Stina Häggström Rudolfsson

Mast Cells Are Novel Independent Prognostic Markers in Prostate Cancer and Represent a Target for Therapy

Mast cells affect growth in various human tumors, but their role in prostate cancer (PC) is unclear. Here, we identify mast cells as independent prognostic markers in PC using a large cohort of untreated PC patients with a long follow-up. By analyzing mast cells in different tissue compartments, our data indicate that intratumoral and peritumoral mast cells have anti- opposed to protumor properties. Intratumoral mast cells negatively regulate angiogenesis and tumor growth, whereas peritumoral mast cells stimulate the expansion of human prostate tumors. We also observed mast cell recruitment particularly to the peritumoral compartment in men during the formation of castrate-resistant prostate tumors. In our ortothopic rat model, mast cells accumulated in the peritumoral tissue where they enhanced angiogenesis and tumor growth. In line with this, prostate mast cells expressed high levels of the angiogenic factor FGF-2. Similar to the situation in men, mast cells infiltrated rat prostate tumors that relapsed after initially effective castration treatment, concurrent with a second wave of angiogenesis and an up-regulation of FGF-2. We conclude that mast cells are novel independent prognostic markers in PC and affect tumor progression in animals and patients. In addition, peritumoral mast cells provide FGF-2 to the tumor micro environment, which may contribute to their stimulating effect on angiogenesis.

Decreased Pigment Epithelium-Derived Factor Is Associated with Metastatic Phenotype in Human and Rat Prostate Tumors

Pigment epithelium-derived factor, a potent angiogenesis inhibitor in the eye, is also expressed in the prostate. Prostate size and angiogenesis is increased in pigment epithelium-derived factor knockout mice, and pigment epithelium-derived factor is down-regulated in some prostate cancers. To investigate whether pigment epithelium-derived factor expression correlates with tumor progression, we examined 5 Dunning rat prostate sublines with different growth rates, differentiation, androgen dependence, vascular density, and metastatic ability and 26 human prostate cancers of Gleason score 8–10 obtained from patients at transurethral resection selected to represent two groups, with and without metastases at diagnosis. By Western blot, real-time quantitative reverse transcription-PCR, and immunostaining, pigment epithelium-derived factor was detected in highly differentiated, nonmetastatic, androgen-sensitive Dunning tumors and in the anaplastic, androgen insensitive but nonmetastatic Dunning tumors. In contrast, the metastatic Dunning tumor sublines showed very low pigment epithelium-derived factor expression levels. In human cancer tissues, by immunohistochemistry and real-time quantitative reverse transcription-PCR, patients without metastases at diagnosis had higher tumor pigment epithelium-derived factor levels than tumors from patients with metastases at diagnosis. In both the rat model and in the human tumors, the proliferation index and vascular count, as determined by Ki-67 staining and endoglin and/or factor VIII-related antigen staining, inversely correlated with pigment epithelium-derived factor mRNA levels. These observations indicate that loss of pigment epithelium-derived factor expression could be associated with the progression toward a metastatic phenotype in prostate cancer.

Low Levels of Phosphorylated Epidermal Growth Factor Receptor in Nonmalignant and Malignant Prostate Tissue Predict Favorable Outcome in Prostate Cancer Patients

Purpose: To explore if the expression of phosphorylated epidermal growth factor receptor (pEGFR) in nonmalignant and malignant prostate tissue is a potential prognostic marker for outcome in prostate cancer patients. Experimental Design: We used formalin-fixed tissues obtained through the transurethral resection of the prostate from 259 patients diagnosed with prostate cancer after the transurethral resection of the prostate, and patients were then followed with watchful waiting. Tissue microarrays of nonmalignant and malignant prostate tissue were stained with an antibody against pEGFR. The staining pattern was scored and related to clinicopathologic parameters and to outcome. Results: Low phosphorylation of EGFR in prostate epithelial cells, both in the tumor and surprisingly also in the surrounding nonmalignant tissue, was associated with significantly longer cancer-specific survival in prostate cancer patients. This association remained significant when Gleason score and local tumor stage were added together with pEGFR to a Cox regression model. Tumor epithelial pEGFR immunoreactivity was significantly correlated to tumor cell proliferation, tumor vascular density, and nonmalignant epithelial pEGFR immunoreactivity. Patients with metastases had significantly higher immunoreactivity for tumor and nonmalignant epithelial pEGFR compared with patients without metastases. Conclusions: Low pEGFR immunoreactivity is associated with the favorable prognosis in prostate cancer patients and may provide information about which patients with Gleason score 6 and 7 tumors that will survive their disease even without treatment. Changes in the nonmalignant tissue adjacent to prostate tumors give prognostic information. Clin Cancer Res; 16(4); 1245–55

Characterization of prostate cancer bone metastases according to expression levels of steroidogenic enzymes and androgen receptor splice variants.

Background Intra-tumoral steroidogenesis and constitutive androgen receptor (AR) activity have been associated with castration-resistant prostate cancer (CRPC). This study aimed to examine if CRPC bone metastases expressed higher levels of steroid-converting enzymes than untreated bone metastases. Steroidogenic enzyme levels were also analyzed in relation to expression of constitutively active AR variants (AR-Vs) and to clinical and pathological variables. Methodology/Principal Findings Untreated, hormone-naïve (HN, n = 9) and CRPC bone metastases samples (n = 45) were obtained from 54 patients at metastasis surgery. Non-malignant and malignant prostate samples were acquired from 13 prostatectomy specimens. Transcript and protein levels were analyzed by real-time RT-PCR, immunohistochemistry and immunoblotting. No differences in steroidogenic enzyme levels were detected between CRPC and HN bone metastases. Significantly higher levels of SRD5A1, AKR1C2, AKR1C3, and HSD17B10 mRNA were however found in bone metastases than in non-malignant and/or malignant prostate tissue, while the CYP11A1, CYP17A1, HSD3B2, SRD5A2, and HSD17B6 mRNA levels in metastases were significantly lower. A sub-group of metastases expressed very high levels of AKR1C3, which was not due to gene amplification as examined by copy number variation assay. No association was found between AKR1C3 expression and nuclear AR staining, tumor cell proliferation or patient outcome after metastases surgery. With only one exception, high AR-V protein levels were found in bone metastases with low AKR1C3 levels, while metastases with high AKR1C3 levels primarily contained low AR-V levels, indicating distinct mechanisms behind castration-resistance in individual bone metastases. Conclusions/Significance Induced capacity of converting adrenal-gland derived steroids into more potent androgens was indicated in a sub-group of PC bone metastases. This was not associated with CRPC but merely with the advanced stage of metastasis. Sub-groups of bone metastases could be identified according to their expression levels of AKR1C3 and AR-Vs, which might be of relevance for patient response to 2nd line androgen-deprivation therapy.

Different vascular endothelial growth factor (VEGF), VEGF-receptor 1 and -2 mRNA expression profiles between clear cell and papillary renal cell carcinoma.

To examine vascular endothelial growth factor (VEGF), VEGF‐receptor‐(R)1, and R2 mRNA levels in renal cell carcinoma (RCC), a tumour generally refractory to most medical therapy, but for which a potentially useful therapeutic alternative is inhibition of angiogenesis.

Hormonal Regulation and Functional Role of Vascular Endothelial Growth Factor A in the Rat Testis

Abstract Vascular endothelial cell growth factor (VEGF-A) is synthesized in the testis but its role and regulation in this organ have not been examined. VEGF and its receptors (VEGF-R) were quantified using reverse transcription-polymerase chain reaction and Western blot. VEGF, VEGF-R1, and VEGF-R2 mRNAs and VEGF protein were increased after treatment with 50 IU hCG. Injection of 100 ng human recombinant VEGF 165 into the testis caused an increase in endothelial cell proliferation, but only a moderate increase in testicular interstitial fluid volume. In contrast with systemic hCG treatment, local VEGF injection did not increase the permeability to intravenously injected colloidal carbon particles. However, if VEGF was given locally in the testes of animals pretreated with hCG 4 or 8 h earlier, VEGF acted in synergy with hCG to increase vascular carbon leakage by forming interendothelial cell gaps. Testicular blood flow was unaffected by local VEGF 165 injection. Treatment with a specific VEGF-R2 tyrosine kinase inhibitor blocked the hCG-induced increase in endothelial cell proliferation but did not affect the hCG-induced accumulation of polymorphonuclear leukocytes in testicular blood vessels or the increase in the testicular interstitial space. The present study demonstrated that testicular VEGF secretion is increased by hormonal stimulation of Leydig cells and that VEGF, through effects mediated via VEGF-R2, regulates endothelial cell proliferation in the rat testis. VEGF does not appear to regulate testicular blood flow and it is not involved in inducing the hCG-induced inflammation-like response in the testicular microvasculature. The permeability-increasing effect of VEGF is low in the testis under basal conditions but is apparently up-regulated by hCG treatment.

Localized Expression of Angiopoietin 1 and 2 May Explain Unique Characteristics of the Rat Testicular Microvasculature

Abstract The testicular vasculature is unique in several ways. The unfenestrated endothelial cells constitute one part of the blood-testis barrier, and testicular microvessels are normally resistant to inflammation mediators. At the same time that angiogenic factors and inflammation mediators are constitutively produced, the proportion of proliferating endothelial cells is considerably higher than in other organs, but new blood vessels are not formed. Hormonal stimulation of the testis with hCG increase endothelial cell proliferation, vascular permeability, and sensitivity to locally injected inflammation mediators. In the present study, we examined whether local expression of angiopoietin (ang) 1, an inhibitor of vascular leakage and sprouting angiogenesis, and its antagonist, ang 2, could be involved in establishing this vascular phenotype. Using reverse transcription-polymerase chain reaction and immunohistochemistry, we demonstrate that testicular vascular endothelial growth factor-A (VEGF-A), ang 1, ang 2, and the ang-receptor tie 2 are expressed in the testis and that hormonal stimulation with hCG is accompanied by increased expression of VEGF-A and ang 2. The ang 1 protein is expressed in testicular microvessels under basal conditions, and it is largely unaffected after hCG stimulation. Expression of ang 2 in microvessels, in contrast, is low under basal conditions and is up-regulated by hCG. Intratesticular injection of human recombinant ang 1 protein inhibits hCG-induced increase in vascular permeability. Injection of ang 2 in the testis increases endothelial cell proliferation and the volume of the interstitial space. We therefore suggest that ang 1 stabilizes testicular microvessels under basal conditions and that a shift in this balance caused by increased ang 2, together with increased VEGF-A, allows vascular leakage, high endothelial cell proliferation, and presumably, vascular growth after hormonal stimulation.

Testosterone-stimulated growth of the rat prostate may be driven by tissue hypoxia and hypoxia-inducible factor-1alpha.

Testosterone-stimulated growth of the ventral prostate (VP) in castrated rats is preceded by angiogenesis, but the mechanisms coordinating vascular and tissue growth are unknown. Adult rats were castrated and some treated with testosterone. Tissue hypoxia was studied morphologically using the hypoxia marker pimonidazole (Hypoxyprobe), hypoxia-inducible factor-1 (HIF-1) alpha, vascular endothelial growth factor (VEGF), and carbonicanhydrase 9 (CA-9) levels by western blotting and quantitative RT-PCR. In the intact untreated prostate, most glands were unstained by the hypoxia marker but already 1 day after castration most epithelial cells in the VP were stained. Seven days after castration prostate glands were apparently normoxic again, and HIF-1alpha, VEGF, and CA-9 were decreased. Treatment of 7-day castrated rats with testosterone resulted in increased epithelial hypoxyprobe staining and increased HIF-1alpha, VEGF, and CA-9 levels. The transient increase in tissue hypoxia after testosterone treatment is probably caused by a temporary mismatch between oxygen consumption and supply. Treatment of prostate epithelial cells in vitro under normoxic conditions also increased HIF-1alpha, and this could be blocked if epidermal growth factor receptor (EGFR) signaling was blocked with gefitinib. In vivo gefitinib could, however, not block the testosterone induced increase in HIF-1alpha. Testosterone may thus induce HIF-1alpha and its downstream angiogenesis promoting genes by at least two mechanisms, hypoxia and EGFR signaling. Transient epithelial cell hypoxia could by rapidly increasing HIF-1alpha and VEGF be an essential coordinator of testosterone-stimulated vascular and glandular growth.

Hypoxia drives prostate tumour progression and impairs the effectiveness of therapy, but can also promote cell death and serve as a therapeutic target.

Hypoxia is common in prostate tumours, promoting tumour progression and impairing treatment responses. Hypoxia stimulates angiogenesis but blood vessels formed in tumours are functionally abnormal so the tissue remains hypoxic. Castration treatment is the standard therapy for advanced prostate cancer. In non-malignant prostate tissue castration-induced epithelial cell death is in part mediated by vascular insult and acute hypoxia, but in prostate tumours the cell death response is less prominent and the tumours will eventually relapse. The effect of androgen ablation therapy should therefore be enhanced by additional targeting of the vasculature and hypoxic tumour cells. However if castration fails to kill a sufficiently large number of cells it could by inducing hypoxia make the situation worse. Androgen ablation treatment, may, after the initial vascular insult, result in temporary vascular normalisation and transiently increased tissue oxygen levels. During this time window, which needs to be better defined, the efficacy of cytotoxic drug and radiation treatments are probably enhanced. In order to allow development of more effective treatment strategies for advanced prostate cancer we need to understand the role of hypoxia in prostate cancer progression and treatment responses. With this knowledge we can properly tailor and time additional treatments with androgen ablation.

Inhibitory effects of castration in an orthotopic model of androgen-independent prostate cancer can be mimicked and enhanced by angiogenesis inhibition.

Purpose: 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. Experimental Design: 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. Results: 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. Conclusion: 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.