Targeting treatment resistant breast cancer stem cells with FKBPL and its peptide derivative, AD-01

FKBPL and its peptide derivative, AD-01, have already demonstrated well-established inhibitory effects on breast cancer growth and CD44 dependent anti-angiogenic activity1, 2, 3. Since breast cancer stem cells (BCSCs) are CD44 positive, we wanted to explore if AD-01 could specifically target BCSCs. FKBPL stable overexpression or AD-01 treatment were highly effective at reducing the BCSC population measured by inhibiting mammosphere forming efficiency (MFE) in cell lines and primary breast cancer samples from both solid breast tumours and pleural effusions. Flow cytometry, to assess the ESA+/CD44+/CD24- subpopulation, validated these results. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed across three generations of mammospheres, where mammospheres were completely eradicated by the third generation (p<0.001). Clonogenic assays suggested that AD-01 mediated BCSC differentiation, with a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones. In support of this, the stem cell markers, Nanog and Oct4 were significantly reduced following AD-01 treatment, whilst transfection of FKBPL-targeted siRNAs led to an increase in these markers and in mammosphere forming potential, highlighting the endogenous role of FKBPL in stem cell signalling. The clinical relevance of this was confirmed using a publically available microarray data set (GSE7390), where, high FKBPL and low Nanog expression were independently associated with improved overall survival in breast cancer patients (log rank test p=0.03; hazard ratio=3.01). When AD-01 was combined with other agents, we observed synergistic activity with the Notch inhibitor, DAPT and AD-01 was also able to abrogate a chemo- and radiotherapy induced enrichment in BCSCs. Importantly, using ‘gold standard’ in vivo limiting dilution assays we demonstrated a delay in tumour initiation and reoccurrence in AD-01 treated xenografts. In summary, AD-01 appears to have dual anti-angiogenic and anti-BCSC activity which will be advantageous as this agent enters clinical trial.

Targeting treatment resistant cancer stem cells with FKBPL and its peptide therapeutics

FKBPL and its peptide derivatives have already demonstrated well-established inhibitory effects on cancer growth and CD44-dependent anti-angiogenic activity. Since cancer stem cells (CSCs) are CD44 positive, we wanted to explore if these therapeutics could specifically target CSCs in breast and ovarian cancer. In a tumoursphere assay, FKBPL stable overexpression or FKBPL-based peptide (AD-01, preclinical peptide or ALM201, clinical peptide candidate) treatment were highly effective at reducing the CSC population measured by inhibiting tumoursphere forming efficiency in breast and ovarian cancer cell lines and primary breast cancer samples from both solid breast tumours and pleural effusions. Flow cytometry, to assess the ESA+/CD44+/CD24- and ALDH+ cell subpopulations representative of CSCs, validated these results. The ability of AD-01 and ALM201 to inhibit the self-renewal capacity of CSCs was confirmed across three generations, eradicating CSC completely by the third generation (p<0.001). Furthermore, clonogenic assay demonstrated that FKBPL-based peptides mediated CSC differentiation, with a significant decrease in the number of CSCs or holoclones and an associated increase in differentiated cancer cells or meroclones/paraclones. In addition, AD-01 treatment in vitro and in vivo led to a significant reduction in the stem cell markers, Nanog, Sox2 and Oct4 protein and mRNA levels; whilst transfection of FKBPL-targeted siRNAs led to an increase in these markers and in tumoursphere forming potential, highlighting the endogenous role of FKBPL in stem cell signalling. The clinical relevance of this was confirmed using a publically available microarray data set (GSE7390), where, high FKBPL and low Nanog expression were independently associated with improved overall survival in breast cancer patients (log rank test p=0.03; hazard ratio=3.01). Additionally, when AD-01 was combined with other agents, we observed additive activity with the Notch inhibitor, DAPT and AD-01 was also able to abrogate a chemo- and radiotherapy induced enrichment in CSCs. Importantly, using gold standard in vivo limiting dilution assays we demonstrated a delay in tumour initiation and reoccurrence in AD-01 treated xenografts. In summary, FKBPL-based peptides appear to have dual anti-angiogenic and anti-CSC activity which will be advantageous as this agent enters clinical trial.

Polyomavirus enhancer activator 3 protein promotes breast cancer metastatic progression through Snail-induced epithelial-mesenchymal transition.

Polyomavirus enhancer activator 3 protein (Pea3), also known as ETV4, is a member of the Ets-transcription factor family, which promotes metastatic progression in various types of solid cancer. Pea3-driven epithelial-mesenchymal transition (EMT) has been described in lung and ovarian cancers. The mechanisms of Pea3-induced EMT, however, are largely unknown. Here we show that Pea3 overexpression promotes EMT in human breast epithelial cells through transactivation of Snail (SNAI1), an activator of EMT. Pea3 binds to the human Snail promoter through the two proximal Pea3 binding sites and enhances Snail expression. In addition, knockdown of Pea3 in invasive breast cancer cells results in down-regulation of Snail, partial reversal of EMT, and reduced invasiveness in vitro. Moreover, knockdown of Snail partially rescues the phenotype induced by Pea3 overexpression, suggesting that Snail is one of the mediators bridging Pea3 and EMT, and thereby metastatic progression of the cancer cells. In four breast cancer patient cohorts whose microarray and survival data were obtained from the Gene Expression Omnibus database, Pea3 and Snail expression are significantly correlated with each other and with overall survival of breast cancer patients. We further demonstrate that nuclear localization of Pea3 is associated with Snail expression in breast cancer cell lines and is an independent predictor of overall survival in a Chinese breast cancer patient cohort. In conclusion, our results suggest that Pea3 may be an important prognostic marker and a therapeutic target for metastatic progression of human breast cancer. © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Solid phase synthesis of Smac/DIABLO-derived peptides using a 'Safety-Catch' resin:Identification of potent XIAP BIR3 antagonists

The N-terminal sequence of the Smac/DIABLO protein is known to be involved in binding to the BIR3 domain of the anti-apoptotic proteins IAPs, antagonizing their action. Short peptides and peptide mimetics based on the first 4-residues of Smac/DIABLO have been demonstrated to re-sensitize resistant cancer cells, over-expressing IAPs, to apoptosis. Based on the well-defined structural basis for this interaction, a small focused library of C-terminal capped Smac/DIABLO-derived peptides was designed in silico using docking to the XIAP BIR3 domain. The top-ranked computational hits were conveniently synthesized employing Solid Phase Synthesis (SPS) on an alkane sulfonamide 'Safety-Catch' resin. This novel approach afforded the rapid synthesis of the target peptide library with high flexibility for the introduction of various C-terminal amide-capping groups. The library members were obtained in high yield (>65%) and purity (>85%), upon nucleophilic release from the activated resin by treatment with various amine nucleophiles. In vitro caspase-9 activity reconstitution assays of the peptides in the presence of the recombinant BIR3-domain of human XIAP (500nM) revealed N-methylalanyl-tertiarybutylglycinyl-4-(R)-phenoxyprolyl-N-biphenylmethyl carboxamide (11a) to be the most potent XIAP BIR3 antagonist of the series synthesized inducing 93% recovery of caspase-9 activity, when used at 1µM concentration. Compound (11a) also demonstrated moderate cytotoxicity against the breast cancer cell lines MDA-MB-231 and MCF-7, compared to the Smac/DIABLO-derived wild-type peptide sequences that were totally inactive in the same cell lines.