Membrane metalloendopeptidase suppresses prostate carcinogenesis by attenuating effects of gastrin-releasing peptide on stem/progenitor cells

ABSTRACT Aberrant neuroendocrine signaling is frequent yet poorly understood feature of prostate cancers. Membrane metalloendopeptidase (MME) is responsible for the catalytic inactivation of

neuropeptide substrates, and is downregulated in nearly 50% of prostate cancers. However its role in prostate carcinogenesis, including formation of castration-resistant prostate

carcinomas, remains uncertain. Here we report that MME cooperates with PTEN in suppression of carcinogenesis by controlling activities of prostate stem/progenitor cells. Lack of MME and PTEN

results in development of adenocarcinomas characterized by propensity for vascular invasion and formation of proliferative neuroendocrine clusters after castration. Effects of MME on

prostate stem/progenitor cells depend on its catalytic activity and can be recapitulated by addition of the MME substrate, gastrin-releasing peptide (GRP). Knockdown or inhibition of GRP

receptor (GRPR) abrogate effects of MME deficiency and delay growth of human prostate cancer xenografts by reducing the number of cancer-propagating cells. In sum, our study provides a

definitive proof of tumor-suppressive role of MME, links GRP/GRPR signaling to the control of prostate stem/progenitor cells, and shows how dysregulation of such signaling may promote

formation of castration-resistant prostate carcinomas. It also identifies GRPR as a valuable target for therapies aimed at eradication of cancer-propagating cells in prostate cancers with

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PROMOTES LINEAGE PLASTICITY AND PROGRESSION TO NEUROENDOCRINE PROSTATE CANCER Article 19 August 2022 INTRODUCTION Prostate cancer is the most frequently diagnosed cancer and is the second

leading cause of cancer-related death in men in the United States1. While most prostate cancers are adenocarcinomas, a significant percentage also have dysregulation of neuroendocrine

signaling, such as excessive accumulation of cells with neuroendocrine differentiation and/or overproduction of neuropeptides2,3,4. A large amount of data demonstrate neuropeptides, such as

gastrin-releasing peptide (GRP), are associated with accelerated prostate cancer progression and inferior prognosis5,6,7. GRP can promote cell proliferation and accelerate migration and

invasion of prostate cancer cells5,8,9,10. Targeting of the GRP receptor suppresses growth in cell culture and xenograft models11. However, specific mechanisms by which neuropeptide

dysregulation contributes to the pathogenesis of prostate cancer remain insufficiently elucidated. Local concentration of neuropeptides is regulated in part by membrane metalloendopeptidase

(MME, aka Neutral endopeptidase). MME is a cell-surface peptidase member of the M13 family of zinc peptidases, which also includes endothelin converting enzymes (ECE-1 and ECE-2), KELL and

PEX. MME cleaves peptide bonds on the amino side of hydrophobic amino acids and is the key enzyme in processing of a variety of physiologically active peptides, such as GRP, neurotensin

(NT), and vasoactive intestinal peptide (VIP)10,12. MME is downregulated in nearly 50% of primary and metastatic prostate cancers, independently predicting an inferior prognosis13,14,15. In

addition to its downregulation by androgen withdrawal16,17, MME expression is also downregulated by methylation, suggesting its tumor-suppressive effects15,18. Indeed, MME expression reduces

growth, motility9, and survival19,20 of prostate cancer cells in cell culture. Consistent with these observations, replacements of MME inhibit tumorigenicity of prostate cancer cells in

xenograft experiments21,22. Nevertheless, mice lacking _Mme_ show no prostate cancer-related phenotype23, and the role of MME in prostate cancer progression remains uncertain. At least part

of MME effects are mediated by the PI3K/AKT pathway that plays a key role in multiple cellular processes, including cell survival, proliferation, and cell migration reviewed in ref. 24. MME

associates with and stabilizes the PTEN tumor suppressor protein, resulting in increased PTEN phosphatase activity, thereby inhibiting AKT activating phosphorylation25. MME may also have

PTEN-independent mechanisms of AKT inhibition by processing neuropeptides, such as GRP, which are known to activate AKT20. Consistent with a possibility of potential cooperation between MME

and PTEN in suppression of carcinogenesis, downregulation of MME is observed in 42% and 63% of PTEN-deficient cases of human primary and metastatic prostate cancers, respectively26. However,

it remains unknown if catalytically dependent neuropeptide-based mechanisms of MME tumor suppression play a role in prostate cancer progression. The mouse prostate is composed of a series

of branching ducts, each containing distal and proximal regions relative to the urethra27. Proliferating, transit-amplifying cells are preferentially located in the distal region of the

prostatic ducts, whereas cells with stem cell-like properties, such as low cycling rate, self-renewal ability, high ex vivo proliferative potential, and androgen withdrawal resistance,

mainly reside in the proximal region of the prostatic ducts28,29,30,31,32. Thus, approaches based on the isolation of cells according to their displayed stem cell-specific markers can be

complemented by careful evaluation of stem cell compartments in situ. In the current study, we used autochthonous mouse model of prostate neoplasia associated with deficiency of _Pten_ tumor

suppressor gene. In this model, prostate carcinogenesis is initiated by the prostate epithelium-specific inactivation of _Pten_ driven by PB-_Cre4_ transgene (_Pten_PE−/−

mice33,34,35,36,37,38). The majority of mice show early stages of prostate cancer, such as high-grade prostatic intraepithelial neoplasms (HG-PINs) and few animals show early adenocarcinomas

characterized by stromal invasion. Thus, it is well suitable for testing if additional genetic alterations, such as _Mme_ inactivation, may accelerate cancer progression. We report that

lack of both MME and PTEN leads to aggressive prostate cancers manifesting frequent vascular invasion and increased neuroendocrine differentiation after castration. Formation of such cancers

is preceded by morphologically detectable neoplastic lesions at the prostate stem/progenitor cell compartment. The effect of MME deficiency on stem/progenitor cells can be recapitulated by

its substrate GRP and is abrogated by either GRP receptor (GRPR) antagonist or _GRPR_ siRNA knockdown. Knockdown or inhibition of GRP receptor (GRPR) delay growth of human prostate cancer

xenografts by reducing the pool of cancer-propagating cells. RESULTS MME COOPERATES WITH PTEN IN SUPPRESSION OF PROSTATE CANCER IN AUTOCHTHONOUS MOUSE MODEL To test the cooperation of _Mme_

and _Pten_ genes in suppression of prostate cancer in vivo we first evaluated MME expression in HG-PINs and early invasive adenocarcinomas typical for _Pten_PE−/− mice. While irregular MME

expression was observed in the majority of neoplastic lesions, MME was absent in the areas of stromal invasion (Fig. 1, Supplementary Fig. 1). No significant alterations in MME expression

were detected in the proximal regions of prostatic ducts, consistent with the lack of neoplastic lesions in that part of the prostate in _Pten_PE−/− mice (Fig. 2a, Supplementary Fig. 2a,

Supplementary Table 1). Consistent with the reported regulation of MME by the androgen receptor (AR)16,17,39, castration of both WT and _Pten_PE−/− mice resulted in downregulation but not

complete abrogation of MME expression (Fig. 1). Next, we tested if MME deficiency can accelerate prostate carcinogenesis in _Pten_PE−/− mouse model. We crossed _Mme_−/− and _Pten_PE−/− mice,

and evaluated prostates of age-matched wild-type (WT), _Mme_−/−, _Pten_PE−/−, and _Mme_−/−_Pten_PE−/− strains (Fig. 2, Supplementary Fig. 2, Supplementary Table 1). Consistent with previous

observations23, _Mme_−/− mice did not develop any neoplastic lesions by 16 months of age, while _Pten_PE−/− mice showed low- and high-grade PINs at 3 months. At 16 months 29% of _Pten_PE−/−

mice developed early invasive adenocarcinomas, characterized by separate nests of neoplastic cells in desmoplastic stroma (Fig. 2, Supplementary Table 1). All neoplastic lesions were in the

distal regions of prostatic ducts. Eighty-six percent of _Mme__−/−__Pten_PE−/− mice developed adenocarcinomas in the same location (Fisher’s exact test _P_ = 0.0025). Furthermore, contrary

to _Pten_PE−/− mice, _Mme_−/−_Pten_PE−/− mice developed dysplastic lesions followed by adenocarcinomas in the proximal regions of the prostatic ducts (Fig. 2a, Supplementary Fig. 1,

Supplementary Table 1). Some adenocarcinomas of _Mme__−/−__Pten_PE−/− mice showed distinct vascular invasion, a feature not characteristic for prostatic lesions in _Pten_PE−/− mice (Fig. 2a,

Supplementary Figs. 2 and 3). Consistent with these histological observations, prostatic epithelium of _Mme__−/−__Pten_PE−/− mice was characterized by significantly higher proliferative

rate according to Ki67 staining (Fig. 2, Supplementary Fig. 2), expressed higher amounts of epigenetic reprogramming factor EZH2 (Fig. 2, Supplementary Fig. 4), and showed increased number

of CK5 and p63 positive cells (Supplementary Fig. 2) as compared to prostatic lesions in _Pten_PE−/− mice. Prostatic epithelium lesions of _Mme__−/−__Pten_PE−/− mice also showed higher

levels of pAKT (Fig. 2, Supplementary Fig. 4), confirming additional MME-dependent mechanisms of regulation of this downstream target of PTEN20,25. Consistent with our previous

observation35, prostatic neoplastic lesions of _Pten_PE−/− mice had increased number of synaptophysin-positive neuroendocrine cells in the distal regions of prostatic ducts (Fig. 2,

Supplementary Fig. 2). However, this number was not additionally elevated in _Mme__−/−__Pten_PE−/− mice. In sum, lack of both MME and PTEN not only promoted lesions typically observed in the

prostates of _Pten_PE−/− mice but also resulted in a distinct new neoplasms located in the proximal regions of prostatic ducts. MME LOSS LEADS TO MORE AGGRESSIVE TUMOR PHENOTYPE WITH

INCREASED PROLIFERATION OF NEUROENDOCRINE CLUSTERS AFTER CASTRATION As compared to _Pten_PE−/− mice, recurrent tumors in all castrated _Mme__−/−__Pten_PE−/− mice (_n_ = 8) had increased

levels of phosphorylated MET, and reduced expression of E-cadherin, signatures of more aggressive phenotype (Fig. 3). As previously reported35, recurrent tumors in castrated _Pten_PE−/− mice

show a decreased expression of AR and an increased number of neuroendocrine cells. MME deficiency in castrated _Pten_PE−/− mice did not further affect AR expression levels (Fig. 3a).

However, recurrent tumors in _Mme__−/−__Pten_PE−/− mice had a higher frequency of neuroendocrine clusters (≥ 3 cells) and such clusters were more proliferative according to Ki67 staining

(Fig. 3b, c). Thus, MME deficiency promotes the increase in neuroendocrine differentiation of neoplastic cells after castration. MME LOSS PROMOTES ACTIVITIES OF PTEN-DEFICIENT MOUSE PROSTATE

STEM/PROGENITOR CELLS The proximal regions of prostatic ducts are particularly enriched in prostate epithelium stem/progenitor cells28,29,30,31,32. Thus we evaluated potential effects of

MME and PTEN deficiency on prostate stem/progenitor cells, isolated as the CD49fhi/Sca-1+ fraction by fluorescence-activated cell sorting (FACS). _Mme_−/− mice had the same number of

stem/progenitor cells as age-matched WT mice (5.7% vs 5.6%). In contrast, _Pten_PE−/− mice showed a significant increase of the stem cell pool (8.4%) consistent with previous reports

suggesting PTEN’s involvement in regulation of prostate stem/progenitor cells40,41,42. The pool of CD49fhi/Sca-1+ cells deficient for both PTEN and MME, constituted 12.1% of the prostate

epithelium, representing an additional 44% increase as compared to PTEN-deficient CD49fhi/Sca-1+ cells (Fig. 4a, _P_ < 0.0001). Formation of prostaspheres is used as a functional cell

culture test for presence, growth, and self-renewal potential of prostate stem/progenitor cells. Consistent with FACS results, CD49fhi/Sca-1+ cells isolated from prostates of

_Mme__−/−__Pten_PE−/− mice showed the highest frequency of prostaspheres in multiple consecutive sphere dissociation and regeneration passages (Fig. 4b). Furthermore, prostaspheres deficient

for both genes had larger size, as compared to WT, MME, or PTEN-deficient stem/progenitor cells (Fig. 4c). In all groups CD49flo/Sca-1− luminal cells formed very few spheres after the first

plating and no spheres were observed after the first passage (Fig. 4d). Thus, it is unlikely that the increase in prostaspheres in PTEN and MME deficient cells resulted from a reprograming

of differentiated cells towards a stem cell state. To directly test if the observed results represent direct effects of MME and/or PTEN on prostate stem/progenitor cells, we isolated

CD49fhi/Sca-1+ stem/progenitor cells and CD49flo/Sca-1− luminal cells from prostates of WT, _Mme_−/−, _Pten__loxP/loxP_, and _Mme_−/−_Pten__loxP/loxP_ mice and infected them with adenovirus

expressing Cre recombinase (Ad-_Cre_). Consistent with our previous experiments, lack of both _Pten_ and _Mme_ had the most pronounced effect on frequency of CD49fhi/Sca-1+ stem/progenitor

cells in consecutive passages (Fig. 4e). Luminal cells formed only few spheres with the same frequency in all groups (Fig. 4f). Taken together, these results showed that MME cooperates with

PTEN in regulation of prostate stem/progenitor cell functions. GRP PROMOTES ACTIVITIES OF PTEN-DEFICIENT MOUSE PROSTATE STEM/PROGENITOR CELLS To identify mechanisms by which MME may affect

regulation of prostate stem/progenitor cells, we next examined the expression of MMEs main substrates, GRP, NT, and VIP in the prostates of WT, _Mme__−/−_, and _Pten_PE−/− and

_Mme__−/−__Pten_PE−/− strains. Strong GRP expression was detected only in prostates of _Mme__−/−__Pten_PE−/− mice, while NT and VIP were not detected in all cases (Supplementary Fig. 5). To

test if GRP could recapitulate the effects of MME deficiency, we isolated prostate cells from _Mme__−/−__Pten_PE−/− mice, and either performed knockdown of GRP receptor (GRPR, Fig. 5a) or

administered GRPR antagonist [Tyr4, d-Phe12]-Bombesin. Both approaches reversed effects of MME deficiency on size and formation frequency of prostaspheres (Fig. 5b, c). Consistent with the

observation of frequent vascular invasion by prostate adenocarcinomas in _Mme__−/−__Pten_PE−/− mice, we detected increased cell motility and invasion of prostate cells isolated from

_Mme__−/−__Pten_PE−/− mice, as compared to those prepared from _Pten_PE−/− mice (Fig. 5d, e). This effect was reversed by either GRPR knockdown or treatment with [Tyr4, d-Phe12]-Bombesin. We

next isolated prostate cells form _Pten_PE−/− mice, and treated them with the MME enzyme inhibitor Thiorphan. Sphere size (Fig. 5f), sphere-forming capacity (Fig. 5g), cell motility (Fig.

5h), and invasion (Fig. 5l) were each increased by MME catalytic inhibition; and more importantly _Grpr_ siRNA knockdown or a GRPR antagonist abrogated the stimulated functions of MME enzyme

inhibition on the prostate cells form _Pten_PE−/− mice. Finally, to test if GRP directly affects the function of PTEN-deficient prostate stem/progenitor cells, CD49fhi/Sca-1+ prostate stem

cells isolated from _Pten__loxP/loxP_ were infected with Ad-_Cre_ followed by treatment with GRP and/or [Tyr4, d-Phe12]-Bombesin. GRP addition reproduced the effects of MME deficiency on

formation frequency and size of prostasphere, while these effects were abrogated by [Tyr4, d-Phe12]-Bombesin (Fig. 5j, k). Taken together, these data suggest that the MME substrate GRP

mediates in part the growth and invasive effect of PTEN-deficient prostate cells observed in association with loss or catalytic inhibition of MME, and GRP is a key MME target responsible for

stimulating prostate stem/progenitor cells. GRP PROMOTES EXPANSION OF HUMAN PROSTATE CANCER-PROPAGATING CELLS To further assess the relevance of our observations to human disease we tested

the effects of GRP and [Tyr4, d-Phe12]-Bombesin on PTEN-deficient androgen-refractory human prostate cancer cells DU145 and PC3. Consistent with previous reports5,7,9, GRP promoted cell

proliferation, migration, and invasion, and these effects were negated by addition of [Tyr4, d-Phe12]-Bombesin in both cell lines (Supplementary Fig. 6). Similar effects cell migration and

invasion were also observed in androgen-sensitive human prostate adenocarcinoma cells LNCaP (Supplementary Fig. 7). In agreement with our observations on mouse prostate epithelium cells,

GRPR knockdown rescued the stimulating effects of GRP and inhibitory effects of [Tyr4, d-Phe12]-Bombesin, suggesting the crucial role of GRP/GRPR in control of the above parameters in both

species (Supplementary Figs. 6 and 7). GRP treatment also increased expression of synaptophysin in DU145, PC3 cells, and LNCaP cells, thereby suggesting that GRP/GRPR signaling may influence

neuroendocrine commitment (Supplementary Fig. 8). Next we tested if GRP/GRPR signaling has an immediate effect on prostate cancer cells with stem cell-like properties. Such cells, called

cancer propagating or cancer stem cells29,43, are characterized by their ability for long-term self-renewal, high potential for proliferation, high tumorigenicity, capacity to generate the

whole spectrum of heterogeneity of their original tumors, and resistance to androgen withdrawal. Prostate cancer-propagating cells can be isolated by either high enzymatic activity of ALDH

(ALDEFLUOR assay)44 or by detection of CD44 expression45. In both approaches cancer-propagating cells isolated from DU145, PC3, and LNCaP cells were increased in number after GRP treatment

(Fig. 6a–d, Supplementary Fig. 7e, f). Cancer-propagating cells also formed spheres in greater number and size. These changes were diminished by [Tyr4, d-Phe12]-Bombesin and/or GRPR siRNA

(Fig. 6a–g). To study the effect of GRP on human prostate cancer xenografts in vivo, immunodeficient NSG mice were subcutaneously injected with PC3 cells and treated with [Tyr4,

d-Phe12]-Bombesin when the tumor size reached 10 mm3 (Fig. 6h–j). Daily intraperitoneal injections of [Tyr4, d-Phe12]-Bombesin reduced both tumor volume and weight by 70% by the time of

animal euthanasia at 26 days after the beginning of treatment. Importantly, according to the ALDEFLUOR assay, a fraction of prostate cancer-propagating cells was significantly reduced in

tumors treated with [Tyr4, d-Phe12]-Bombesin as compared to controls treated with vehicle alone. The same outcomes have been observed after transplantation of PC3 cells infected with

lentivirus-expressing GRPR shRNA (Supplementary Fig. 9). Thus, the effects of GRP/GRPR signaling abrogation in inhibiting prostate cancer cell tumorigenicity result from diminishing the

population of cancer-propagating cells. DISCUSSION Our study provides direct genetic support to previous reports proposing _Mme_ role as a tumor suppressor gene. Our findings show that _Mme_

cooperates with _Pten_ in suppression of prostate carcinogenesis. Lack of _Mme_ leads to formation of advanced adenocarcinomas characterized by intravascular invasion, a feature not

observed adenocarcinomas of _Pten_PE−/− mice. In agreement with their more aggressive behavior adenocarcinomas of composite mice also have higher proliferative rate, increased number of CK5

and p63 positive cells, and elevated levels of EZH2 expression. It has been reported previously that _Pten_ conditional deletion in the prostate epithelium leads to basal cell proliferation

with concomitant expansion of the prostate stem/progenitor-cell-like Sca-1+ and BCL-2+ subpopulation42. CD49fhi/Sca-1+ _Pten_-deficient neoplastic cells exhibits cancer-propagating cell

properties, such as high sphere-forming capacity, sustained self-renewal, increased proliferation, and tumorigenic potential, as compared with other isogenic subpopulations40,41. According

to our studies, the combined _Mme_ and _Pten_ deficiency leads to further increase of number and growth potential of prostate stem/progenitor cells. These observations suggest that _Mme_ and

_Pten_ cooperate in controlling stem/progenitor cells. However, since lack of _Pten_ may also stimulate a basal to luminal transdifferentiation in conjunction with proliferation46,47,48,

further studies are needed to evaluate the role of cell plasticity as an additional factor influencing effects of combined _Mme_ and _Pten_ deficiency. Our findings also show that the lack

of both _Mme_ and _Pten_ leads to the formation of dysplastic lesions and adenocarcinomas in the proximal regions of prostatic ducts, the structures particularly enriched in stem/progenitor

cells. Thus, _Mme_ may playing a particularly critical role at the site of prostate stem/progenitor cell niche. Our findings also show that effects of MME on prostate stem/progenitor cells

depend on the presence of its downstream effector, GRP, Furthermore, we have observed that abrogation of GRP/GRPR signaling may diminish the pool of cancer-propagating cells, thereby

highlighting an important role of neuroendocrine signaling in the regulation of cell stemness. Previously, prostate cancer-propagating cells have been shown to be regulated by the

PTEN/PI3K/AKT pathway40. Our study suggests that this effect is induced by neuropeptide signaling regulated by MME and is potentiated by MME loss. This provides a rationale for using the

MME/GRP pathway for targeting of cancer-propagating cells, perhaps as a part of combinatorial therapy approaches. Our study shows that accelerated progression of prostate carcinomas

associated with MME deficiency is not associated with increase in the number of neuroendocrine cells. Neuroendocrine cells are important for prostate development49. The majority of human

prostate adenocarcinomas and mouse models associated with _Pten_ deficiency contain neuroendocrine cells35,50. However, with the exception of highly aggressive neuroendocrine/small cell

carcinomas, it remains debatable if increased number of neuroendocrine cells, as defined by their expression of neuroendocrine markers, such as synaptophysin and calcitonin gene-related

peptide, are essential for the accelerated progression of pre-castrate prostate adenocarcinoma6,51,52. Our study suggests that accumulation of un-cleaved neuropeptides may mitigate the

requirement for neuroendocrine cell expansion or lead to a feedback inhibition of neuroendocrine cell differentiation in MME-deficient prostate cancers. The recent introduction of therapies

that better target the androgen axis has led to a significant increase in frequency of castrate-resistant prostate cancer with neuroendocrine differentiation2. It has been shown that

inactivation of the _RB1_ tumor suppressor gene increases cell plasticity along with the promotion of an aggressive neuroendocrine phenotype3,4,53. In castrated mice with combined _Pten_ and

_Trp53_ deficiency some prostate tumors arising from luminal cells had regions of highly proliferative cells with overt neuroendocrine differentiation. However, other tumors showed only

limited foci of neuroendocrine differentiation without any detectable proliferation50. In this report _Pten_ inactivation alone resulted in modest increase in neuroendocrine foci, consistent

with our findings. In our model associated with _Pten_ and _Mme_ deficiency we observed increased number of neuroendocrine neoplastic clusters and increased proliferation in such clusters

after castration. Furthermore, as compared to _Pten_PE−/− mice, recurrent tumors in castrated _Mme__−/−__Pten_PE−/− mice had more aggressive phenotype based on increased levels of

phosphorylated MET and reduced expression of E-cadherin. The mechanisms by which MME deficiency facilitates neuroendocrine transdifferentiation of the prostate epithelium or leads to

expansion neuroendocrine cell lineage after castration remain to be investigated. It also remains to be investigated if MME deficiency represents an alternative to _RB1_ loss for progression

of some castrate-resistant prostate cancer. Our mouse model based on inactivation of _Mme_ and _Pten_ should offer an important tool for answering these important questions of prostate

cancer pathogenesis and testing new therapeutic approaches. MATERIALS AND METHODS MICE _ARR__2__PB-Cre_ transgenic male mice on FVB/N background (_PB-Cre4_)37 were crossed with

_Pten__loxP/loxP_54 female mice on the 129/BALB/c background. Resulting _PB-Cre4Pten__loxP/loxP_ male mice were designated as _Pten_PE−/− mice. _Pten_PE−/− male mice were crossed with _Mme_

null female mice on C57BL6 background (_Mme__−/−_)23. Offspring with _PB-Cre4 Mme__−/−_ _Pten__loxP/loxP_ genotype were designated as _Mme__−/−__Pten__PE−/−_ mice. To minimize the

confounding effects of genetic background _Pten_PE−/− and _Mme__−/−__Pten_PE−/− mice were backcrossed to FVB/N for at least 10 crosses and all control experiments were performed on age and

sex-matched randomized mice of the same background. All animal experiments were carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory

Animals of the National Institutes of Health. The protocol was approved by the Institutional Laboratory Animal Use and Care Committee at Cornell University. All efforts were made to minimize

animal suffering. IMMUNOHISTOCHEMISTRY AND QUANTITATIVE IMAGE ANALYSIS Immunoperoxidase staining of paraffin sections of paraformaldehyde-fixed tissue was performed by a modified Elite

avidin-biotin-peroxidase (ABC) technique28. Antigen retrieval was done by boiling the slides in 10 mM citric buffer (pH 6.0) for 10 min. The primary antibodies to MME (Santa Cruz; Dallas,

TX; #sc-80021, 1:100), Ki67 (Leica Microsystems; Bannockburn, IL; #NCLKi67p, 1:1000), EZH2 (Cell Signaling Technology; Danvers, MA; #5246, 1:200), keratin 5 (CK5, Covance; Dallas, TX,

#PRB-160P, 1:2500), keratin 8 (CK8, Developmental Studies Hybridoma Bank; Iowa City, IA; #TROMA-I, 1:10), p63 (Santa Cruz, #sc-8431, 1:1000), PTEN (Cell Signaling Technology, #9559 S,

1:800), pAKT (Cell Signaling Technology, # 3787 S, 1:50), SYP (BD Biosciences; #611880, 1:500), GRP (Santa Cruz, #sc-7788, 1:100), NT (Santa Cruz, #sc-20806, 1:1000), and VIP (Abcam;

Cambridge, MA; #ab8556, 1:200), E-cadherin (Cell Signaling Technology #3195 S, 1:200), phospho-MET (Cell Signaling Technology, #3077, 1:50) were incubated with deparaffinized sections at 4 

°C overnight, followed by incubation with secondary biotinylated antibody (1 h, room temperature) and modified theh avidin-biotin-peroxidase (ABC) technique. Methyl green was used as the

counterstain in immunoperoxidase stainings. Slides were scanned by a ScanScope CS (Leica Biosystems, Vista, CA) with ×40 objective followed by lossless compression. For double fluorescence

antibodies against SYP (BD Biosciences, #611880) and Ki67 (Abcam, #16667) were used at a concentration of 1:40 and 1:50, respectively, for overnight incubation at 4 °C, followed by the

incubation with secondary antibodies (Alexa Fluor 568 donkey-anti-mouse #A10037, Alexa Fluor 488 donkey-anti-rabbit #21206, Life Technologies, 1:200 each, 2 h, room temperature) and

counterstaining with 4′6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich #D9542). Immunofluorescent sections were observed and imaged using a Leica TCS SP2 confocal laser scanning microscope

system (Leica Microsystems). Quantitative analysis of immunohistochemistry (IHC) was performed with the ImageJ software (W. Rasband, National Institutes of Health, Bethesda, MD).

TUMORIGENICITY EXPERIMENTS Human prostate cancer PC3 cells (5 × 106 cells) were suspended in the mixture of 200 µl phosphate-bufferd saline (PBS) and 200 µl Matrigel (BD Biosciences,

#356237) and injected subcutaneously into 5-week-old NSG (NOD.Cg-_Prkdc_scid_Il2rg_tm1Wjl/SzJ4; The Jackson Laboratory, stock number #005557) male mice. Intraperitoneal injection of GRPR

antagonist (0.8 μg/g body weight/day in PBS) was started when the tumor size reached 10 mm3. After 26 days of treatment with GRPR antagonist, mice were euthanized and subjected to necropsy.

Tumor xenografts were collected pathological evaluation and ALDEFLUOR assay followed by FACS analysis. CASTRATION EXPERIMENTS Male mice were weighed and anesthetized using isoflurane.

Pressure was applied to the abdomen of each mouse to push both testes down into the scrotal sac. An approximately 1 cm incision was made across the midline of the scrotal sac to expose the

testicular membrane. The midline between the left and right testes sacs were located and a small incision is made on the left side of the membrane midline. The testes, attached fatty tissue,

the vas deferens, and associated blood vessels are carefully pulled out through the incision. The blood vessels were cauterized and the testes was removed by severing below the

cauterization. Same procedure was repeated to remove testes from the right side of the midline. Remaining tissue was pushed back into incision which was then closed with wound clips. Post

procedure, the mouse was placed on a heating pad till it recovered from the effects of the anesthetic followed by placement in a clean cage with fresh chow and water. PATHOLOGIC ASSESSMENT

Moribund mice, as well as those sacrificed according to schedule, were anesthetized with avertin and, if necessary, subjected to cardiac perfusion at 90 mm Hg with PBS. After macroscopic

evaluation during necropsy, lung, liver, prostate, and lymph nodes were fixed in phosphate-buffered 4% paraformaldehyde tissues, embedded in paraffin, and 4-μm-thick sections were stained

with hematoxylin (Mayer’s haemalum) and eosin. Striated muscle layer was used for identification of proximal (periurethral) and distal regions of prostatic ducts in transverse sections, as

previously described32. Mouse prostatic intraepithelial neoplasia (PIN) and adenocarcinoma were defined according to earlier publications28,34,38,55. Briefly, distal PIN1 has 1 or 2 layers

of atypical cells; PIN2 has 3 or more layers of atypical cells, PIN3 occupies the near entire glandular lumen; and PIN 4 fills and distorts the glandular profile, and is frequently marked by

pronounced desmoplastic reaction. In agreement with the recent consensus report34, PIN1 and PIN2 represent low-grade PIN (LG-PIN) and PIN3 and 4 represent high-grade PIN (HG-PIN). Due to

different architecture of the proximal regions of prostatic ducts, current PIN classification cannot be carefully applied to atypical proliferative lesions found in those structures. Thus we

named those lesions as proximal duct dysplasia to stress their dissimilarity to PIN of distal regions of prostatic ducts. Given the complexity and controversial nature of the interpretation

of stromal microinvasion we used term early adenocarcinoma only for neoplasms with invasive stromal growth confirmed by serial sections followed by 3D reconstruction. We used term advanced

adenocarcinoma for neoplasms invading blood and lymphatic vessels. All pathological evaluations were performed in blinded fashion. CELL CULTURE DU145, PC3, and LNCaP cell lines were obtained

from the American Type Culture Collection (ATCC) and cultured in minimum essential medium (Cellgrow, #10-010-CV), F-12K (Cellgrow, #10-025-CV) and RPMI 1640 (VWR, cat # 4500-396),

respectively, supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO, #16141-079) and penicillin–streptomycin (Cellgrow, #30-002-Cl). The cultures were maintained at 37 °C in

a 5% CO2 incubator. All cell lines were confirmed to be free of mycoplasma. Primary mouse prostate cells were isolated following described procedures28,56. For GRP experiments, 5 nM GRP

(Sigma, #G8022) and 4 μM GRPR antagonist ([Tyr4, d-Phe12]-Bombesin, Sigma, #B0650) were used for cell culture experiments. Media was changed every 3 days. Lipofectamin 2000 reagent

(Invitrogen; Carlsbad, CA, #11668-030) was used for the transfection following the manufacturer’s recommendations. Control siRNA (Santa Cruz, #sc-37007) and two independent human _GRPR_

siRNAs (Santa Cruz, # sc-106924 and Life Technologies, #145216), human GRPR shRNA in Lentiviral Vector (Genomics Online; Limerick, PA, # ABIN3479889) and mouse _Grpr_ siRNAs (Life

Technologies, #157912 and #157914) were used for all knockdown experiments. MME inhibitor dl-thiorphan, (1 μM, Sigma, #T6031)57 was used to treat cells at 37 °C for 20 min, followed by

subsequent experiments. 5-Bromo-2′deoxyuridine (BrdU) staining, cell motility, and invasion assays were performed as previously described28,58. ALDEFLUOR ASSAY AND FACS For detection of

aldehyde dehydrogenase (ALDH) enzymatic activity, 106 cells were placed in ALDEFLUOR buffer and processed for staining with the ALDEFLUOR Kit (Stem Cell, #01700) according to the

manufacturer’s protocol. Unstained and ALDH inhibitor (diethylaminobenzaldehyde, DEAB)-treated cells served as controls. For detection of CD44-expressing cancer cells, prostate cancer cells

were stained for CD44 (BD Bioscience, #553134) to sort out CD44-positive and CD44-negative cells. Cell sorting and data analysis were performed on a FACS Aria II sorter equipped with the

FACS DiVa software (BD Bioscience). PROSTASPHERE ASSAY The preparation of prostate epithelial cell suspensions, stem/basal cells, and luminal cells from male mice were performed based on

previously described prostrate sphere assays28,56. Briefly, 104 mouse prostate stem/basal cells, mouse prostate luminal cells, human prostate cancer cells, and human prostate

cancer-propagating cells were resuspended in 120 µl of a 1:1 mixture of Matrigel (BD Biosciences, #354234) and PrEGM (Lonza, #CC-3166), and plated around the rim of a well of a 12-well

tissue culture plate. Matrigel mix was alloµwed to solidify at 37 °C for 15 min, and 1 ml of PrEGM was added per well. Media was changed every 3 days. To recover the spheres, each well was

treated with enzyme mixture: 750 µl collegenase/dispase 4 mg/ml (Roche, #10269638001), 30 mg BSA (Sigma, #A3311), and 1 µl DNase1 10 mg/ml (Sigma, D4513), followed by Trypsin 0.25% EDTA

(Cellgrow, 25-052-Cl) to make cell suspensions, which were ready for passage. The sphere-forming efficiency was calculated as previously described28. A constant number of cells was used for

each passaging. QUANTITATIVE REAL-TIME PCR RNA was extracted using TRIzol reagent according to the manufacturer’s instructions (Thermo Fisher). cDNA was produced using the SuperScript III

First-Strand Synthesis kit (Thermo Fisher). Real-time PCR was performed using PerfeCTa SYBR Green Super Mix Reagent (Quanta bio) on C1000 Touch Thermal Cycler PCR machine (Bio-Rad). _SYP_

expression was assessed using forward primer 5′-TGCGCTAGAGCATTCTGGG-3′ and reverse primer 5′-CTTAAAGCCCTGGCCCCTTCT-3′. WESTERN BLOT For western blot cell lysates were prepared using RIPA

buffer (50 mM Tris-HCl, (pH 7.4), 1% Nonidet P-40, 0.25% Na-deoxicholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, Aprotinin, leupeptin, pepstatin: 1 μg/ml each, 1 mM Na3VO4, 1 mM NaF), followed

by sonication for 10 s five times on ice. Lysates were then separated by 12% SDS-PAGE and transferred to PVDF membrane (Millipore #IPVH00010). The membrane was incubated overnight at 4 °C

with antibodies to detect GRPR (Santa Cruz, #sc-32903, 1:1000) and GAPDH (Advanced Immunohistochemical Inc.; Long Beach, CA; #2-RGM2,1:5000), followed by incubation for 1 h at room

temperature with corresponding horseradish peroxidase-conjugated anti-rabbit secondary antibodies (Santa Cruz, #sc-2004, 1:2000) or anti-mouse secondary antibodies (Santa Cruz, #sc-2005,

1:2000) and developed using chemiluminescent substrate (Thermo Scientific, Rockford, IL, #34077). STATISTICS Statistical analyses were performed with InStat 3.10 and Prism 7 software.

(GraphPad, Inc., San Diego, CA). Two-tailed unpaired _t_-test, direct Fisher’s tests, and log-rank Mantel–Haenszel test were used as appropriate. To ensure adequate power to detect a

pre-specified effect size all sample sizes were chosen based on initial pilot experiments, including animal studies. No samples or animals were excluded from the analysis. The variance was

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ACKNOWLEDGEMENTS We would like to thank David Dupee, Aditi Iyengar, and Elaina Wang for expert technical assistance; Lavanya Sayam (NYSTEM supported FACS Core) for her help with

fluorescence-activated cell sorting; and all members of the Nikitin Lab for their advice and support. This work has been supported by NIH (CA096823 and CA197160) and NYSTEM (C023050 and

C028125) grants to A.Y.N., NIH (CA72717) and the Genitourinary Oncology Research fund (Weill Cornell) to D.M.N., and fellowship funding from the Cornell Comparative Cancer Biology Training

Program to C.-Y.C. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Biomedical Sciences, and Cornell Stem Cell Program, Cornell University, Ithaca, NY, 14850, USA Chieh-Yang

Cheng, Zongxiang Zhou, Meredith Stone, Andrea Flesken-Nikitin & Alexander Yu. Nikitin * Harvard Medical School, Children’s Hospital, Boston, MA, 02115, USA Bao Lu * Department of

Medicine, Weill Cornell Medicine and Meyer Cancer Center, New York, NY, 10021, USA David M. Nanus Authors * Chieh-Yang Cheng View author publications You can also search for this author

inPubMed Google Scholar * Zongxiang Zhou View author publications You can also search for this author inPubMed Google Scholar * Meredith Stone View author publications You can also search

for this author inPubMed Google Scholar * Bao Lu View author publications You can also search for this author inPubMed Google Scholar * Andrea Flesken-Nikitin View author publications You

can also search for this author inPubMed Google Scholar * David M. Nanus View author publications You can also search for this author inPubMed Google Scholar * Alexander Yu. Nikitin View

author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Alexander Yu. Nikitin. ETHICS DECLARATIONS CONFLICT OF INTEREST The

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prostate carcinogenesis by attenuating effects of gastrin-releasing peptide on stem/progenitor cells. _Oncogenesis_ 9, 38 (2020). https://doi.org/10.1038/s41389-020-0222-3 Download citation

* Received: 29 January 2020 * Revised: 02 March 2020 * Accepted: 05 March 2020 * Published: 23 March 2020 * DOI: https://doi.org/10.1038/s41389-020-0222-3 SHARE THIS ARTICLE Anyone you share

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