suppress mammary tumor growth in vivo. Collectively our data demonstrate that manipulation of MCL-1 protein expression by CDK inhibition or inhibition of MCL-1 sequestering function by Obatoclax renders breast cancer cells more susceptible to BAX/BAK-dependent mitochondrial dysfunction and tumor cell death