Article. 12 May 2025. Research Service, Bruce W. Carter Veterans Affairs Medical Center, Miami, FL 33125, USA. Geriatric Research, Education, and Clinical Center, Bruce W. Carter Veterans Affairs Medical Center, Miami, FL 33125, USA. Division of Gerontology & Palliative Medicine, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA. Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
The increased use of potent androgen receptor antagonists has resulted in a rise in advanced prostate cancers resistant to androgen deprivation therapy with few treatment options. GHRH expression in cancers has led to the development of peptide antagonists (e.g., MIA-602 and -690) for therapeutic treatment. However, MIA-602/690 GHRH antagonists alone are not likely to be effective against advanced prostate cancer. We identified the novel combination of PI3K inhibitors + MIA-602/690 that increased cell death in all types of prostate cancer cells, including ones resistant to androgen deprivation therapy. The ability of MIA-602/690 and PI3K inhibitors to affect multiple signaling pathways may enhance cell death and optimize therapeutic benefit.
Background. Antagonists of GHRH have experimental therapeutic value, but as single agents are not likely to improve clinical outcomes, especially in advanced prostate cancer resistant to androgen deprivation therapy. Our objective is to identify anti-cancer drugs that, in combination with MIA-602 or -690 GHRH antagonists, increase cell death in all types of prostate cancer. Methods/Results. We identified inhibitors of PI3Kα or PI3Kβ that consistently increased cell death when combined with MIA-602/690. The PI3K family is critical in mediating upstream signals from receptors to downstream AKT/mTOR signaling pathways and has an important role in cancer progression. The results revealed that MIA-602/690 alone decreased androgen receptors and likely enhanced PI3K (negative feedback), which was then countered by the addition of PI3K inhibitors. Furthermore, the MIA-602/690 + PI3K inhibitor combination affected multiple signaling pathways, including apoptosis (anti-apoptotic Mcl-1L switching to pro-apoptotic Mcl-1S), proliferation (E2F1, cyclin A), PI3Kα/β, AKT, and ERK. Similar results were obtained with a more clinically relevant acetate salt form of MIA-602/690. The identification of PI3K as a drug target for prostate cancer is significant because PTEN (negative regulator of PI3K) loss of function occurs in 40–50% and PIK3CA mutation/amplification occurs in 60% of prostate cancer patients, leading to a poor prognosis. Conclusion. The ability of the MIA-602/690 + PI3K inhibitor combination to alter multiple signaling pathways may weaken the activation of adaptive mechanisms resulting from each drug and improve efficacy.
Prostate cancer (PCa) is initially responsive to androgen deprivation therapy (ADT) but can develop resistance, leading to the progression of castration-resistant PCa (CRPC) [1,2]. New and more potent ADT drugs (e.g., androgen receptor [AR] antagonist enzalutamide) have been successfully used against CRPC; however, resistance eventually develops. A proposed mechanism of acquired resistance to ADT is an adaptive response where CRPC cells switch from being sensitive to the drug target (AR) to a CRPC cell type which is not dependent on the drug target (e.g., neuroendocrine PCa [NEPC]), leading to a reduction or loss of AR [3,4,5]. There are no effective treatments for late-stage advanced CRPC/NEPC resistant to current ADT strategies, suggesting that new approaches are required [3,4,5].
Standard treatments for androgen-sensitive PCa are gonadotropin-releasing hormone (GnRH) agonists such as goserelin and leuprolide, approved in the 1980s by the FDA [6]. These are based on the Nobel Prize discovery of Andrew Schally and others that hormones secreted from the hypothalamus stimulate the release of pituitary hormones (e.g., luteinizing hormone), which regulate androgen synthesis. Since the 1990s, the Schally group has focused their research on the role of growth hormone-releasing hormone (GHRH) in cancer, prompting efforts to develop synthetic antagonists of GHRH that can be used therapeutically [7]. GHRH is a neuropeptide secreted from the hypothalamus that regulates the secretion of growth hormone (GH) from the pituitary, which then stimulates the liver to produce insulin growth factor 1 (IGF1), a potent mitogen for multiple cancers [7,8]. GHRH and its receptor GHRHR (a member of the G protein-coupled receptor [GPCR] family) are also produced in multiple tissues and cancers to modulate cell proliferation and apoptosis, including PCa [7].
The early development of GHRH peptide antagonists improved upon pharmacokinetic properties, target binding, and anti-tumor effects [7]. More recently, the MIA series, especially MIA-602 and -690, have emerged as one of the most promising antagonists by binding to GHRH expressed on tumor cells, blocking GHRHR-mediated signaling pathways, and inhibiting tumor growth [7] (see ref. 67 in [7]). In PCa, GHRH peptide antagonists (including MIA-602) reduced the growth of CRPC xenograft tumors, possibly by decreasing ERK and AKT signaling [9] (see ref. 13 in [9]. However, it is unlikely that the treatment of advanced CRPC/NEPC with GHRH antagonists as single agents will be sufficient for optimal efficacy. There are a few studies that have identified potential drugs that, in combination with GHRH antagonists, can improve efficacy, including DNA-damaging agents (doxorubicin, 5-flourouracil, irinotecan, cisplatin), anti-mitotic docetaxel, and the EGFR inhibitor gefitinib [10]. We searched a series of anti-cancer drugs that, in combination with MIA-602 or -690 GHRH antagonists, increase PCa/CRPC/NEPC cell death.
In this report, we identified phosphatidylinositol 3-kinase (PI3K) isoform inhibitors that, when combined with MIA-602 or -690, increased cell death in all types of PCa, including CRPC and NEPC. PI3K is a family of lipid kinases that increases phosphatidylinositol (3,4,5)-trisphosphate (PIP3) lipids, which are important in mediating signals from receptor tyrosine kinase (RTK) and GPCR to downstream AKT/mTOR signaling pathways [11]. Since the loss of PTEN, a negative regulator of the PI3K pathway, occurs in 40% to 50% of patients with PCa and results in PI3K hyperactivation [12,13], there have been significant efforts to identify PI3K inhibitors that can improve efficacy [11,14,15,16]. Our results showed MIA-602 or -690 + PI3K isoform inhibitors altered multiple signaling pathways including apoptosis, proliferation, PI3Kα/β, AKT, ERK, and AR. The use of MIA-602 and -690 converted into a more clinically relevant acetate salt form had similar results. Overall, the MIA-602 or -690 + PI3K isoform inhibitor combination may improve the therapeutic efficacy in PCa/CRPC/NEPC.
GHRH antagonists MIA-602 (PhAc-Ada 0, Tyr 1, d-Arg 2, 5FPhe 6, Ala 8, Har 9, Tyr(Me)10, His 11, Orn 12, Abu 15, His 20, Orn 21, Nle 27, d-Arg 28, Har 29-NH2) and MIA-690 (PhAc-Ada 0-Tyr 1, d-Arg 2, Cpa 6, Ala 8, Har 9, 5FPhe 10, His 11, Orn 12, Abu 15, His 20, Orn 21, Nle 27, d-Arg 28, Har 29-NH2) were synthesized and purified as previously described [7] (see ref. 67 in [7]). Changes from the bioactive wild-type human GHRH (1–29) amino acid peptide are provided above. Dried preparations were resuspended in DMSO and small aliquots were stored at −20 °C. The abbreviations are as follows: PhAc, phenylacetate; Ada, 12-aminododecanoyl; 5FPhe, pentafluoro-phenylalanine; Har, homoarginine; Tyr (Me), O-methyltyrosine; Orn, ornithine; Abu, alpha-aminobutanoyl; Nle, norleucine; and Cpa, 4-chloro-phenyalanine. Peptides were eluted from the resin with a solvent containing trifluoroacetic acid (TFA), which is not acceptable for human studies due to its potential subcutaneous toxicity [17]. To remove residual TFA, peptides were passed through a carbonate ion-exchange resin column (VariPure IPE; Agilent, Santa Clara, CA, USA), diluted acetic acid was added, and the samples were lyophilized (referred to as MIA-602Ac [Ac, Acetate salt] and MIA-690Ac). GHRH antagonist activity was confirmed using the GH release assay in rats, as previously described [7] (see ref. 67 in [7]).
PI3K inhibitors alpelisib (PI3Kα), AZD8186 (PI3Kβ/δ), and duvelisib (PI3Kδ/γ); AKT1-3 inhibitor AZD5363 (capivasertib); proteasome inhibitor ixazomib (MLN9708); and mTOR inhibitor rapamycin were obtained from APExBIO (Houston, TX, USA); pan-PI3K inhibitor LY294002 and NFκB inhibitor parthenolide were from Sigma-Aldrich (St. Louis, MO, USA); anti-mitotic docetaxel and CDK inhibitor flavopiridol were from Sanofi-Aventis (Bridgewater, NJ, USA); anti-mitotic cabazitaxel was from LC Laboratories (Woburn, MA, USA); Bcl-2 inhibitor ABT-737 was from Abbott Laboratories (Abbott Park, IL, USA); AR antagonist enzalutamide was from Selleckchem (Houston, TX, USA); and Coomassie blue and trypan blue (0.4%) were from Thermo Fisher Scientific (Waltham, MA, USA).
Human AR+ androgen-sensitive PCa (LNCaP), AR+ CRPC (22Rv1), AR—CRPC (PC3, DU145), AR—NEPC (NCI-H660, LASCPC), and human non-cancer RWPE-1 (prostate epithelial) cells were obtained from the American Type Culture Collection (ATCC) and were used within 6 months of the resuscitation of the original cultures. The molecular characteristics of the PCa/CRPC/NEPC cell lines are summarized in Table 1. The LNCaP, 22Rv1, PC3, and DU145 cells were maintained in RPMI 1640 medium (Thermo Fisher Scientific) and 5% fetal bovine serum (R&D Systems, Minneapolis, MN, USA). The H660 and LASCPC cells were maintained in Advanced DMEM/F12, B27 supplement, Glutamax (Thermo Fisher Scientific), EGF, and bFGF (R&D Systems) [18,19]. RWPE-1 was maintained in Keratinocyte-SFM media (Thermo Fisher Scientific). All cells were grown with 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin (Thermo Fisher Scientific).
Cells were cultured in media containing MIA-602, MIA-690 (5 μM, TFA and Ac forms), alpelisib (10 μM), AZD8186 (0.025–10 μM), duvelisib (10 μM), capivasertib (0.025–10 μM), docetaxel (0.25–1 nM), ABT-737 (1 μM), LY294002 (10 μM), cabazitaxel (1 nM), flavopiridol (100 nM), ixazomib (50 nM), rapamycin (0.05 nM), and parthenolide (0.5 μM). In all the experiments, floating and trypsinized attached cells were pooled for further analysis.
Treated and control cells were harvested, resuspended in PBS, diluted 1:1 in 0.4% trypan blue, the dead blue and live non-blue cells were immediately counted using a hemacytometer, and the % of dead blue cells was determined from at least 2–3 independent experiments performed in triplicate.
The CellTiter Aqueous colorimetric method from Promega (Madison, WI, USA) was used to determine the cell proliferation of LNCaP and PC3 cells in media containing MIA-602/690 (TFA, Ac; 1, 2.5, 5 μM), alpelisib (1, 2.5, 5 μM), AZD8186 (5, 25 nM), or the control (0.1% DMSO). Cell proliferation was normalized against DMSO control and the data expressed as a percentage of the control from three independent experiments performed in triplicate. Whether drug interactions were synergistic, additive, or antagonistic was determined using the CalcuSyn Version 2 software program from Biosoft (Cambridge, UK). This program is no longer available from Biosoft. CI ≤ 0.7 was synergistic.
Cell pellets were resuspended in NP40 cell lysis buffer (1% NP40, 50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 2 mmol/L EGTA, 2 mmol/L EDTA, Halt Protease Inhibitor Cocktail, and Pierce Phosphatase Inhibitor [Thermo Fisher Scientific]), lysed by vortex, left on ice for 30 min, centrifuged, and the protein concentrations of the supernatant were determined with the Bio-Rad Laboratories (Hercules, CA, USA) protein assay. After the separation of 25 to 50 μg of protein by SDS-PAGE, the proteins were transferred by electrophoresis to Immobilon-P membrane and incubated in 5% nonfat dry milk, PBS, and 0.25% Tween 20 for 1 h. The following antibodies were used: GHRHR (28692) from Abcam (Waltham, MA, USA); PI3Kα (C73F8), PI3Kβ (C33D4), cl-PARP (9541), phospho (P)-AKT (Ser473; 587F11), AKT (9272), ERK1/2 (9102), and P-ERK1/2 (9101) from Cell Signaling Technology (Danvers, MA, USA); and Mcl-1 (S-19), AR (441), cyclin A (H432), E2F1 (KH59), mouse anti-rabbit IgG-HRP (2357), and m-IgG-Fc BP-HRP (525409) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Precision Plus Protein Dual Color Standards (Bio-Rad Laboratories) was used to estimate the molecular weights in kDa. Markers were used to cut the blots into horizontal strips so high, medium, and low molecular weight targets could be analyzed separately with the appropriate antibodies. In some cases, after analysis, the strips were pretreated with methanol for 1 min, washed, treated with Ponceau S Staining Solution (Thermo Fisher Scientific) for 15 min to strip the antibody signal, and analyzed with a different antibody. After immunodetection, our preference for loading controls was the staining of total proteins transferred to the membrane with Coomassie blue, because drug treatments often affect the levels of typical housekeeping proteins such as actin or tubulin. Blot images were cropped for the clarity of the presentation. Quantification of protein bands from images (Chem Doc MP Imaging System, Bio-Rad Laboratories) was performed using the UN-SCAN-IT digitizing software version 5.1 from Silk Scientific (Provo, UT, USA) (normalized to the protein signal from Coomassie blue staining). Measurements (pixels) from the specific protein signal were divided by the Coomassie blue stain protein signal and the final fold changes were determined by dividing this over the control cells (=1).
Statistical differences between drug-treated and control cells were determined by two-tailed Student’s t-test (unequal variance) from 2 to 3 independent experiments performed in duplicate or triplicate with p< 0.05 considered significant
