Summary The thioether phospholipid ilmofosine (BM 41 440) is a new anti-cancer drug presently undergoing phase 11 clinical trials. Because resistance to anti-tumour drugs is a major problem in cancer treatment, we investigated the resistance of different cell lines to this compound.Here we report that the multidrug-resistant cell lines MCF7/ADR, CCRFNCR1000, CCRF/ADR500, CEMNLB100 and HeLa cell lines transfected with a wild-type and mutated (gly/vall 85) multidrug resistance 1 gene (MDR1) are cross-resistant to ilmofosine compared with the sensitive parental cell lines. In CEMNM-1 cells, in which the resistance is associated with an altered topoisomerase 11 gene, no crossresistance to ilmofosine was observed. Ilmofosine is not capable of modulating multidrug resistance and neither does it reduce the labelling of the P-glycoprotein (P-gp) by azidopine nor alter ATPase activity significantly. The resistance to ilmofosine in multidrug-resistant CCRFNCR1000 cells cannot be reversed by the potent multidrug resistance modifier dexniguldipine-HCI (B8509-035). A tenfold excess of ilmofosine does not prevent the MDR-modulating effect of dexniguldipine-HCI. Treatment of cells with ilmofosine does not alter the levels of MDR1 mRNA. Long-term treatment of an ilmofosine-resistant Meth A subline with the drug does not induce multidrug resistance, indicating that ilmofosine does not increase the level of P-gp. Determination of the MDR2 mRNA levels in the cells revealed that the resistance pattern to ilmofosine is not correlated with the expression of this gene. It is concluded, therefore, that multidrug-resistant cells are cross-resistant to ilmofosine and that the compound is not a substrate of Pgp. No association between the expression of the MDR2-encoded P-gp and resistance to ilmofosine was observed. It is supposed that MDR1-associated alterations in membrane lipids cause resistance to ilmofosine.

The problem of tumor cell drug resistance remains a barrier to II) and resistance to epipodophyllotoxins or anthracyclines; 8 the successful treatment of many neoplastic diseases. Prob-(3) dihydrofolate reductase (dhfr) and methotrexate resistlems of tumor cell heterogeneity and expression of multiple ance; 9 and (4) methylguanine methyltransferase (mgmt) and mechanisms of drug resistance complicate treatment straalkylating agent resistance. 10 In all these cases, it is or will be tegies. Indeed, even that form of resistance to natural product important to detect reliably the expression and/or activity of RNA expression (Northern, slot-blot, RT-PCR, in situ PCR) and

Apoptosis is coordinated by a family of cysteine proteases, the caspases, that dismantle the cell by targeting a panoply of proteins for limited proteolysis. The mammalian caspase family contains 14 members, a subset of which participate in apoptosis, with the remainder likely to be involved in the processing of pro-inflammatory cytokines. Our current research is focused upon establishing the repertoire and order of caspase activation events that occur during the signalling and demolition phases of apoptosis. Apical caspase activation events are typically initiated by adaptor molecules that promote caspase aggregation and facilitate caspase autoactivation. In contrast, distal caspase activation events are controlled by caspases activated earlier in the cascade. Many cellular stresses provoke apoptosis by damaging mitochondria which results in the release of factors (such as cytochrome c and Smac/Diablo) that trigger caspase activation and cell death as a consequence. Here, we discuss the hierarchical nature of the caspase cascade that is triggered upon release of mitochondrial cytochrome c into the cytoplasm and the role of specific caspases within this cascade in targeting proteins for degradation. Finally, feedback amplification loops and important control points within the caspase cascade will also be discussed.

Background: Cancer-specific alternative splicing as well as aberrant splicing factor expression in tumors have been reported (Korner M et al, Am. J. Pathol. 175:461-472, 2009). Splicing factor SRp20 belongs to a family of highly conserved serine-arginine-rich proteins with multiple functions in RNA processing such as spliceosome assembly and alternative splicing (Graveley BR, RNA. 6(9):1197-211, 2000). Materials and Methods: We established stable MDA-MB-231, MCF-7, and T47D cell lines carrying doxycycline (Doxy)-inducible SRp20shRNA and assessed Doxy-induced knockdown (KD) of SRp20 by western blot. We performed cell growth curves in triplicate using the MTT assay to determine the effect of SRp20 KD in these cell lines after Doxy induction of SRp20shRNA, and compared these to controls. For apoptosis assays, we stained SRp20shRNA sublines with Hoechst 33342 and counted apoptotic cells under the microscope. We also carried out western blots to examine the expressions of Bcl-2 and cleaved caspases-3, -6, -7, and -9. Results and Discussion: We found that the expression of SRp20 was upregulated in immortalized human mammary epithelial cells (HMECs), compared to controls, and that the level of SRp20 correlated with the transformation state. Knockdown of SRp20 was seen to be 88%, 58%, and 52% in MDA-MB-231, MCF-7, and T47D cells, respectively. However, SRp20 KD suppressed breast cancer cell growth in a cell line-dependent manner. Cell growth after 7 days was decreased in estrogen receptor-negative (ER-) MDA-MB-231 cells (91.4%), and estrogen receptor-positive (ER+) MCF-7 (73.2%), while SRp20 KD had no effect on growth of ER+ T47D cells. The differences between MCF-7 and T47D were notable, since SRp20 was suppressed by -50% in both lines. We found that the number of apoptotic cells in MDA-MB-231 line remarkably increased after Doxy treatment, indicating that the inhibition of cell growth was due to apoptosis. Further, immunoblot analysis of Bcl-2 and cleaved caspases in MDA-MB-231 cells indicated that the SRp20 KD activates the intrinsic apoptotic pathway in these breast cancer cells. We are presently assessing the expressions of these proteins in MCF-7 and T47D cells and investigating the mechanistic basis for this cell line-dependent effect of SRp20 KD. In summary, we report here a novel finding that inhibition of splicing factor SRp20 inhibits cell growth in some breast cancer cell lines. The intrinsic apoptotic pathway appears to be the main mechanism of cell growth inhibition in these cells. Our results indicate that SRp20 plays a major role in the survival of some breast cancer cell lines. This splicing factor may represent a novel therapeutic target for the treatment of some breast cancers. (Supported in part by CA40570 and CA138762 from NCI to WTB, in part by IDPH to XH, and in part by UIC) Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-11.

correlated with radiation survival. Androgen receptor is detected in a majority of breast carcinomas in a proportion largely determined by the assay used for detection and the subtype of breast carcinoma. In triplenegative breast carcinomas (TNBC), it is estimated that AR is expressed in 15e35% of cases, representing a significant proportion of this currently un-targetable and aggressive subtype. We collated data from the CCLE to determine the relative mRNA levels of AR across lineages. Unsurprisingly, prostate cancers had the highest mean value of AR expression among 28 tumor lineages. This was followed by osteosarcoma and then breast cancer. Using TCGA RNAseq data, we determined the level of AR mRNA and its correlation with ER. We showed that a subset of breast carcinoma tumor samples have elevated AR and not ER (or PR). To demonstrate the role of AR in therapeutic resistance, we showed that the androgen receptor antagonist, enzalutamide, only sensitized cell lines with elevated AR expression. Conclusion: It has been previously demonstrated that AR signaling promotes the radioresistance of prostate cancer. This resistance has heretofore not been described in breast carcinoma. This data indicates that antagonism of AR is a promising therapeutic strategy in patients with AR expression and nominates enzalutamide, a drug currently in clinical use in prostate and breast cancer, as a potent selective sensitizer of therapy.