Anti-tumor Effects of Targeted Follicle-stimulatingHormone-lytic Peptide Conjugates in ProstateCancer (PC-3) Xenograft Mouse Model

Conjugates of membrane disrupting lytic peptides with a 15-amino acid segment of the β chain of chorionic gonadotropin (CG) or luteinizing hormone releasing hormone (LHRH) target and destroy cancer cell xenografts in nude mouse model. Follicle stimulating hormone receptors (FSHR) have been detected in tumor neo-vascular endothelial cells of a variety of cancers and tumor tissue in prostate and ovarian cancers. In the present study, we have conjugated a lytic peptide (Phor18) to each of three segments of the β chain of FSH that bind to FSHR, and tested these conjugates (FSH90-95-Phor18, FSH81-95-Phor18 and FSH33-53-Phor18) for their ability to target and inhibit growth of prostate cancer cells in vivo. We found that intravenous administration of FSH90-95-Phor18, FSH81-95-Phor18 and FSH33-53-Phor18 significantly (p<0.05) inhibited the growth of prostate cancer (PC-3) xenografts in nude mice. The minimal effective dose of FSH81-95-Phor18 was 0.1 mg/kg and 1 mg/kg for FSH9095-Phor18 and FSH33-53-Phor18. Immunohistochemical analyses of tumor sections at necropsy showed dense expression of FSHR on vascular endothelial cells in control mice, reduced expression in tumors of mice treated with FSH90-95-Phor18, FSH81-95-Phor18 or FSH33-53-Phor18. FSH8195-Phor18 had the most potent anti-angiogenic activity of the three conjugates tested. These data show that a lytic peptide conjugated to FSH β chain segments bind to FSHR and are capable of inhibiting prostate cell tumor growth by targeting and destroying endothelial cells that express


Introduction
Aside from non-melanoma skin cancer, prostate cancer is the most common cancer among men of all races. In the USA, prostate cancer is the second leading cause of cancer death in men and is associated with high morbidity and mortality [1]. The Cancer Statistics estimated new prostate cancer cases of 220,800 and 27,540 men died from the disease in 2015 [2]. Though currently used chemotherapeutic drugs are effective in reducing the mortality and morbidity caused by prostate cancer; but disease progression is inevitable [3][4][5][6]. One of the major problems associated with cancer treatment is dose-limiting toxicity that limits the doses of the chemotherapeutics that are systemically administered and do not specifically target the cancer cells. These facts led to attempts to develop more effective drugs that are able to specifically target cancer cells. It is widely accepted that luteinizing hormone/human chorionic gonadotropin (LH/hCG) and LHRH receptors are highly expressed in various tumor cells. Several lytic peptide drug conjugates that target LH/hCG and LHRH receptors expressed by prostate, breast, ovarian, pancreatic and testicular cancer cells have been tested in our laboratory in the past few years [7,8].
Among these drugs is a class of membrane disrupting peptides [9,10] that are abundantly present in nature. Tumor cells are 50 times more sensitive to these membrane-disrupting peptides than normal cells [11], suggesting their usefulness in destroying various cancers. In previous reports, we showed that conjugates of membrane disrupting lytic peptides with either luteinizing hormone releasing hormone (LHRH) or with a 15-amino acid segment of the β chain of hCG target and destroy human prostate cancer cells, breast cancer cells, ovarian cancer cells and pancreatic cancer cells in tumor bearing nude mice [12,13]. A LHRH receptor targeting membrane disrupting peptide conjugate, EP-100, has been developed by Esperance Pharmaceuticals and completed Phase 2 clinical studies [14].
Like LH, follicle-stimulating hormone (FSH) is a key gonadotropin hormone in mammalian reproduction. FSH is produced mainly in the anterior pituitary gland, and the target organs are the ovary and testis. FSH is a heterodimer consisting of a common α-subunit non-covalently associated with β-subunit. All mammalian β subunits of FSH have 111 amino acids. FSH receptor is a glycosylated transmembrane protein that binds FSH and belongs to the family G-protein-coupled receptors. In adult humans and animals, the FSH receptor is expressed only in the testicular Sertoli cells and the ovarian granulosa cells [15,16] and in low levels in the endothelial cells of the ovary [17] and testis [18]. Recently, it has been reported that the FSH receptor is expressed in vascular endothelial cells of at the periphery in a wide range of human tumors, including prostate cancer [19]. Two previous studies confirmed the presence of FSH receptors in prostate adenocarcinoma by immunohistochemical detection [20,21]. The role of FSH as a marker of extra prostatic extension in human prostate cancer is significant. Therefore, these results suggest that FSH receptor and its ligand FSH may play an important role in prostate carcinogenesis [22].
Given these recent reports and earlier findings from our laboratory showing effectiveness of lytic peptide conjugates in inhibiting tumor

ISSN 2381-3318
Subcutaneous implantation of PC-3 cells: PC-3 cells were harvested from subconfluent cultures after a brief exposure to 0.25% trypsin and 0.2% EDTA. Trypsinization was stopped by adding medium containing 10 % FBS. The cells were washed once in serum-free medium and resuspended in PBS. Only suspensions consisting of single cells with >90% viability were used for the injections. PC-3 cells (1 × 10 6 ) in 100 µL PBS suspended in Matrigel (0.1 ml) (Collaborative Biomedical Products Becton Dickinson Labware, Bedford, MA) were injected subcutaneously into the interscapular area using a 27-gauge needle. On day 21, after tumor cell implantation, mice bearing tumors between 100-200 mm 3 , were randomized into the following treatment groups (n=8); Vehicle controls; FSH 90-95 peptide (1 mg/kg), FSH 81-95 peptide (1 mg/kg), FSH 33-53 peptide (1 mg/kg), FSH 90-95 -Phor18 (0.1 mg/kg, and 1 mg/kg), FSH 81-95 -Phor18 (0.1 mg/kg, and 1 mg/kg), and FSH 33-53 -Phor18 (0.1 mg/kg, and 1 mg/kg) treatments. Peptides and peptide conjugates were dissolved in USP saline and further diluted with saline to achieve a dose concentration of 1 mg/kg (0.2 mg/ml) dose and 0.1 mg/kg (0.02 mg/ml) dose. Volumes of 100 µL were given intravenously by tail vein injections twice weekly and given five injections in total. The vehicle treated group received an equivalent amount of USP saline. Tumor volumes were measured with calipers twice a week and body weights of mice were measured once a week. Tumor volumes were calculated by the formula: V (mm 3 )=(length × width 2 )/2, in which width was the shortest measurement in millimeters. Mice were necropsied one week after the last treatment on day 36 th . At necropsy, tumors were dissected, weighed, photographed and portions were fixed by immersion in 5% neutral buffered formalin. The histology tissue samples were processed into paraffin blocks, and 5 μm sections were cut for further studies.

Immunohistochemistry
Immunoperoxidase staining was performed for FSH receptors on sections from the tumor tissue samples obtained from nude mouse xenografts. Sections were deparaffinized with xylene (3 times for 10 min. each) and rehydrated through ascending ethanol concentrations (30%, 50%, 70%, 95% and 99.7-100%) followed by an antigen retrieval step. In this procedure, slides were placed in boiling 0.01 M sodium citrate and left standing at room temperature for a further 35 minutes to unmask epitopes. The slides were then rinsed in phosphate buffer saline (PBS) twice for 5 min each. Endogenous peroxidase activity was inactivated by immersing slides in 1% (v/v) hydrogen peroxide in methanol for 20 min. The slides were washed twice for 5 min each in PBS. Subsequently, sections were blocked for 60 min in a humidified chamber using a blocking solution, comprised of 1% normal horse serum (Vectastain progression, we conceptualized that development of conjugates of lytic peptides with FSH could be an effective approach to target and destroy human prostate cancer cells. To date, a number of lytic peptide conjugates have been synthesized and their activities have been tested [8,13]. One such membrane-disrupting lytic peptide is Phor18, an 18-amino acid peptide that was developed at Esperance Pharmaceuticals for targeting LHRH receptors in cancers by conjugating Phor18 to LHRH (EP-100). EP-100 has been successfully administered intravenously to 76 patients, and demonstrated activity and safety [23]. In the present study, we synthesized a bio conjugate of Phor18 to each of three segments of the β chain of FSH that are known to bind to the FSH receptor. The three segments of the β chain of FSH used were 6 amino acids (90-95), 15 amino acids (81-95) and 21 amino acids (33-53) resulting in three conjugates FSH 90-95 -Phor18, FSH 81-95 -Phor18 and FSH 33-53 -Phor18. Subsequently, we assessed the ability of all three conjugates to target and lyse prostate cancer xenografts and assessed their effects on tumor growth and neovasculature.
A comprehensive understanding of the molecule's characteristics, including its destruction of prostate metastases in animal models is important for further development of FSH-Phor18 as a novel therapeutic option.

Indirect immunofluorescence confocal microscopy
Double immunoperoxidase staining was performed for FSH receptors and anti-von Willebrand factor, a specific marker of vascular endothelial cells, in sections from the tumor tissue samples obtained from nude mouse xenografts. The preparation of samples was conducted as described above including antigen retrieval and blocking. The slides were incubated overnight at 4℃ in a humidified chamber with the FSH receptor (323) monoclonal antibody at a dilution of 1:50 and the rabbit polyclonal antivon Willebrand factor (AbCam, USA), a specific marker of vascular endothelial cells at a dilution of 1:50. The negative control sections were treated in the same manner with PBS and biotin mixture in the absence of primary antibodies. The slides were then washed thoroughly with PBS (3 times for 10 min.) and incubated for 1 hour with a mixture of goat antirabbit Alexa 594 (Molecular Probes, USA) and goat anti-mouse Alexa 488 (Molecular Probes, USA) secondary antibodies. In the same experiment, nuclei were detected by incubating slides for 15 min. with Hoechst stain (Sigma, USA). The slides were mounted with a drop of clear mount (Electron Microscopy Sciences, USA). Pictures were taken with Leica Zeiss Confocal Laser Scanning Microscope at 63X with water emersion.

Statistical analysis
All results were expressed as mean ± S.E. unless otherwise noted. Statistical significance was assessed by Student's t-test or one-way analysis of variance was applied to compare the means of two or more groups simultaneously, p<0.05 was considered to be statistically significant.

Synthesis of the three conjugates, FSH 90-95 -Phor18, FSH 81-95 -Phor18, and FSH 33-53 -Phor18 was achieved
Mass spectra are shown in Figure 1(A-C). Purity of each of the FSH-Phor18 conjugate was obtained on mass spectra. The purity was >95% for all three conjugates.

FSH-Phor18 conjugates inhibit growth of prostate cancer cell xenografts in nude mice
Results of the in vivo experiment, in which doses of 0.1 mg/kg, and 1 mg /kg of each of the three FSH-Phor18 conjugates were tested for their abilities to inhibit growth of PC-3 xenografts in nude mice are shown in

Figures 2(A-C) and 3(A-C)
. The data illustrate that administration of FSH 90-95 -Phor18 and FSH 81-95 -Phor18 significantly (P<0.05) inhibited prostate cancer cell growth in vivo, as measured by tumor weights Figure 2(A-C) and tumor volumes Figure 3 (A-C) at necropsy when compared with vehicle group. The effective dose for FSH 90-95 -Phor18 was 1 mg/kg body weight, while the effective dose for FSH 81-95 -Phor18 was 0.1 mg/kg. The FSH 35-53 -Phor18 conjugate caused a significant reduction of tumor volume at necropsy at a dose of 1 mg/kg, but the reduction in tumor weight was not statistically significant when compared with vehicle group. None of the segments of the β chain of FSH had any effect on tumor weight or volume when administered alone and compared with vehicle group.

Tumor volume changes during treatment
To further investigate the effects of the lytic peptide conjugates on prostate tumor development, we measured tumor volume twice every week. As depicted in Figure 4 (A-C), vehicle treated mice bearing prostate tumor xenografts had a time-related increase in tumor volume. FSH 90-95 -Phor18, FSH 81-95 -Phor18 and FSH 33-53 -Phor18 caused significant reduction in tumor growth at the dose of 1 mg/kg from day 25 when compared to vehicle treated group and each of the FSH-peptide fragment treated groups. No side effects in FSH-Phor18 treated groups were found as no changes were found in liver and kidney function test in treated groups (data not shown). There was no significant difference in the mice weight at necropsy between the vehicle and treated groups (Figure 4(E-F)).

FSH-Phor18 conjugates reduce expression of FSHR in the neovasculature supplying tumors in mice bearing human prostate cancer cell xenografts
We next examined whether FSH-Phor18 conjugates reduce FSH receptors on the neovasculature of the tumors. To examine this, immunohistochemical analysis was performed on paraffin-embedded tissue sections by the use of the anti-FSH-receptor monoclonal antibody (323), followed by a secondary peroxidase-coupled antibody visualized with the use of the red-brown peroxidase-reaction product. Sections were stained with hemotoxylin. When compared to tumors of vehicle treated ( Figure 5A), fewer FSH-receptor-positive vascular endothelial cells were present in tumors of nude mice treated with FSH 90-95 -Phor18 ( Figure 5B) FSH 81-95 -Phor18 ( Figure 5C), and FSH 33-53 -Phor18 ( Figure 5D).

FSH receptors are present in vascular endothelial cells in prostate tumor xenografts
In order to confirm the presence of FSH receptors on the neovasculature of the tumors, we stained paraffin embedded sections of tumor xenografts using double immunofluorescence staining. We stained the sections using antibodies against FSH receptor (green) and von Willebrand factor (vascular endothelial cell marker; red). FSH receptors were co-localized with von Willebrand factor, as indicated by yellow fluorescence in the merged images ( Figure 6; panel D). Hoechst stained nuclei are shown in Figure 6; Panel A. These results clearly show the presence of FSH receptors in vascular endothelial cells of the prostate tumor xenografts in nude mice. Negative controls ( Figure B. Supplementary data).

Discussion
The goal of the current study was to synthesize conjugates of the membrane disrupting lytic peptide (Phor18) and one of the three segments of the beta chain of FSH (6 to 21 amino acids) and to test each of    ability of conjugates consisting of the membrane disrupting lytic peptide, Phor18 and selected FSH segments to inhibit the prostate tumor growth in nude mice bearing human prostate tumor xenografts. FSH 81-95 -Phor18 was the most potent conjugate of the three FSH-Phor18 conjugates tested.
Additionally, our results demonstrate that the anti-tumor effects of these conjugates are mediated by specific binding to FSH receptors expressed on the blood vessels supplying the prostate tumors and on FSH receptors on tumor cells. Our results are in agreement with previous studies that have demonstrated that FSH receptors selectively expressed on the surface of the blood vessels of a wide range of human tumors including prostate cancer [19][20][21].
Non tumoral tissues and their vasculature of pleura, lung, liver, bone, and lymph nodes do not express FSHR [19]. Several markers have been described that are preferentially expressed on blood vessels in tumors (e.g., prostate specific membrane antigen, α v β 3 -integrin, vascular endothelial growth factor and its receptors) and in the extracellular matrix surrounding newly formed blood vessels (matrix metalloproteinases, Robo-4) [24]. However, as suggested previously, targeting FSHR of the tumor neovasculature and tumor cells could enhance the potency of the FSH-Phor18 in killing tumors and can be more effective than targeting other known markers because many of these markers (e.g. integrins) do not allow highly specific targeting of tumors [25]. It has recently been reported that FSHR levels in primary renal cell carcinoma tumors correlate strongly with the response of metastatic tumors in patients treated with sunitinib, an antiangiogenic receptor tyrosine kinase inhibitor suggesting a direct relation between FSHR levels and extent of metastasis [26,27]. Our current results are significant from a clinical perspective because FSHR are absent in most healthy tissues; hence, targeting FSHR could minimize the damage that anti-cancer drugs do to surrounding tissues or organs. The lytic peptide conjugates have the added advantage that they are not antigenic because they are small and are metabolized with minimal side effects such as, probable inhibition of spermatogenesis in males and ovulation in females [28]. A limitation of our study is increased risk for animal's fertility. However, it was previously shown that the function of the pituitary gonadotrophs was completely recovered within 2 weeks after treatment with a targeted cytotoxic analog of LHRH [29]. Furthermore, the recombinant FSH treatment restored serum inhibin-B levels and partially restored spermatogenesis in gonadotropin-deficient rats [30].
The present study demonstrates the effectiveness of the FSH-Phor18 conjugates against primary prostate tumors through targeting neovasculature and tumors. Because the lytic peptides used in the present study are specifically bound by the targeting segment (βFSH), we suggest that our FSH-Phor18 conjugates would not only destroy primary tumors but also metastatic tumors. Reinforcing this concept, previous reports from our laboratory have shown that lytic peptides bound with LH or hCG prevent metastasis. Nonetheless, it would be interesting to investigate whether these conjugates are equally effective in targeting metastatic tumors, and these conjugates would overall increase survival rate of the treated mice, could be a focus for future studies. Taken together, these results suggest that FSH-Phor18 conjugates could be novel therapeutic options for the treatment of prostate cancers in humans.