Castration-induced Prostate Research Articles

Abstract B092: Myokine signaling blockade prevents androgen deprivation therapy-induced sarcopenia and promotes prostate tumor regression

Abstract One of the most significant side effects of androgen-deprivation therapy (ADT), the first-line therapy for recurrent and metastatic prostate cancer (PrCa), is sarcopenia (loss of skeletal muscle mass and function). ADT produces a significant disability syndrome in PrCa patients known as obese frailty. Use of ADT has been increasing such that side effects increasingly impact patient quality of life, and result in nursing home placement. The sarcopenia and functional loss of strength is recapitulated following castration of “middle-aged,” but not younger, male mice. In this model all five muscle-regulating TGFß superfamily members, known as myokines, are upregulated in distinctive temporal patterns during sarcopenia development. Complete myokine blockade fully restores muscle mass and strength in castrated mice. We castrated 6- to 9-month-old PbCre4 x PTENfl/fl PrCa-bearing mice and measured tumor volume (by high-frequency ultrasound), body composition (by NMR), muscle mass, fat mass, grip strength, and expression of TGFß family members. Tumor-bearing mice were treated with ActRIIB-Fc (or vehicle) to block myokine signaling; additional tumor-bearing mice were sacrificed biweekly to monitor myokine levels. In an autochthonous mouse model of PrCa, castration-induced changes closely resemble the loss of skeletal muscle strength and body composition that occurs in ADT treated patients. The reduction in muscle strength is comparable to that seen in patients. ADT reduces the mass of individual skeletal muscles, overall LBM, and grip strength. We examined the expression of myokines following castration and found that muscle levels of free dimeric myostatin, the activins, and GDF11 all increase over time, but with distinctive kinetics. Functionally, ActIIRB-Fc, which binds and blocks myokines, inhibits sarcopenia in our model, indicating that the myokines mediate ADT-induced sarcopenia. Surprisingly, this myokine inhibition also induced prostate tumor regression in the absence of ADT, suggesting a protumorigenic effect of myokines. Myokine inhibition also increased the rate of castration-induced prostate tumor regression in this model. In the tumor tissue and in the circulation, as in the muscle tissue of these mice, some of the myokines also increase post-castration. Myokines mediate the induction of sarcopenia following castration, and may also mediate tumor growth. These results suggest that myokines might be both biomarkers and potential targets for therapy to reduce this comorbidity in PrCa patients and may also control tumor growth directly. Citation Format: Chunliu Pan, Yanni Zulia, Kent L. Nasituk. Myokine signaling blockade prevents androgen deprivation therapy-induced sarcopenia and promotes prostate tumor regression [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B092.

Desquamation is a novel phenomenon for collective prostate epithelial cell deletion after castration

The mechanism underlying castration-induced prostate regression, which is a classical physiological concept translated into the therapeutic treatment of advanced prostate cancer, involves epithelial cell apoptosis. In searching for events and mechanisms contributing to prostate regression in response to androgen modulation, we have frequently observed the collective deletion of epithelial cells. This work was undertaken to characterize this phenomenon hereafter named desquamation and to verify its presence after 17β-estradiol (E2) administration. Electron microscopy revealed that the desquamating cells had preserved cell-cell junctions and collapsed nuclear contents. The TUNEL reaction was negative for these cells, which were also negative for cleaved caspases-8, -9, -3 and nuclear apoptosis-inducing factor. Detailed analyses revealed that the condensed chromatin was first affected detaching from the nuclear lamina, which was observable after lamin A immunohistochemistry, suggesting the lack of lamin A degradation. A search in animals treated with supraphysiological E2 employed as an alternative anti-androgen treatment revealed no desquamation. The combined treatment (Cas+E2 group) caused changes particular to each treatment, including desquamation. In conclusion, desquamation appeared as a novel phenomenon contributing to collective prostate epithelial cell deletion, distinct from the classical castration-induced apoptosis and particular to the androgen deprivation resulting from surgical castration, and should be considered as part of the mechanisms promoting organ regression.

The role of autophagy in castration-induced prostate atrophy

Objective To investigate the role of autophagy in castration-induced proatate atrophy.Methods Seventy-two healthy male SD rats were randomly divided into 3 groups:sham operation group,castration group and hydroxychloroquine group.At 24 h,72 h,7 days,10 days,14 days and 21 days after operation,we took out four rats randomly from each group and the protaste tissues were removed.The morphological changes of the prostates were observed under a light microscope and the changes of cytolysosomes and apoptotic bodies in the prostates were observed under the electron microscopy.The protein expression of Bechn1,P62,B lymphocytes/leukemia-2 (bcl-2) and bax was detected by detected by using immunohistochemistry.The mRNA expression of microtabule-associated protein 1 light chain 3 (LC3),and autophagy related gene 5 (Atg5) was detected by using reverse transcription-polymerase chain reaction (RT-PCR).Restults The prostate tissues shranked remarkably in the castrated rats.Cytolysosomes were observed under the electron micreseopy.The expressoin of Beelin1 (0.0665 ±0.0137) and bax (0.0456 ±0.0122) was increased,and that of P62 (0.0103 ±0.0042) and bcl-2 (0.0340 ±0.0048) was reduced.After treatment with hydroxychloroquine,the expressoin of Beclin1 (0.0103 ±0.0042) and bcl-2 (0.0090 ±0.0032) was reduced,and that of P62 (0.0526 ±0.0096),and Bax (0.0828±0.0098) was increased.RT-PCR revealed that the mRNA expression of LC3 and Atg5 was enhanced.After treatment with hydroxychloroquine,the mRNA expression of LC3,and Atg5 was weakened.Conclusion The expression of autophagy was increased in prostates after castration.After treatment with hydroxychloroquine,the expression of autophagy was decreased,but apoptosis increased,which suggested that the autophagy can protect the prostates to shinking. Key words: Cell autophagy; Castration; Prostate; Hydroxychloroquine

Abstract C10: Tumor necrosis factor is necessary but not sufficient for castration-induced prostate regression

Abstract TNF, a pro-inflammatory and immune-regulatory cytokine, is a potent apoptotic stimulus in vitro. There are few examples of a physiologic role for TNF induced apoptosis in vivo, but here we describe a novel role for TNF in prostate epithelial cell apoptosis following androgen withdrawal. We demonstrate, using high resolution serial MRI (magnetic resonance imaging) to measure mouse prostate volume changes over time, that castration induced prostate regression is significantly reduced in mice null for either the Tnf or Tnfr1 genes, but not mice deficient for TRAIL or Fas signaling. Wild type mice treated with soluble TNFR2 (to bind TNF and block signaling) prior to castration showed a similar reduction. Additionally, consistent with a paracrine role for TNF in prostate regression, membrane-bound TNF protein and stromal cell specific TNF mRNA levels increase in rat prostate following castration. Injection of soluble TNF restores normal levels of prostate regression of Tnf −/− mice. Together, these data indicate that uniquely among these extrinsic death signals, TNF is required for castration induced prostate regression. However, wild type mice receiving soluble TNF in the absence of castration do not exhibit prostate regression, indicating that TNF alone is not sufficient, but acts in the context of additional castration induced signals. A preliminary investigation using ALK5 inhibitors prior to castration suggests that TGFβ signaling may also be important. Understanding the physiologic role for TNF, and the interaction with other signaling pathways active in prostate regression following androgen withdrawal may lead to novel therapies for prostate cancer. Citation Format: Jennifer S. Davis, Kent L. Nastiuk. Tumor necrosis factor is necessary but not sufficient for castration-induced prostate regression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C10.

TNF Is Necessary for Castration-Induced Prostate Regression, Whereas TRAIL and FasL Are Dispensable

TNF Is Necessary for Castration-Induced Prostate Regression, Whereas TRAIL and FasL Are Dispensable

TNF Is Necessary for Castration-Induced Prostate Regression, Whereas TRAIL and FasL Are Dispensable

TNF, a proinflammatory and immune-regulatory cytokine, is a potent apoptotic stimulus in vitro. However, there have been few examples of a physiologic role for TNF-induced apoptosis in vivo. Here, we describe a novel role for TNF in prostate epithelial cell apoptosis after androgen withdrawal. Employing high-resolution serial magnetic resonance imaging to measure mouse prostate volume changes over time, we demonstrate that the extent of castration-induced prostate regression is significantly reduced in mice null for either the Tnf or Tnfr1 genes but not mice deficient for TNF-related apoptosis-inducing ligand or Fas signaling. Wild-type mice receiving soluble TNF (sTNF) receptor 2 (to bind TNF and block signaling) before castration exhibit an identical reduction of prostate regression. Together, these data indicate that uniquely among known extrinsic death signals, TNF is required for castration-induced prostate regression. Additionally, membrane-bound TNF protein and stromal cell specific TNF mRNA levels increase in rat prostate after castration. This is consistent with a paracrine role for TNF in prostate regression. When injected into the peritoneum of Tnf(-/-) mice at the time of castration, sTNF restores normal levels of prostate regression. However, wild-type mice receiving sTNF in the absence of castration do not exhibit prostate regression, indicating that TNF alone is not sufficient but acts in the context of additional castration-induced signals. These findings support a physiologic role for TNF in prostate regression after androgen withdrawal. Understanding this role may lead to novel therapies for prostate cancer.

Generation of a prostate from a single adult stem cell

The existence of prostate stem cells (PSCs) was first postulated from the observation that normal prostate regeneration can occur after repeated cycles of androgen deprivation and replacement in rodents. Given the critical role of PSCs in maintaining prostate tissue integrity and their potential involvement in prostate tumorigenesis, it is important to define specific markers for normal PSCs. Several cell-surface markers have been reported to identify candidate PSCs, including stem cell antigen-1 (Sca-1, also known as Ly6a), CD133 (Prom1) and CD44 (refs 3-10). However, many non-PSCs in the mouse prostate also express these markers and thus identification of a more defined PSC population remains elusive. Here we identify CD117 (c-kit, stem cell factor receptor) as a new marker of a rare adult mouse PSC population, and demonstrate that a single stem cell defined by the phenotype Lin(-)Sca-1(+)CD133(+)CD44(+)CD117(+) can generate a prostate after transplantation in vivo. CD117 expression is predominantly localized to the region of the mouse prostate proximal to the urethra and is upregulated after castration-induced prostate involution-two characteristics consistent with that of a PSC marker. CD117(+) PSCs can generate functional, secretion-producing prostates when transplanted in vivo. Moreover, CD117(+) PSCs have long-term self-renewal capacity, as evidenced by serial isolation and transplantation in vivo. Our data establish that single cells in the adult mouse prostate with multipotent, self-renewal capacity are defined by a Lin(-)Sca-1(+)CD133(+)CD44(+)CD117(+) phenotype.

Inhibition of the epidermal growth factor receptor enhances castration‐induced prostate involution and reduces testosterone‐stimulated prostate growth in adult rats

The epidermal growth factor receptor (EGFR) mediates regulatory signals in the normal prostate, but the functional importance of this is unclear. Adult male rats were castrated, or castrated + treated with gefitinib (Iressa, ZD1839), an EGFR tyrosine kinase inhibitor, for 3 days. Seven-day castrated rats were treated with testosterone, or testosterone + gefitinib, for 3 days. Both castration alone and testosterone treatment in castrated animals increased the mRNA and protein levels of EGFR and phospho-EGFR in the ventral prostate. Inhibition of EGFR during castration and during testosterone-stimulated prostate growth resulted in a decrease in total epithelial weight, epithelial cell proliferation, endothelial cell proliferation, and increased epithelial cell apoptosis. This study suggests that increased EGFR signaling during castration mediates stimulatory effects balancing castration-induced prostate regression, and that EGFR signaling is a necessary component in testosterone-stimulated prostate growth.

Effect of Castration on Extracellular Matrix Remodeling and Angiogenesis of the Prostate Gland

This study was conducted to evaluate the long term effect of castration on the prostate gland proliferation, extracellular matrix remodeling and angiogenesis. Prostate gland proliferation was assessed by immunolocalization of proliferating cell nuclear antigen (PCNA). The expression level of vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-beta) and metaloprotenase-13 (MMP-13) by the prostate gland were assessed by immunohistochemistry and quantitative real-time PCR. The expression of the above mentioned parameters by the prostate gland of mature intact dogs were compared to that of castrated dogs six months post-castration. The results showed that castration induced a remarkable atrophy of the prostate gland which was associated with a highly significant decrease in the PCNA proliferation index. Although TGF-beta protein was immunolocalized to the epithelial and stroma cells of the prostate gland from both intact and castrated dogs, castration induced a significant up-regulation of TGF-beta mRNA expression. VEGF mRNA expression and its encoded protein immunolocalization were decreased significantly by the prostate gland from castrated dogs as compared to that of intact dogs. Castration, on the other hand, resulted in no significant change in MMP-13 mRNA expression despite an effect on its cellular immunolocalization which appeared to be localized to the epithelial and stromal cells of the prostate gland from castrated dogs as compared to epithelial cells of the prostate gland from intact dogs. These results indicated that castration-induced prostate gland regression continued to exert a potent suppressive effect on prostate gland proliferation which might be mediated by the elevated level of TGF-beta. Moreover, the low expression level of VEGF might reflect a reduced blood flow demand by the regressed and growth-dormant prostate after castration.

Inhibitory Effects of Castration in an Orthotopic Model of Androgen-Independent Prostate Cancer Can Be Mimicked and Enhanced by Angiogenesis Inhibition

Today, the most important treatment of advanced prostate cancer is castration; unfortunately, however, the long-term effect of this therapy is insufficient. Recent studies suggest that castration-induced prostate involution could be caused by primary effects in the prostate vasculature; therefore, we examined if antivascular treatments could mimic the effects of castration. Androgen-independent AT-1 prostate cancer cells were grown inside the ventral prostate in adult rats. Tumor-bearing animals were treated with an inhibitor of vascular endothelial growth factor receptor 2 and epidermal growth factor receptor signaling, N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine (ZD6474, AstraZeneca, Södertälje, Sweden), and short-term effects (after 3 days) were compared with those induced by castration. Castration caused decreased vascular density in the normal tissue surrounding the tumor and consequently increased tumor hypoxia and apoptosis, and moderately decreased tumor growth. ZD6474 treatment resulted in decreased tumor vascular density accompanied by increased tumor hypoxia, apoptosis, and decreased tumor growth, suggesting that castration and antiangiogenic therapy work through similar mechanisms. Interestingly, castration or ZD6474 alone worked by reducing vascular density in the surrounding normal tissue and ZD6474 also in the tumor. Combined treatment with castration + ZD6474 was more effective than castration and ZD6474 alone in inducing tumor hypoxia, apoptosis, necrosis, and decreasing tumor vascular density. These findings show that a drug that targets the vasculature in the tumor and in the surrounding ventral prostate lobe could mimic and even enhance the effects of castration. Our present findings thus suggest that castration + ZD6474 could be a particularly effective way to treat prostate tumors.

Castration‐induced epithelial cell death in human prostate tissue is related to locally reduced IGF‐1 levels

Castration rapidly reduces stroma insulin-like growth factor (IGF)-1 synthesis and action in mouse prostate epithelium. We explore if similar changes are of importance for castration-induced prostate regression in humans. Epithelial and surrounding stroma cells were micro-dissected from patient biopsies obtained before and shortly after castration. IGF-1 mRNA levels were quantified by RT-PCR and related to epithelial apoptosis and IGF-1, IGF-1 receptor, and androgen receptor (AR) immunoreactivity. IGF-1 mRNA was principally produced in the stroma and IGF-R1 in the epithelium. Stroma IGF-1 mRNA levels were significantly decreased after castration in non-malignant but not malignant tissue. Lack of stroma IGF-1 reduction after castration was associated with low stroma AR expression before therapy. Reduction of IGF-1 mRNA levels in the tumor stroma and/or epithelium was associated with epithelial apoptosis after therapy. Low AR expression and maintained stroma IGF-1 synthesis may result in limited tumor cell death after castration therapy.

Castration rapidly decreases local insulin-like growth factor-1 levels and inhibits its effects in the ventral prostate in mice

The mechanisms by which castration induces prostate involution are largely unknown. Early responses to castration in mouse ventral prostate (VP) were explored by quantitative microscopy, cDNA array expression, quantitative RT-PCR, and Western blot analysis. As several changes occurred in the insulin-like growth factor (IGF) system this was studied in more detail. Laser micro-dissection was used to localize sites of IGF-1 and IGF-1 receptor (IGF-R1) production. IGF-1 protein levels and IGF-R1 mediated signaling via insulin regulated substrate 1 and 2 (IRS-1 and 2) were examined. IGF-1 was injected into the VP in intact, and castrated mice and effects studied 1 day later. IGF-1 and IGF binding protein 2 (IGFBP-2) mRNA were rapidly reduced whereas IGFBP-3 and IGF-R1 mRNA were increased after castration. IGF-1 was principally produced in the stromal compartment, while IGF-R1 was produced in both epithelial and stromal cells. IGF-1 and IRS-1 protein levels were decreased 1 and 3 days after castration, respectively, while IRS-2 was unchanged. Inactivating phosphorylation of IRS-1 at serine 307 was increased 1 day after castration, and activating phosphorylation at tyrosine 612 was decreased 2 days later. These changes were accompanied by decreased cell proliferation and increased cell death in the glandular and vascular compartment. Local injection of IGF-1 increased vascular density and epithelial cell proliferation in intact mice, but had no effect in castrated animals. Decreased IGF-1 levels and action may mediate some of the key features of castration-induced prostate involution.

Fas antigen/CD-95 upregulation and activation during castration-induced regression of the rat ventral prostate gland

Fas antigen/CD 95 is a 45-kDa transmembrane protein that can initiate intracellular signaling pathways, leading to apoptosis when it is clustered on the cell surface. A recent report claiming that the ventral prostate glands of lpr -/- mutant mice (lacking functional fas antigen) do not regress following castration prompted our analysis of the regressing rat ventral prostate gland for evidence that fas antigen might participate in the molecular process leading to prostate cell apoptosis after castration. An RNase protection assay and Western blotting analysis were used to quantify fas antigen mRNA and protein expression in the regressing rat ventral prostate gland. Immunoprecipitates of fas antigen from membrane preparations made from control or castrated rat prostates were analyzed for coprecipitation of FADD and RIP proteins to assess the activation state of the fas antigen before and after castration. Finally, prostate tissues obtained from two different strains of lpr -/- mutant mice were analyzed for induced apoptosis after castration by the TUNEL staining method. Rat ventral prostate gland fas antigen mRNA and protein expression was upregulated approximately 3-5-fold in the 3-day castrated rat as compared to hormonally intact rats. Immunoprecipitates of fas antigen from membranes of ventral prostates from castrated rats contained significantly increased amounts of both FADD and RIP proteins when compared to those of intact or control operated rats. However, counts of TUNEL-labeled cells in the ventral prostate glands of castrated lpr -/- mice were not significantly different from those in castrated, genetically normal controls. Likewise, the morphology of apoptotic bodies formed in the prostates of castrated lpr -/- mice was indistinguishable from that in control animals. Fas antigen/CD-95, a protein that is involved in some forms of apoptosis, is upregulated during regression of the rat ventral prostate gland and becomes functionally "activated." However, our inability to distinguish any difference in the apoptosis rate or in the morphology of the apoptotic bodies formed in response to castration between lpr -/- mice and genetically normal controls indicates that, contrary to the prior report, functional fas protein is not required for castration-induced prostate cell apoptosis.

Electron microscopic analysis of adrenalectomy-induced hippocampal granule cell degeneration in the rat: apoptosis in the adult central nervous system.

As described in the preceding paper, adrenalectomy triggers hippocampal granule cell degeneration that begins within days after adrenalectomy, continues for months, and is the only apparent cell death anywhere within the brain. At the light microscopic level, granule cell degeneration is characterized by coalescing of nuclear chromatin into numerous spherical bodies. Since the morphology at the light microscopic level resembled the nuclear morphology characteristic of "apoptosis" rather than "necrosis," we undertook this ultrastructural study to determine if adrenalectomy induces the morphological features characteristic of apoptosis. Electron microscopy revealed coalescing of nuclear chromatin, compaction of cytoplasm, and the budding-off of cytoplasmic bodies that were engulfed by glia. Mitochondria, the Golgi apparatus, and rough endoplasmic reticulum appeared relatively normal early in the process of granule cell degeneration when nuclear changes were prominent. Presynaptic terminals innervating degenerating granule cells appeared normal. Electron-dense degeneration of granule cell axon terminals in association with normal postsynaptic elements of CA3 pyramidal cells highlighted the extraordinary selectivity of adrenalectomy-induced granule cell death. Ten weeks after adrenalectomy, astrocytes were filled with abnormally abundant glial fibrils and neuronal debris. This "apoptotic" morphology produced by adrenalectomy was clearly distinct from the "necrotic" granule cell morphology produced by intrahippocampal injection of the neurotoxin volkensin. These results indicate that, in a manner possibly analogous to castration-induced prostate cell death, loss of adrenal hormone triggers a process in dentate granule cells that causes the morphological changes characteristic of "apoptosis." Thus, adrenal steroids may be obligatory growth factors for dentate granule cells and their loss may initiate a selective process in the mature brain that is unique or that may normally occur only in the developing brain.