Kent L. Nastiuk

Common mutations in BRCA1 and BRCA2 do not contribute to early prostate cancer in Jewish men.

Families with a high incidence of hereditary breast cancer, and subsequently shown to have terminating mutations in BRCA1 or BRCA2, appear to have a higher incidence of prostate cancer among male relatives. We aimed to determine whether the common germline mutations of BRCA1 or BRCA2 in Ashkenazi Jewish men predisposed them to prostate cancer.

Opportunities and challenges in combination gene cancer therapy.

Treatment for solid tumor malignancies, which constitute the majority of human cancers, is still dominated by surgery and radiotherapies. This is especially true for many localized solid tumors, which are often curable with these treatments. However, metastatic cancers are beyond the reach of these therapies, and many localized cancers that are initially treated with surgery and radiation will recur and metastasize. Thus, for over 60years there has been a concerted effort to develop effective drug treatments for metastatic cancers. Combination therapies are an increasingly important part of the anti-cancer drug armamentarium. In the case of cytotoxic chemotherapy, multi-drug regimens rapidly became the norm, as the earliest single agents were relatively ineffective. In contrast to chemotherapy, where combination therapies were required in order to achieve treatment efficacy, for both hormonal and targeted therapies the impetus to move toward the use of combination therapies is to prevent or reverse the development of treatment resistance. In addition, emerging evidence suggests that combination therapy may also improve cancer treatment by neutralizing an emerging treatment side effect termed therapy-induced metastasis, which accompanies some effective single agent therapies. Finally, although gene therapy is still far from use in the clinic, we propose that combination therapies may enhance its effectiveness.

FLIP-ping out: Death receptor signaling in the prostate

Prostate cancer is a leading cause of cancer related death. The growth of normal prostate epithelial cells is under the tight control of various growth factors, most notably androgens, such that castration leads to apoptosis of this cell population. Androgen-depletion has a similar effect on prostate cancers; however, following initial regression tumors often return in an androgen-depletion independent form that is frequently lethal. Thus, castration induced prostate regression in rodents has been a valuable model for identifying cell signaling pathways that control the proliferation and apoptosis of both normal and neoplastic prostate epithelial cells. For example, studies of normal prostate regression demonstrated the critical role of paracrine (stromal produced) transforming growth factor-ß. This review examines the role of the TNF-family death receptors and caspases-8 and -10 in prostate epithelial cell death. There is significant evidence that expression of the caspase-8 inhibitor FLIP (FLICE-like inhibitory protein) is androgen regulated and that this protein is one of the key regulators of androgen withdrawal induced cell death. However, it is not yet known which of the death receptor pathways is required for prostate apoptosis in vivo, and this remains an active topic of research.

Convenient, nonradioactive, heteroduplex-based methods for identifying recurrent mutations in the BRCA1 and BRCA2 genes.

The ability to identify inviduals who are predisposed to specific malignant tumors is a promising molecular diagnostic byproduct of over two decades of intensive research into the genetic pathogenesis of human cancer. Approximately 2% of Ashkenazi Jews carry recurrent germline mutations in either the BRCA1 or BRCA2 genes that may predispose these individuals to the development of breast and ovarian cancer. We have developed a nonisotopic method, based on the formation of heteroduplexes between polymerase chain reaction (PCR) amplified wild-type and mutant alleles, which can be used to identify the BRCA1 185delAG and the BRCA2 6174delT mutations. The same assay can also be used to verify the loss of heterozygosity in a tumor sample arising in an individual with a germline mutation. The four steps described in this report (PCR amplification, heteroduplex formation, acrylamide gel electrophoresis, and ethidium bromide staining/UV-fluorescence photography) can be readily and reproducibly performed in the course of a single day, making this a useful method for the routine identification of these mutations.

Variant allele of HSD3B1 increases progression to castration‐resistant prostate cancer

3β‐hydroxysteroid dehydrogenase type 1 (3βHSD1), which is a rate‐limiting enzyme that catalyzes the conversion of adrenal‐derived steroid dehydroepiandrosterone to dihydrotestosterone (DHT), may be a promising target for treating castration‐resistant prostate cancer (CRPC).

TNF signaling mediates an enzalutamide-induced metastatic phenotype of prostate cancer and microenvironment cell co-cultures

The dramatic responses tumors display to targeted therapies are limited by acquired or pre-existing mechanisms of therapy resistance. We recently discovered that androgen receptor blockade by the anti-androgen enzalutamide paradoxically enhanced metastasis and that these pro-metastatic effects were mediated by the chemoattractant CCL2. CCL2 is regulated by TNF, which is negatively regulated by androgen signaling. Thus, we asked if TNF mediates the pro-metastatic effects of enzalutamide. We found that androgen withdrawal or enzalutamide induced TNF mRNA and protein secretion in castration resistant prostate cancer (C4-2) cells, but not in macrophage-like (THP1) or myofibroblast-like (WPMY1) cells. Androgen deprivation therapy (ADT) induced autocrine CCL2 expression in C4-2 (as well as a murine CRPC cell line), while exogenous TNF induced CCL2 in THP1 and WPMY1. TNF was most potent in myofibroblast cultures, suggesting ADT induces CCL2 via paracrine interactions within the tumor microenvironment. A soluble TNF receptor (etanercept) blocked enzalutamide-induced CCL2 protein secretion and mRNA, implying dependence on secreted TNF. A small molecule inhibitor of CCR2 (the CCL2 receptor) significantly reduced TNF induced migration, while etanercept inhibited enzalutamide-induced migration and invasion of C4-2. Analysis of human prostate cancers suggests that a TNF-CCL2 paracrine loop is induced in response to ADT and might account for some forms of prostate cancer therapy resistance.

Sequential proteolytic processing of an interferon-alpha receptor subunit by TNF-alpha converting enzyme and presenilins.

It is well established that interferons trigger tyrosine-kinase-dependent signaling via JAK kinases and STAT transcription factors. However, we have observed both IFNaR2 receptor cleavage and functional activity of the liberated intracellular domain (ICD), suggesting that interferon-alpha (IFN-alpha) can also signal via regulated intramembrane proteolysis (RIP), an evolutionarily conserved mechanism of receptor-mediated signaling. Sequential cleavage of the receptor ectodomain and transmembrane domain is a hallmark of the most common class of RIP. To investigate the mechanisms of IFNaR2 RIP signaling, we examined IFNaR2 cleavage by TNF-alpha converting enzyme (TACE) and presenilin proteases. We tracked the fate of epitope-tagged and fusion variants of IFNaR2 in cells expressing wild-type, mutant, or null versions of TACE and presenilins 1 and 2. Cleavage and subcellular location were determined by immunoblot, fluoresence microscopy, and reporter assays. We found that both TACE and presenilin 1/2 cleave IFNaR2, in a sequential manner that allows the ICD to move to the nucleus. TACE cleavage was induced by IFN-alpha but was not consistently required for the anti-proliferative effects of IFN-alpha. In conclusion, IFNaR2 is cleaved by TACE and Presenilin 1/2, suggesting that interferons signal by both kinase and RIP-mediated pathways.

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

This article appears in Molecular Endocrinology, published February 3, 2011, 10.1210/me.2010-0312

Metastasis to scrotal skin as the initial manifestation in a patient with rectal adenocarcinoma: a rare case report and literature review.

adenocarcinomas have a higher occurrence to give rise to cutaneous scrotum metastases. The first case of cutaneous scrotum metastasis was reported in 1939. Until now, only 40 such cases had been published in the literature written in Chinese and English (Supplementary Table 1). The mean reported patient age was 55.5 years, range from 2 years old to 84 years old. The most frequent symptom was cutaneous nodules, which happened in 24 patients. Another manifestation included papules, plaques, edema, and ulcer. Skin lesions have been reported in seven patients. Some patients complained of pain associated with ulcer, while others were just painless ulcer or nodules. The gastrointestinal system was the most common organ system metastasized to scrotum skin (17 patients). The urogenital system was the second most frequent sites of cancer origin responsible for cutaneous scrotum metastasis (14 patients), which included prostate, bladder, kidney and urethra. The median interval between diagnosis of the primary tumor and subsequent metastasis to the scrotum was just 6 months (range 1–27 months). In addition to single case reports, we reviewed larger case series, which contained data regarding cutaneous metastases. In two series of patients performed by Lookingbill et al.3,5 1420 (10.4%) of 4020 patients were found to have skin involvement. However, just one case had metastasized to the scrotum skin. Reingold4 analyzed that 32 of their 2300 patients with carcinoma had cutaneous metastases. However, no case metastasized to the scrotum skin. The scrotum and penile skin are organs that rarely develop metastases. Maestro et al.6 speculates there are two theories have been postulated to explain this low frequency of metastasis, while their rich vascularization (blood and lymphatic) should result in the opposite. First, perhaps the scrotum and penis have a distinctive defense mechanism, which is not yet discovered (similar to the spleen). Second, these patients with advanced stages of disseminated neoplastic disease pay little attention to the exploration and evaluation of the scrotum and penis, even where a nodule is discovered. The diagnosis of cutaneous scrotum metastases is based on clinical suspicion and physical examination, but the best diagnostic method is biopsy or surgical specimen.6 Treatment depends on the location, size, symptomatology and patient’s prognosis and so on, but average survival in these patients is generally short due to metastatic progression. Treatment may consist of local tumor excision, radiation therapy, and radical surgical procedures for cutaneous scrotum metastatic Dear Editor, We present here a rare case of rectal adenocarcinoma metastasized to scrotum skin that was detected in a 36-year-old man. In addition, we have reviewed the Chinese and English literature for reports of internal malignancy secondary to scrotum skin. To our best knowledge, there are forty cases reported in literature. Rectal adenocarcinoma mainly metastasizes to the lymph nodes, liver, lung and bone.1 Cutaneous metastasis of rectal carcinoma is rare, which mostly occur in the skin of the abdomen and crissum. Scrotum skin is rarely involved. Cutaneous metastasis is generally an indication of widespread disease.2 A 36-year-old male patient was admitted to our hospital due to the swelling of the scrotum in July 2012. The patient had been undergone with lymphadenectomy due to bubo when he was a child. Some scars could be seen in his bilateral legs (Figure 1a). In March 2010, he had been treated with rectal resection and Colocutaneous colostomy due to colorectal adenocarcinoma in another hospital. The patient complained of dysuria as well as the pain of scrotum and penis more than 1-month prior to the visit. The physical examination revealed the multiple papules in the scrotum with obvious tenderness, and enlargement of the inguinal lymph nodes (Figure 1a). About 2 weeks later, the scrotum papules and plaques began to ulcerate and involved in the penis. Biopsies of the ulcer confirmed metastatic adenocarcinoma that was infiltrating the scrotum skin (Figure 1b). Tumor is immunoreactive for CK7 and CK20 (Figure 1c and 1d). No other distance organs were involved by CT. The patient was subsequently treated with suprapubic urinary diversion and two cycles of chemotherapy consisting of gemcitabine and docetaxel. Then, he refused to accept treatment further and was dead due to liver metastasis after discovering scrotum skin metastasis for 1 year. Cutaneous metastases originating from internal malignancies are uncommon.3 The most probable sites of cutaneous metastases are the skin of the anterior chest, followed by the abdomen and back.4 Scrotum skin is unusual invaded. Compared with other histologic subtypes, LETTER TO THE EDITOR

Laminates for miniaturized integrated bioelectronic protein analysis systems

We report the use of microelectronic laminate-based manufacturing to produce integrated devices capable of performing both electronic and biological analysis, particularly for protein assays. There is currently a significant push to develop miniaturized biofluidic devices for portable, point-of-care medical applications. Such “lab-on-a-chip” devices are typically built using lithographic and stamping processes borrowed from semiconductor and other industries. However, these devices suffer from severe lack of integration, especially with electronic components. Electronic wiring and electrical components are typically added post-facto, usually off the device, in a clumsy non-integrated way. In this research, we have developed an innovative packaging approach to system miniaturization which incorporates fluidic, electronics, and mechanical components into a laminate-based device. The use of lamination allows one to readily integrate many different types of technologies and materials into a single device, making the production of a truly integrated unit. Furthermore, this approach, which borrows heavily from the printed circuit board and microelectronics packaging industry, has great potential for large scale, low cost manufacturing. Our demonstration system reproduces protein separation in a hybrid fluidic-gel-electronic circuit board. The assay (SDS-PAGE), which is built into a single laminate structure, allows one to do an important and sensitive protein analysis using only a small quantity of protein. The device consists of printed circuit board layers laminated against fluidic and acrylamide gel layers to produce a single hybrid device. Since the electrical traces and components are on-board, the device can be plugged into a power or data connector without the need for extra wiring and interconnects to drive the device. We describe the manufacturing of this device and demonstrate the ability to perform protein separation using this device. We also discuss some of the issues associated with integrating highly dissimilar laminates together for the purpose of building a true “lab-on-a-board”.