A novel role for microphthalmia-associated transcription factor-regulated pigment epithelium-derived factor during melanoma progression.

Microphthalmia-associated transcription factor (MITF) acts via pigment epithelium-derived factor (PEDF), an antiangiogenic protein, to regulate retinal pigment epithelium migration. PEDF expression and/or regulation during melanoma development have not been investigated previously. Using immunohistochemistry, we determined expression of PEDF in common and dysplastic melanocytic nevi, melanoma in situ, invasive melanoma, and metastatic melanoma (n = 102). PEDF expression was consistently decreased in invasive and metastatic melanoma, compared with nevi and melanoma in situ (P < 0.0001). PEDF was lost in thicker melanomas (P = 0.003), and correlated with depth of invasion (P = 0.003) and distant metastasis (P = 0.0331), but only marginally with mitotic index, AJCC stage, nodal metastasis, or blood vascular density (0.05 < P < 0.10). Quantitative real-time PCR and microarray analyses confirmed PEDF down-regulation at the mRNA level in several melanoma lines, compared with melanocytes. MITF positively correlated with PEDF expression in invasive melanomas (P = 0.0003). Searching for PEDF regulatory mechanisms revealed two occupied conserved E-boxes (DNA recognition elements) in the first intron of the human and mouse PEDF promoter regions, confirmed by binding assays. Dominant-negative and siRNA approaches in vivo demonstrated direct transcriptional influence of MITF on PEDF, establishing the PEDF gene (SERPINF1) as a MITF target in melanocytes and melanoma cells. These findings suggest that loss of PEDF expression promotes early invasive melanoma growth.

89Zr-huJ591 immuno-PET imaging in patients with advanced metastatic prostate cancer.

PURPOSE: Given the bone tropism of prostate cancer, conventional imaging modalities poorly identify or quantify metastatic disease. (89)Zr-huJ591 positron emission tomography (PET) imaging was performed in patients with metastatic prostate cancer to analyze and validate this as an imaging biomarker for metastatic disease. The purpose of this initial study was to assess safety, biodistribution, normal organ dosimetry, and optimal imaging time post-injection for lesion detection. METHODS: Ten patients with metastatic prostate cancer received 5 mCi of (89)Zr-huJ591. Four whole-body scans with multiple whole-body count rate measurements and serum activity concentration measurements were obtained in all patients. Biodistribution, clearance, and lesion uptake by (89)Zr-huJ591 immuno-PET imaging was analyzed and dosimetry was estimated using MIRD techniques. Initial assessment of lesion targeting of (89)Zr-huJ591 was done. Optimal time for imaging post-injection was determined. RESULTS: The dose was well tolerated with mild chills and rigors seen in two patients. The clearance of (89)Zr-huJ591 from serum was bi-exponential with biological half-lives of 7 ± 4.5 h (range 1.1-14 h) and 62 ± 13 h (range 51-89 h) for initial rapid and later slow phase. Whole-body biological clearance was 219 ± 48 h (range 153-317 h). The mean whole-body and liver residence time was 78.7 and 25.6 h, respectively. Dosimetric estimates to critical organs included liver 7.7 ± 1.5 cGy/mCi, renal cortex 3.5 ± 0.4 cGy/mCi, and bone marrow 1.2 ± 0.2 cGy/mCi. Optimal time for patient imaging after injection was 7 ± 1 days. Lesion targeting of bone or soft tissue was seen in all patients. Biopsies were performed in 8 patients for a total 12 lesions, all of which were histologically confirmed as metastatic prostate cancer. One biopsy-proven lesion was not positive on (89)Zr-huJ591, while the remaining 11 lesions were (89)Zr-huJ591 positive. Two biopsy-positive nodal lesions were noted only on (89)Zr-huJ591 study, while the conventional imaging modality was negative. CONCLUSION: (89)Zr-huJ591 PET imaging of prostate-specific membrane antigen expression is safe and shows good localization of disease in prostate cancer patients. Liver is the critical organ for dosimetry, and 7 ± 1 days is the optimal imaging time. A larger study is underway to determine lesion detection in an expanded cohort of patients with metastatic prostate cancer.

Effect of dietary polyunsaturated fatty acids on castration-resistant Pten-null prostate cancer.

A common treatment of advanced prostate cancer involves the deprivation of androgens. Despite the initial response to hormonal therapy, eventually all the patients relapse. In the present study, we sought to determine whether dietary polyunsaturated fatty acid (PUFA) affects the development of castration-resistant prostate cancer. Cell culture, patient tissue microarray, allograft, xenograft, prostate-specific Pten knockout and omega-3 desaturase transgenic mouse models in conjunction with dietary manipulation, gene knockdown and knockout approaches were used to determine the effect of dietary PUFA on castration-resistant Pten-null prostate cancer. We found that deletion of Pten increased androgen receptor (AR) expression and Pten-null prostate cells were castration resistant. Omega-3 PUFA slowed down the growth of castration-resistant tumors as compared with omega-6 PUFA. Omega-3 PUFA decreased AR protein to a similar extent in tumor cell cytosolic and nuclear fractions but had no effect on AR messenger RNA level. Omega-3 PUFA treatment appeared to accelerate AR protein degradation, which could be blocked by proteasome inhibitor MG132. Knockdown of AR significantly slowed down prostate cancer cell proliferation in the absence of androgens. Our data suggest that omega-3 PUFA inhibits castration-resistant prostate cancer in part by accelerating proteasome-dependent degradation of the AR protein. Dietary omega-3 PUFA supplementation in conjunction with androgen ablation may significantly delay the development of castration-resistant prostate cancer in patients compared with androgen ablation alone.

ß-Catenin is a useful adjunct immunohistochemical marker for the diagnosis of pulmonary lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare multisystem disease leading to cystic destruction of the lung parenchyma and is associated with abnormal smooth muscle proliferation affecting airways, lymphatics, and blood vessels. LAM occurs sporadically or in association with the tuberous sclerosis complex (TSC). Recent evidence demonstrates the role of aberrant ß-catenin signaling in TSC. To further understand the pathogenesis of LAM and to examine the diagnostic usefulness of ß-catenin, we examined protein expression in 28 pulmonary LAM cases and 10 cases of renal angiomyolipoma resected from patients with sporadic LAM. Immunohistochemical analysis was performed for established markers of LAM cells (HMB45, estrogen receptor [ER]-α, and progesterone receptor [PR]) and ß-catenin. All LAM cases were positive for ß-catenin and demonstrated high specificity with overall immunoreactivity superior to HMB45, ER-α, and PR. Similar expression was demonstrated in renal angiomyolipoma. Our results indicate that ß-catenin is a useful marker of LAM and may be clinically useful in the diagnostic setting.

BRAF activation initiates but does not maintain invasive prostate adenocarcinoma.

Prostate cancer is the second leading cause of cancer-related deaths in men. Activation of MAP kinase signaling pathway has been implicated in advanced and androgen-independent prostate cancers, although formal genetic proof has been lacking. In the course of modeling malignant melanoma in a tyrosinase promoter transgenic system, we developed a genetically-engineered mouse (GEM) model of invasive prostate cancers, whereby an activating mutation of BRAF(V600E)--a mutation found in approximately 10% of human prostate tumors--was targeted to the epithelial compartment of the prostate gland on the background of Ink4a/Arf deficiency. These GEM mice developed prostate gland hyperplasia with progression to rapidly growing invasive adenocarcinoma without evidence of AKT activation, providing genetic proof that activation of MAP kinase signaling is sufficient to drive prostate tumorigenesis. Importantly, genetic extinction of BRAF(V600E) in established prostate tumors did not lead to tumor regression, indicating that while sufficient to initiate development of invasive prostate adenocarcinoma, BRAF(V600E) is not required for its maintenance.

The oncogenic properties of mutant p110alpha and p110beta phosphatidylinositol 3-kinases in human mammary epithelial cells.

The PIK3CA gene encoding the p110alpha subunit of Class IA phosphatidylinositol 3-kinases (PI3Ks) is frequently mutated in human tumors. Mutations in the PIK3CB gene encoding p110beta, the only other widely expressed Class IA PI3K, have not been reported. We compared the biochemical activity and transforming potential of mutant forms of p110alpha and p110beta in a human mammary epithelial cell system. The two most common tumor-derived alleles of p110alpha, H1047R and E545K, potently activated PI3K signaling. Human mammary epithelial cells expressing these alleles grew efficiently in soft agar and as orthotopic tumors in nude mice. We also examined a third class of mutations in p110alpha, those in the p85-binding domain. A representative tumor-derived p85-binding-domain mutant R38H showed modestly reduced p85 binding and weakly activated PI3K/Akt signaling. In contrast, a deletion mutant lacking the entire p85-binding domain efficiently activated PI3K signaling. When we constructed in p110beta a mutation homologous to the E545K allele of p110alpha, the resulting p110beta mutant was only weakly activated and allowed minimal soft-agar growth. However, a gene fusion of p110beta with the membrane anchor from c-Src was highly active and transforming in both soft-agar and orthotopic nude mouse assays. Thus, although introduction of activating mutations from p110alpha at the corresponding sites in p110beta failed to render the enzyme oncogenic in human cells, the possibility remains that other mutations might activate the beta isoform.

Prospective evaluation of AMACR (P504S) and basal cell markers in the assessment of routine prostate needle biopsy specimens.

Distinguishing benign prostate glands from malignant ones, based purely on morphology, on prostatic core needle biopsy specimens (PNBs) may prove difficult, particularly if the suspicious focus is small. In recent years, several immunohistochemical markers, including the basal cell cocktail (BCC), 34betaE12 and p63, and the prostate cancer (PCa) biomarker alpha-methylacyl-CoA-racemase (AMACR), have been used as adjuvants to morphology, in these diagnostically challenging cases. We prospectively address the diagnostic utility of using the BCC, in combination with the commercially available AMACR monoclonal antibody, P504S, on PNBs that required immunohistochemistry (IHC) studies to make a diagnosis. The goals of this prospective study were to assess the day-to-day practice in an academic setting, to determine how often these IHC tests were used on routine PNBs, and to establish how often a combination of the BCC and P504S were helpful in diagnosing prostate cancer. A total of 772 prospectively collected PNB cases were examined over a 7-month period. IHC staining was performed in 171 cases (22%); 123 cases were stained with the BCC in addition to the commercially available monoclonal AMACR antibody. In 86 of these 123 cases (70%), both stains contributed to the final diagnosis: PCa in 44 cases, benign in 33 cases and high-grade prostatic intraepithelial neoplasia in 9 cases. Of the remaining 37 cases (30%), 18 were called benign or PCa, based solely on appropriate staining with the BCC, with AMACR being noncontributory because the focus of interest had been cut through (12 cases), there was negative staining with AMACR (in 4 PCa cases), or there was positive staining with AMACR (in 2 benign cases showing atrophy). Nineteen of 37 cases were diagnosed as atypical small acinar proliferation. In these 19 cases either the focus had been cut through on one or both of the stains (11 cases), both AMACR and BCC failed to work (2 cases), AMACR was positive in the presence of patchy BCC staining (1 cases), AMACR was negative in the absence of BCC staining (3 cases), or despite appropriate staining the focus consisted of 1 gland and was considered too small to call carcinoma (2 cases). Additional IHC stains were performed in 171 of 772 cases; of these, 123 had sufficient material to perform both the BCC and P504S. The BCC when used in combination with AMACR rendered a diagnosis in almost 70% of cases. Using these stains in combination may be a better approach in diagnostically difficult cases as it increases the likelihood that a definitive diagnosis can be rendered while decreasing the likelihood of an equivocal diagnosis. However, a limitation of this approach is the loss of tissue in these small lesions, suggesting that combining AMACR and the BCC on a single slide would be superior to using either marker separately.

Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease.

The anemia of chronic disease is a prevalent, poorly understood condition that afflicts patients with a wide variety of diseases, including infections, malignancies, and rheumatologic disorders. It is characterized by a blunted erythropoietin response by erythroid precursors, decreased red blood cell survival, and a defect in iron absorption and macrophage iron retention, which interrupts iron delivery to erythroid precursor cells. We noted that patients with large hepatic adenomas had severe iron refractory anemia similar to that observed in anemia of chronic disease. This anemia resolved spontaneously after adenoma resection or liver transplantation. We investigated the role of the adenomas in the pathogenesis of the anemia and found that they produce inappropriately high levels of hepcidin mRNA. Hepcidin is a peptide hormone that has been implicated in controlling the release of iron from cells. We conclude that hepcidin plays a major, causative role in the anemia observed in our subgroup of patients with hepatic adenomas, and we speculate that it is important in the pathogenesis of the anemia of chronic disease in general.