Targeting Treatment-Resistant Breast Cancer Stem Cells with FKBPL and Its Peptide Derivative, AD-01, via the CD44 Pathway

Abstract

Purpose: FK506-binding protein like (FKBPL) and its peptide derivative, AD-01, have already shown tumor growth inhibition and CD44-dependent antiangiogenic activity. Here, we explore the ability of AD-01 to target CD44-positive breast cancer stem cells (BCSC).Experimental Design: Mammosphere assays and flow cytometry were used to analyze the effect of FKBPL overexpression/knockdown and AD-01 treatment AE other anticancer agents on BCSCs using breast cancer cell lines (MCF-7/MDA-231/ZR-75), primary patient samples, and xenografts. Delays in tumor initiation were evaluated in vivo. The anti-stem cell mechanisms were determined using clonogenic assays, quantitative PCR (qPCR), and immunofluorescence.Results: AD-01 treatment was highly effective at inhibiting the BCSC population by reducing mammosphere-forming efficiency and ESAtions in vitro and tumor initiation in vivo. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed; mammospheres were completely eradicated by the third generation. The mechanism seems to be due to AD-01-mediated BCSC differentiation shown by a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones; the stem cell markers, Nanog, Oct4, and Sox2, were also significantly reduced. Furthermore, we showed additive inhibitory effects when AD-01 was combined with the Notch inhibitor, DAPT. AD-01 was also able to abrogate a chemo-and radiotherapyinduced enrichment in BCSCs. Finally, FKBPL knockdown led to an increase in Nanog/Oct4/Sox2 and an increase in BCSCs, highlighting a role for endogenous FKBPL in stem cell signaling. Conclusions: AD-01 has dual antiangiogenic and anti-BCSC activity, which will be advantageous as this agent enters clinical trial.

RALA-Mediated Delivery of FKBPL Nucleic Acid Therapeutics

“…Samples from the same tumors were subsequently processed for mammosphere assay. The results from this assay were previously published in McClements et al 2013 [23] in graphical format, where we clearly demonstrate that FKBPL's ability to target CD44 leads to a reduction in stemness by targeting CD44+ breast cancer stem cells. As shown in Figure 4C, FKBPL knockdown resulted in a significant increase (56% increase) in mammosphere forming efficiency compared with control.…”

“…The remaining half of the excised tumor was excised and assayed using the ex vivo mammosphere assay, as described previously [23]. Briefly, the tumor was cut into less than 1 mm pieces, disaggregated overnight using DMEM/F12 (Gibco) medium containing 10% collagenase/hyaluronidase (Stem Cell Technologies) and filtered through 100, 70 and 40 μm cell strainers.…”

“…FKBPL is emerging as a key player in the DNA damage response [16], steroid receptor signaling [17,18] and more recently, control of tumor growth where it regulates response to endocrine therapy [19] as well as acting as a novel secreted antiangiogenic protein, where it inhibits endothelial cell migration and has potent antitumor activity in vivo [20,21]; this effect is dependent on the cell surface receptor CD44 [22]. More recently, we have demonstrated that because of FKB-PL's ability to target the cell surface receptor, CD44, we are also able to reduce cancer stem cells [23]. Finally, in a meta-analysis of 3277 patients from five breast cancer cohorts, FKBPL levels were a significant and independent predictor of breast cancer-specific survival, indicating superiority over current prognostic markers, not surprising giving the antitumor activity of this protein [24].…”

The Role of Peptidyl Prolyl Isomerases in Aging and Vascular Diseases

“…This extracellular role was associated with a potent anti-angiogenic and anti-CSC function which is initiated following binding of FKBPL to the CD44 cell surface receptor [193][194][195] . The region responsible for this interaction is the N-terminal region of FKBPL, which is unique and not homologous to other FKBPs.…”

“…The novel FKBPL-based therapeutic, ALM201, unlike other PPIases, appears to be protective of age-related diseases 147,154,187,193,194,196 ; ALM201 is a peptide mimetic of FKBPL and could essentially correct a deficiency in FKBPL in a number of diseases. It has already completed preclinical evaluation for imminent phase I/II clinical trials in cancer patients 196 ; our unpublished data also suggests that it has activity against a number of other inflammatory conditions.…”