FKBPL and its therapeutic peptides target CSCs and downregulate DLL4 and Notch4 in MDA-MB-231 and MCF-7 cells
We have already demonstrated that FKBPL and its peptide derivatives potentially exert their activity by targeting the CD44 pathway [5, 24]. Nevertheless, when we treated MDA-MB-231 cells with a gamma-secretase inhibitor which inhibits the Notch pathway in combination with AD-01, an additive inhibitory effect on the CSCs was observed [5]. Therefore, we investigated the impact of AD-01, as well as endogenous FKBPL, using cells stably overexpressing FKBPL (A3), on DLL4 and Notch 4 levels, which are implicated in metastasis and CSC fate [11, 14]. When we stably overexpress FKBPL in MDA-MB-231 cells, the number of holoclones, which represent CD44+ CSCs [32], were reduced by over 50% (Fig. 1a, p < 0.001, picture 1 inset), whilst the number of meroclones and paraclones, representing differentiated cells [5], concomitantly increased (Fig. 1b, p < 0.001; picture 2 and 3 inset). Overall the number of colonies was unaffected. FKBPL stable overexpression in MDA-MB-231 cells also led to down-regulation of DLL4 protein (Fig. 1c, p < 0.01) and mRNA levels (Fig. 1d, p < 0.01). Similarly, treatment of MDA-MB-231 cells with AD-01 (100 nM), demonstrated inhibitory effects on both DLL4 protein (Fig. 2a, p < 0.05) and Notch 4 intracellular domain (ICD) protein expression (Fig. 2b, p < 0.05). Treatment with the clinical peptide, ALM201 (100 nM), also led to downregulation of DLL4 mRNA levels (Fig. 2c, p < 0.01). To elucidate whether the FKBPL-mediated effect on mammosphere forming efficiency (MFE) was dependent on CD44, ALM201 was used to treat MDA-MB-231 mammospheres with stable CD44 knockdown. To demonstrate that MDA-MB-231 mammospheres were representative of CSCs, a 10-fold enrichment in the ESA+/CD44+/CD24- subpopulation was observed using flow cytometry within MDA-MB-231 mammospheres (Additional file 1: Figure S1). ALM201 was still able to inhibit the MFE in MDA-MB-231 CD44 knockdown cells at a similar level to parental MDA-MB-231 cells (Fig. 2d, p < 0.01). No difference was observed between ALM201 treated cells with stable CD44 knockdown versus ALM201 treated parental cells (MDA-MB-231; Fig. 2d). This data suggests that ALM201 is not completely dependent on CD44 for its anti-CSC activity, implicating the involvement of DLL4 and Notch4, as demonstrated above.
In ER+ breast cancer, we have shown that FKBPL is in a HSP90-associated chaperone complex with ERα receptor and that it can regulate ER signalling [22]. Using the ER+ MCF-7 cell line, FKBPL overexpression led to a better response to endocrine therapy i.e. tamoxifen and fulvestrant, whereas FKBPL knockdown had the opposite effect [22]. Endocrine therapy resistance is associated with an increase in the number of CSCs through activation of the Notch pathway [13, 33]. Here, we expand on the role of FKBPL in ER+ breast cancer by investigating the effect of stable FKBPL overexpression in MCF-7 cells (D2) on CSC-like colonies, holoclones. Similar to the triple negative breast cancer cell line, MDA-MB-231, FKBPL overexpression in MCF-7 cells resulted in a reduction in the number of holoclones and concomitant increase in the number of differentiated colonies while the overall colony number remained the same (Fig. 3a, p < 0.001). The effect of FKBPL stable overexpression in MCF-7 cells on DLL4 was dramatic, showing over 90% reduction in DLL4 protein expression (Fig. 3b, p < 0.001) and a trend towards a reduction in DLL4 mRNA levels (Fig. 3c, p = 0.057). In support of this, treatment of MCF-7 cells with FKBPL’s peptide derivative, AD-01, also led to downregulation of both DLL4 (p < 0.01) and the Notch4 ICD (p < 0.05) proteins levels (Fig. 3d). Similarly, the clinical peptide, ALM201, also showed downregulation of DLL4 mRNA levels in MCF-7 cells (Fig. 4e, p < 0.001).
AD-01 inhibits migration, invasion and lung metastasis in triple negative MDA-MB-231 experimental models
Based on our previously published work we have established that the FKBPL pre-clinical peptide, AD-01, could inhibit both endothelial and tumour cell migration in a CD44-dependent manner [24, 25]. Furthermore, we have demonstrated that AD-01 could target CSCs in the triple negative MDA-MB-231 cell line [5]. Since CSCs are known to be associated with invasion and metastasis, here we addressed whether the FKBPL-peptide could inhibit invasion in vitro and whether this could be translated in an experimental model of metastasis using the triple negative metastatic MDA-MB-231 breast cancer cells. Treatment of MDA-MB-231 cells for 24 h with AD-01 (1 nM) confirmed inhibition of cell migration, the first step in the invasive process, through an uncoated Boyden chamber (Fig. 4a, n ≥ 3, p < 0.01). Furthermore, we were also able to prevent invasion through a Matrigel coated Boyden chamber (Fig. 4b, n ≥ 3, p < 0.001). Since it has been previously demonstrated by Ebos and colleagues [34] that anti-angiogenic agents can promote metastases, we investigated whether AD-01 pre-treatment can prevent metastatic invasion using two separate in vivo MDA-MB-231 experimental lung metastasis models. In the first experiment, SCID mice were pre-treated daily with the stated dose of AD-01 or PBS for 1 week prior to being inoculated with MDA-MB-231-lucD3H1 cells via tail vein injection. AD-01 was subsequently administered to mice daily via i.p. injection (0.03 and 0.3 mg/kg/day). Control mice received PBS injections daily. AD-01 (0.3 mg/kg/day, p < 0.05; 0.003 mg/kg/day, p = 0.08) inhibited lung colonization of breast cancer cells following 28 days of in vivo treatment in addition to pre-treatment in vitro (Fig. 4c). No difference in the total photon flux was observed at day 0 following intravenous inoculation of MDA-231-lucD3H1 cells from either of the pre-treated groups (PBS or AD-01; Fig. 4c). In the second experiment, MDA-MB-231-lucD3H1 cells were pre-treated for 24 h with the stated dose of AD-01 or PBS, and injected via tail vein (in this experiment the mice were not pre-treated). Following i.v. injection of the cells, mice were treated i.p. in vivo for 26 days using either PBS as a control or AD-01 (0.3 or 0.003 mg/kg/day). Lung metastasis colonization was assessed using non-invasive bioluminescence of total photon flux. AD-01 (0.003 mg/kg/day; p < 0.05) treatment significantly reduced the total photon flux, indicative of the lung cell load compared to the vehicle PBS control (Fig. 4d, p < 0.05). Weight and wellbeing of each mouse was monitored daily and no significant weight reduction (≥15%) was observed. The drug was generally well tolerated and all animals where initial metastatic burden was recorded following MDA-MB-231-lucD3H1 cell inoculation via tail vein were included in the analysis.
FKBPL and its therapeutic peptides target endocrine therapy-resistant CSCs within an ER+ breast cancer context in both cell lines and clinical samples
CSCs within ER+ breast cancer are resistant to endocrine therapy due to the lack of ER expression [35]. In order to demonstrate that FKBPL-based clinical peptide, ALM201, is able to target endocrine therapy resistant CSCs in ER+ breast cancer, we treated the ER+ breast cancer cell line, MCF-7, with estradiol (100 nM) ± tamoxifen (1 μM) ± ALM201 (1 nM) and carried out mammosphere assay. A trend towards increase in the MFE was demonstrated following treatment with tamoxifen alone (MFE = 3.5%, p = 0.17) in the presence of estradiol (Fig. 5a). ALM201 alone, at a very low dose (1 nM) (MFE = 2.2%, p = 0.08) or in combination with tamoxifen (MFE = 1.86%, p < 0.01) reduced the MFE compared to estradiol treatment alone (Fig. 5a); the statistical significance was only observed when tamoxifen and ALM201 were used in combination. Importantly, the combination of ALM201 and tamoxifen seems to be even more effective at inhibiting the MFE (%). To ensure that mammospheres were representative of the CSC population, we were able to demonstrate a two-fold enrichment in the CD44+/CD24− subpopulation of cells within MCF-7 mammospheres (Additional file 2: Figure S2).
Previously, using a range of ER+ and ER- metastatic breast cancer patient samples we demonstrated a modest 20% reduction in the MFE following AD-01 treatment, even though the dose of AD-01 used was low (5 nM) [5]. Here we assessed the effects of ALM201, at a dose of 100 nM using clinically relevant ER+ breast cancer tissue from patients undergoing mastectomy and treated in the neoadjuvant setting with letrozole. We demonstrated up to 12-fold higher MFE (MFE ranged from 1.5–4.8; Fig. 5b- black bars) compared to our previously published data using breast cancer tissue from patients without neoadjuvant treatment (where MFEs of 0.4 were observed) [5]. Importantly, in three of the four patient samples, ALM201 significantly reduced the MFE, up to 70% (Fig. 5b). Sample 029AF, where no significant effect was observed, was negative for the expression of PR unlike the rest of the samples which were all ER+ and PR+ (Additional file 3: Table S1).
The anti-CSC effect of ALM201 was further validated in ER+ metastatic breast cancer samples from pleural effusions. ALM201 (100 nM) was effective at reducing the MFE by over 45% in three patient samples (BB3EC66–45% reduction; BB3RC8–66% reduction; BB3RC90–64% reduction; p < 0.01 or 0.001; Fig. 5c). This is important, since these samples are from patients with end-stage, highly metastatic disease with treatment-resistant tumours; all of these patients were unsuccessfully treated with a wide range of endocrine and chemotherapy regimens (Additional file 4: Table S2).
ALM201 in combination with tamoxifen delays tumour initiation and reduces the number of mammosphere forming tamoxifen-resistant CSCs in ER+ MCF-7 xenografts
In order to validate the results obtained in vitro and in clinical samples, an in vivo tumour initiation assay was carried out. Here, mice carrying established tumours (100–150 mm3) were treated with 1) vehicle control, 2) tamoxifen (12.5 mg/kg/day), 3) ALM201 (0.3 mg/kg/day), and 4) tamoxifen + ALM201, for a period of 21 days. Following three weeks of treatment, tumours were excised and tumour cells used in an ex vivo mammosphere assay or re-implanted into second generation SCID mice without any further treatment to assess the tumour initiating potential. The ex vivo mammosphere assay, using tumour cells from first generation treated MCF-7 xenografts, showed no change in the MFE between control and tamoxifen treated tumours (MFE = 3.5%, control (n = 6) vs. MFE =3.3%, tamoxifen (n = 4); Fig. 6a). ALM201 alone or in combination with tamoxifen led to a significant reduction in the MFE (MFE = 2%, ALM201 (n = 4), p < 0.01; and MFE = 0.5%, ALM201 and tamoxifen (n = 4), p < 0.001; Fig. 6a) compared to tamoxifen treatment. Interestingly, the combination of tamoxifen and ALM201 appeared even more effective at inhibiting the MFE than ALM201 alone (Fig. 6a, p < 0.01). When tumour cells were re-implanted into the second generation untreated mice, there was no delay in the number of days to palpable tumours between vehicle-treated or tamoxifen-treated tumour cells (Fig. 6b), suggesting that tamoxifen does not target the tumour initiating cell population. However, cells derived from the first generation ALM201-treated mice showed a significant delay in tumour recurrence of ~ 12 days compared to control or tamoxifen (Fig. 6b, p < 0.05). Importantly, the time to palpable tumour was even further delayed by 22 days when cells from the first generation tamoxifen and ALM201 treated mice were used in combination compared to tamoxifen alone (Fig. 6b, p < 0.001). Weight and wellbeing of each mouse were monitored closely and no significant weight reductions (≥15%) were observed. Any mouse showing signs of poor wellbeing was euthanized according to the approved protocol. The drugs was generally well tolerated and all animals displaying tumours were included in the analysis; vehicle-treated/control (13/16), tamoxifen (14/15), ALM201 (5/7) and tamoxifen+ALM201 (5/6).
When secondary tumours were excised and tumour cells were subjected to an ex vivo mammosphere assay, no effect on the MFE was observed in the tamoxifen-treated group (n = 4, p = 0.1) compared to control (n = 6; Fig. 6c). However, in combination with ALM201, the MFE appeared reduced compared to tamoxifen alone (n = 3; Fig. 6c, p = 0.15), however not statistically significant. Treatment with ALM201 alone did not lead to any significant change in the MFE compared to control (n = 2; Fig. 6c). This could be due to the small number of tumours excised or interrupted treatment with ALM201 in the second generation mice. Interestingly, ex vivo qPCR analysis of MCF-7 xenografts treated with both ALM201 and tamoxifen also showed a trend towards downregulation of DLL4 mRNA compared to control (Fig. 6d; n = 2).