Breast Neoplasms: Diamandis EP

Sturgeon CM, Duffy MJ, Stenman UH, Lilja H, Brünner N, Chan DW, Babaian R, Bast RC, Dowell B, Esteva FJ, Haglund C, Harbeck N, Hayes DF, Holten-Andersen M, Klee GG, Lamerz R, Looijenga LH, Molina R, Nielsen HJ, Rittenhouse H, Semjonow A, Shih IeM, Sibley P, Sölétormos G, Stephan C, Sokoll L, Hoffman BR, Diamandis EP, Anonymous00039. · Department of Clinical Biochemistry, Royal Infirmary of Edinburgh, Edinburgh, UK. · Clin Chem. · Pubmed #19042984 No free full text.

Abstract: BACKGROUND: Updated National Academy of Clinical Biochemistry (NACB) Laboratory Medicine Practice Guidelines for the use of tumor markers in the clinic have been developed. METHODS: Published reports relevant to use of tumor markers for 5 cancer sites--testicular, prostate, colorectal, breast, and ovarian--were critically reviewed. RESULTS: For testicular cancer, alpha-fetoprotein, human chorionic gonadotropin, and lactate dehydrogenase are recommended for diagnosis/case finding, staging, prognosis determination, recurrence detection, and therapy monitoring. alpha-Fetoprotein is also recommended for differential diagnosis of nonseminomatous and seminomatous germ cell tumors. Prostate-specific antigen (PSA) is not recommended for prostate cancer screening, but may be used for detecting disease recurrence and monitoring therapy. Free PSA measurement data are useful for distinguishing malignant from benign prostatic disease when total PSA is <10 microg/L. In colorectal cancer, carcinoembryonic antigen is recommended (with some caveats) for prognosis determination, postoperative surveillance, and therapy monitoring in advanced disease. Fecal occult blood testing may be used for screening asymptomatic adults 50 years or older. For breast cancer, estrogen and progesterone receptors are mandatory for predicting response to hormone therapy, human epidermal growth factor receptor-2 measurement is mandatory for predicting response to trastuzumab, and urokinase plasminogen activator/plasminogen activator inhibitor 1 may be used for determining prognosis in lymph node-negative patients. CA15-3/BR27-29 or carcinoembryonic antigen may be used for therapy monitoring in advanced disease. CA125 is recommended (with transvaginal ultrasound) for early detection of ovarian cancer in women at high risk for this disease. CA125 is also recommended for differential diagnosis of suspicious pelvic masses in postmenopausal women, as well as for detection of recurrence, monitoring of therapy, and determination of prognosis in women with ovarian cancer. CONCLUSIONS: Implementation of these recommendations should encourage optimal use of tumor markers.

Kulasingam V, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. · Int J Cancer. · Pubmed #18712711 No free full text.

Abstract: Current cancer biomarkers suffer from low diagnostic sensitivity and specificity and have not yet made a major impact on reducing cancer burden. Proteomic methods based on mass spectrometry have matured significantly over the past few years and hold promise to deliver candidate markers for diagnosis, prognosis or monitoring therapeutic response. Because of the complex nature of biological fluids such as plasma, biomarker discovery efforts using proteomics have not as yet delivered any novel tumor markers. Recently, there has been a rise in the number of publications utilizing a cell culture-based model of cancer to identify novel candidate tumor markers. The secretome of cancer cell lines constitutes an important class of proteins that can act locally and systemically in the body. Secreted proteins, in addition to serving as serological markers, play a central role in physiology and pathophysiology. In this review, we focus on the proteomics of breast cancer and the different strategies to mine for biomarkers, with particular emphasis on a cell culture-based model developed in our laboratory.

Emami N, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. · Clin Chem. · Pubmed #18687738 No free full text.

Abstract: BACKGROUND: The human kallikrein-related peptidase (KLK) family consists of 15 highly conserved serine proteases, which are encoded by the largest uninterrupted cluster of protease genes in the human genome. To date, several members of the family have been reported as potential cancer biomarkers. Although primarily known for their biomarker value in prostate, ovarian, and breast cancers, more recent data suggest analogous roles of KLKs in several other cancers, including gastrointestinal, head and neck, lung, and brain malignancies. Among the proposed KLK cancer biomarkers, prostate-specific antigen (also known as KLK3) is the most widely recognized member in urologic oncology. CONTENT: Despite substantial progress in the understanding of the biomarker utility of individual KLKs, the current challenge lies in devising biomarker panels to increase the accuracy of prognosis, prediction of therapy, and diagnosis. To date, multiparametric KLK panels have been proposed for prostate, ovarian, and lung cancers. In addition to their biomarker utility, emerging evidence has revealed a number of critical functional roles for KLKs in the pathogenesis of cancer and their potential use as therapeutic targets. SUMMARY: KLKs have biomarker utility in many cancer types but individually lack sufficient specificity or sensitivity to be used in clinical practice; however, groups of KLKs and other candidate biomarkers may offer improved performance.

Paliouras M, Borgono C, Diamandis EP. · Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada. · Cancer Lett. · Pubmed #17275179 No free full text.

Abstract: Human tissue kallikreins (KLKs) are attracting increased attention due to their role as biomarkers for the screening, diagnosis, prognosis, and monitoring of various cancers including those of the prostate, ovarian, breast, testicular, and lung. Human tissue kallikrein genes represent the largest contiguous group of proteases within the human genome. Originally thought to consist of three genes, the identification of the human kallikrein locus has expanded this number to fifteen. These genes, and their encoded proteins, share a high degree of homology and are expressed in different tissues. Prostate-specific antigen (PSA), the most commonly known kallikrein, is a useful biomarker for prostate cancer. Several other kallikreins, including kallikreins 2 (KLK2) and 11 (KLK11) are emerging as complementary prostate cancer biomarkers. Along with these kallikreins, several others have been implicated in the other cancers. For example, KLK5, 6, 7, 10, 11, and 14 are emerging biomarkers for ovarian cancer. The identification of kallikrein substrates and the development of proteolytic cascade models implicate kallikrein proteins in cancer progression. This review describes the current status of kallikreins as cancer biomarkers.

Obiezu CV, Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ont., Canada M5G 1X5. · Cancer Lett. · Pubmed #15911097 No free full text.

Abstract: Human tissue kallikrein genes, located on the long arm of chromosome 19, are a subgroup of the serine protease family of proteolytic enzymes. Initially thought to consist of three members, the human kallikrein locus has now been extended and includes 15 tandemly located genes. These genes, and their protein products, share a high degree of homology and are expressed in a wide array of tissues, mainly those that are under steroid hormone control. PSA (hK3) is one of the human kallikreins, and is the most useful tumor marker for prostate cancer screening, diagnosis, prognosis and monitoring. hK2, another prostate-specific kallikrein, has also been proposed as a complementary prostate cancer biomarker. In the past 5 years, the newly discovered kallikreins (KLK4-KLK15) have been associated with several types of cancer. For example, hK4, hK5, hK6, hK7, hK8, hK10, hK11, hK13 and hK14 are emerging biomarkers for ovarian, breast, prostate and testicular cancer. New evidence raises the possibility that some kallikreins are directly involved with cancer progression. We here review the evidence linking kallikreins and cancer and their applicability as novel biomarkers for cancer diagnosis and management.

Borgoño CA, Michael IP, Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, M5G 1X5 Canada. · Mol Cancer Res. · Pubmed #15192120 links to  free full text

Abstract: Tissue kallikreins are members of the S1 family (clan SA) of trypsin-like serine proteases and are present in at least six mammalian orders. In humans, tissue kallikreins (hK) are encoded by 15 structurally similar, steroid hormone-regulated genes (KLK) that colocalize to chromosome 19q13.4, representing the largest cluster of contiguous protease genes in the entire genome. hKs are widely expressed in diverse tissues and implicated in a range of normal physiologic functions from the regulation of blood pressure and electrolyte balance to tissue remodeling, prohormone processing, neural plasticity, and skin desquamation. Several lines of evidence suggest that hKs may be involved in cascade reactions and that cross-talk may exist with proteases of other catalytic classes. The proteolytic activity of hKs is regulated in several ways including zymogen activation, endogenous inhibitors, such as serpins, and via internal (auto)cleavage leading to inactivation. Dysregulated hK expression is associated with multiple diseases, primarily cancer. As a consequence, many kallikreins, in addition to hK3/PSA, have been identified as promising diagnostic and/or prognostic biomarkers for several cancer types, including ovarian, breast, and prostate. Recent data also suggest that hKs may be causally involved in carcinogenesis, particularly in tumor metastasis and invasion, and, thus, may represent attractive drug targets to consider for therapeutic intervention.

Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital,and Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada. · Mol Cell Proteomics. · Pubmed #14990683 links to  free full text

Abstract: Serum proteomic profiling, by using surfaced-enhanced laser desorption/ionization-time-of-flight mass spectrometry, is one of the most promising new approaches for cancer diagnostics. Exceptional sensitivities and specificities have been reported for some cancer types such as prostate, ovarian, breast, and bladder cancers. These sensitivities/specificities are far superior to those obtained by using classical cancer biomarkers. In this review, I concentrate more on questions that cast doubt on the results reported and propose experiments to investigate these questions in detail, before the technique is used at the clinic. It is clear that the method needs to be externally and thoroughly validated before clinical implementation is warranted.

Yousef GM, Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada. · Clin Biochem. · Pubmed #12951170 No free full text.

Abstract: Kallikreins are serine proteases with diverse physiologic functions. They are represented by multigene families in many animal species, especially in rat and mouse. Recently, the human kallikrein gene family has been fully characterized and includes 15 members, tandemly localized on chromosome 19q13.4. A new definition has now been proposed for kallikreins, which is not based on function but, rather, on close proximity and structural similarities. In this review, we summarize available information about kallikreins in many animal species with special emphasis on human kallikreins. We discuss the common structural features of kallikreins at the DNA, mRNA and protein levels and overview their evolutionary history. Kallikreins are expressed in a wide range of tissues including the salivary gland, endocrine or endocrine-related tissues such as testis, prostate, breast and endometrium and in the central nervous system. Most, if not all, genes are under steroid hormone regulation. Accumulating evidence indicates that kallikreins are involved in many pathologic conditions. Of special interest is the potential role of kallikreins in the central nervous system. In addition, many kallikreins seem to be candidate tumor markers for many malignancies, especially those of endocrine-related organs.

Luo LY, Yousef G, Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada. · APMIS. · Pubmed #12752266 No free full text.

Abstract: Human tissue kallikreins are fifteen homologous genes encoding for secreted serine proteases and residing tandemly on chromosome 19q13.4. These enzymes are highly expressed in a variety of tissues and participate in diverse physiological processes. Human tissue kallikreins were found to be associated with several malignancies, especially endocrine-related cancers, including prostate, ovarian, breast and testicular cancer. In testicular germ cell tumors, some tissue kallikrein genes, including KLK5, KLK10, KLK13 and KLK14, were found to be significantly down-regulated. Tissue-specific splice variant forms of some kallikreins have been identified in the testis. In this paper, the expression of KLK5, KLK10, KLK13 and KLK14 in testicular cancer and their possible roles during testicular cancer development, as well as their clinical applications are briefly reviewed.

Rosenberg Zand RS, Jenkins DJ, Diamandis EP. · Department of Nutritional Sciences, University of Toronto, Toronto, Canada. · J Chromatogr B Analyt Technol Biomed Life Sci. · Pubmed #12270215 No free full text.

Abstract: Steroid-hormone dependent cancers, including those of the breast, prostate and colon, are leading causes of morbidity and mortality in western countries. In rural Asian areas, these diseases are relatively uncommon. Dietary factors, including low consumption of fruit, vegetables and soy in the west have been shown in various epidemiologic studies as reasons for these differences. This review discusses flavonoids, one component of these plant foods that is being investigated for their role in chemoprevention. Epidemiological, in vitro, animal and human studies shall be explored to look at mechanisms involved, including steroid hormone activity, effects on cell growth, antioxidant activities, inhibition of chemical carcinogenesis and influences on modulators of cancer risk. Although the in vitro and animal models point to several pathways by which flavonoids may reduce incidence of these cancers, the clinical data are still relatively lacking. More research is needed to determine how best to use foods containing these compounds to reduce steroid hormone-dependent cancer risk.

Yousef GM, Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ont, Canada. · Tumour Biol. · Pubmed #12218299 No free full text.

Abstract: The full characterization of the human kallikrein gene locus has allowed identification of all members of this gene family on chromosome 19q13.4 and the establishment of common structural criteria, at both the mRNA and protein level. The human kallikrein gene family now consists of 15 members; their mRNA and protein structure, tissue expression and hormonal regulation patterns have been delineated. In addition to prostate-specific antigen (PSA, hK3), which is an established tumor marker for prostate cancer diagnosis and follow-up, and human glandular kallikrein (hK2), an emerging prostate cancer biomarker, accumulating evidence indicates that many other members of the human kallikrein gene family are also implicated in endocrine-related malignancies. Many kallikreins are differentially regulated in breast, prostate, ovarian and testicular cancers. In addition, preliminary reports indicate that three newly identified kallikreins (hK6, hK10 and hK11) are serum biomarkers for diagnosis and monitoring of ovarian and prostate cancer. The mechanism by which kallikreins might be involved in the pathogenesis and/or progression of cancer is not as yet fully understood. Preliminary reports indicate a possible role of kallikreins in controlling vital processes, like apoptosis, angiogenesis and tumor metastasis by cleavage of critical substrates such as growth factors, hormones or extracellular matrix. In this review, we present data on the differential expression of kallikreins in cancer at both the mRNA and protein levels, and propose future directions of research towards our understanding of the involvement of kallikreins in cancer and their possible diagnostic, prognostic and therapeutic applications.

Diamandis EP, Yousef GM. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada. · Clin Chem. · Pubmed #12142373 links to  free full text

Abstract: Kallikreins are a subgroup of the serine protease enzyme family. Until recently, it was thought that the human kallikrein gene family contained only three members. In the past 3 years, the entire human kallikrein gene locus was discovered and found to contain 15 kallikrein genes. Kallikreins are expressed in many tissues, including steroid hormone-producing or hormone-dependent tissues such as the prostate, breast, ovary, and testis. Most, if not all, kallikreins are regulated by steroid hormones in cancer cell lines. There is strong but circumstantial evidence linking kallikreins and cancer. Prostate-specific antigen (PSA; hK3) and, more recently, human glandular kallikrein (hK2) are widely used tumor markers for prostate cancer. Three other kallikreins, hK6, hK10, and hK11, are emerging new serum biomarkers for ovarian and prostate cancer diagnosis and prognosis. Several other kallikreins are differentially expressed at both the mRNA and protein levels in various endocrine-related malignancies, and they have prognostic value. The coexpression of many kallikreins in the same tissues (healthy and malignant) points to the possible involvement of kallikreins in cascade enzymatic pathways. In addition to their diagnostic/prognostic potential, kallikreins may also emerge as attractive targets for therapeutics.

Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada. · Clin Chem. · Pubmed #10894830 links to  free full text

Abstract: BACKGROUND: Prostate-specific antigen (PSA) is a valuable prostatic cancer biomarker that is now widely used for population screening, diagnosis, and monitoring of patients with prostate cancer. Despite the voluminous literature on this biomarker, relatively few reports have addressed the issue of its physiological function and its connection to the pathogenesis and progression of prostate and other cancers. APPROACH: I here review literature dealing with PSA physiology and pathobiology and discuss reports that either suggest that PSA is a beneficial molecule with tumor suppressor activity or that PSA has deleterious effects in prostate, breast, and possibly other cancers. CONTENT: The present scientific literature on PSA physiology and pathobiology is confusing. A group of reports have suggested that PSA may act as a tumor suppressor, a negative regulator of cell growth, and an apoptotic molecule, whereas others suggest that PSA may, through its chymotrypsin-like activity, promote tumor progression and metastasis. SUMMARY: The physiological function of PSA is still not well understood. Because PSA is just one member of the human kallikrein gene family, it is possible that its biological functions are related to the activity of other related kallikreins. Only when the physiological functions of PSA and other kallikreins are elucidated will we be able to explain the currently apparently conflicting experimental data.

Black MH, Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada. · Breast Cancer Res Treat. · Pubmed #10752675 No free full text.

Abstract: Although prostate-specific antigen (PSA) is the most valuable tumor marker for the diagnosis and management of prostate carcinoma, it is widely accepted that PSA is not prostate specific. Numerous studies have shown that PSA is present in some female hormonally regulated tissues, principally the breast and its secretions. In this review, we summarize the findings of PSA in the breast, and focus on its potential for clinical applications in breast disease. PSA is produced by the majority of breast tumors and is a favorable indicator of prognosis in breast cancer. Low levels of PSA are released into the female circulation, and while the level of serum PSA is elevated in both benign and malignant breast disease, the molecular form of circulating PSA differs between women with and without breast cancer. These findings indicate that PSA may have potential diagnostic utility in breast cancer. PSA may also have a clinical application in benign breast disease, as both the level and molecular form of PSA differ between Type I and II breast cysts. High levels of PSA have been reported in nipple aspirate fluid (NAF) and recent studies have shown that the concentration of PSA in NAF is inversely related to breast cancer risk, indicating that NAF PSA may represent a clinical tool for breast cancer risk assessment. Thus, PSA represents a marker with numerous potential clinical applications as a diagnostic and/or prognostic tool in breast disease.

Diamandis EP, Yousef GM, Luo LY, Magklara A, Obiezu CV. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada. · Trends Endocrinol Metab. · Pubmed #10675891 No free full text.

Abstract: The traditional human kallikrein gene family consists of three genes, namely KLK1 [encoding human kallikrein 1 (hK1) or pancreatic/renal kallikrein], KLK2 (encoding hK2, previously known as human glandular kallikrein 1) and KLK3 [encoding hK3 or prostate-specific antigen (PSA)]. KLK2 and KLK3 have important applications in prostate cancer diagnostics and, more recently, in breast cancer diagnostics. During the past two to three years, new putative members of the human kallikrein gene family have been identified, including the PRSSL1 gene [encoding normal epithelial cell-specific 1 gene (NES1)], the gene encoding zyme/protease M/neurosin, the gene encoding prostase/KLK-L1, and the genes encoding neuropsin, stratum corneum chymotryptic enzyme and trypsin-like serine protease. Another five putative kallikrein genes, provisionally named KLK-L2, KLK-L3, KLK-L4, KLK-L5 and KLK-L6, have also been identified. Many of the newly identified kallikrein-like genes are regulated by steroid hormones, and a few kallikreins (NES1, protease M, PSA) are known to be downregulated in breast and possibly other cancers. NES1 appears to be a novel breast cancer tumor suppressor protein and PSA a potent inhibitor of angiogenesis. This brief review summarizes recent developments and possible applications of the newly defined and expanded human kallikrein gene locus.

Paliouras M, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. · Mol Oncol. · Pubmed #19383315 No free full text.

Abstract: The regulation of gene expression by steroid hormones plays an important role in the normal development and function of many organs, as well as in the pathogenesis of endocrine-related cancers, especially breast cancer. However, clinical data suggest that combined testosterone and estrogen treatments on post-menopausal women increase the risk of breast cancer. Experiments have shown that many, if not all kallikreins are under steroid hormone regulation in breast cancer cell lines. Their implication as prognostic and diagnostic markers has also been well-documented. Thus, we investigated the effect of combined hormone stimulation with androgens and 17beta-estradiol on the ductal caricinoma cell line BT474. This cell line has been shown to be sensitive to both, androgens (secreting PSA) and estrogens (secreting a number of kallikreins including KLK10, 11, and KLK14). We found that PSA expression was downregulated upon combined hormone stimulation, confirming reports that estrogen can antagonize and block the activity of the androgen receptor. Upon analysis of estrogen-sensitive kallikreins 10, 11, and 14, all showed to be synergistically enhanced in their expression three- to fourfold, upon joint hormone treatment versus individual hormone stimulation. The enhancement is dependent upon the action of androgens as treatment with the androgen receptor antagonist cyproterone actetate normalized the expression of KLK10, 11, and KLK14 to estrogen-stimulation levels. The synergistic effects between estrogens and androgens on estrogen-sensitive genes may have implications on the role of the kallikreins in associated risk of breast cancer and progression.

Kulasingam V, Zheng Y, Soosaipillai A, Leon AE, Gion M, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. · Int J Cancer. · Pubmed #19322904 No free full text.

Abstract: Activated leukocyte cell adhesion molecule (ALCAM) has been implicated in tumorigenesis. Our goal was to examine the levels of ALCAM, in addition to the classical breast cancer tumor markers carbohydrate antigen 15-3 (CA15-3) and carcinoembryonic antigen (CEA), in serum by quantitative enzyme-linked immunosorbent assay for diagnosis in breast cancer patients. The 3 proteins were measured in serum of 100 healthy women, 50 healthy men and 150 breast carcinoma patients. The diagnostic sensitivity and specificity of the tests were calculated and the association of serum marker concentrations with various clinicopathologic variables was examined using nonparametric Kruskal-Wallis tests. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic performance of the biomarkers. ALCAM, with area under the curve (AUC) of 0.78 [95% CI: 0.73, 0.84] outperformed CA15-3 (AUC = 0.70 [95% CI: 0.64, 0.76]) and CEA (AUC= 0.63 [95% CI: 0.56, 0.70]). The incremental values of AUC for ALCAM over that for CA15-3 were statistically significant (Delong test, p < 0.05). Combining CA15-3 and ALCAM yielded a ROC curve with an AUC of 0.81 (95% CI [0.75, 0.87]). Serum ALCAM appears to be a new biomarker for breast cancer and may have value for disease diagnosis.

Prassas I, Paliouras M, Datti A, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. · Clin Cancer Res. · Pubmed #18794087 No free full text.

Abstract: PURPOSE: Human tissue kallikreins (KLK) comprise a subgroup of 15 homologous secreted serine proteases. Primarily known for their clinical use as cancer biomarkers (e.g., PSA), KLKs have recently been directly implicated in cancer-related processes, including invasion, angiogenesis, and tumor growth regulation. Therefore, the identification of compounds that would modulate expression of KLKs might be of considerable therapeutic value. EXPERIMENTAL DESIGN: A cell-based high-throughput screening (HTS) of three small molecule libraries ( approximately 4,500 compounds) was undertaken; KLK expression in the breast cancer cell line MDA-MB-468 was assessed with sensitive ELISAs. RESULTS: The initial screening resulted in 66 "putative hits" that decreased KLK5 expression by at least 50% over control. Secondary screening and mini-dose-response assays resulted in 21 "validated hits." These 21 compounds were clustered in only three distinct functional families and were further analyzed in vitro to determine their effectiveness (IC(50)s). Hits that failed to show dose-responsiveness or interfered with the viability of the cells were excluded. Multiple members of the cardiac glycoside family were found to be novel inhibitors of KLK expression, acting at low concentrations (10-50 nmol/L). Furthermore, members of the same family induced marked decreases in c-MYC and c-FOS expression, in a dose-dependent manner that correlated the KLK inhibition, suggesting a transcriptional mechanism of regulation of KLK expression. CONCLUSIONS: We conclude that cardiac glycosides can dramatically suppress the transcription of KLKs and that these effects may be linked to proto-oncogene (c-myc/fos) expression. These findings may partially explain the recently realized antineoplastic actions of cardiac glycosides.

Sardana G, Jung K, Stephan C, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. · J Proteome Res. · Pubmed #18578523 No free full text.

Abstract: Early detection of prostate cancer is problematic due to the lack of a marker that has high diagnostic sensitivity and specificity. The prostate specific antigen (PSA) test, in combination with digital rectal examination, is the gold standard for prostate cancer diagnosis. However, this modality suffers from low specificity. Therefore, specific markers for clinically relevant prostate cancer are needed. Our objective was to proteomically characterize the conditioned media from three human prostate cancer cell lines of differing origin [PC3 (bone metastasis), LNCaP (lymph node metastasis), and 22Rv1 (localized to prostate)] to identify secreted proteins that could serve as novel prostate cancer biomarkers. Each cell line was cultured in triplicate, followed by a bottom-up analysis of the peptides by two-dimensional chromatography and tandem mass spectrometry. Approximately, 12% (329) of the proteins identified were classified as extracellular and 18% (504) as membrane-bound among which were known prostate cancer biomarkers such as PSA and KLK2. To select the most promising candidates for further investigation, tissue specificity, biological function, disease association based on literature searches, and comparison of protein overlap with the proteome of seminal plasma and serum were examined. On the basis of this, four novel candidates, follistatin, chemokine (C-X-C motif) ligand 16, pentraxin 3 and spondin 2, were validated in the serum of patients with and without prostate cancer. The proteins presented in this study represent a comprehensive sampling of the secreted and shed proteins expressed by prostate cancer cells, which may be useful as diagnostic, prognostic or predictive serological markers for prostate cancer.

Paliouras M, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ont., Canada. · Tumour Biol. · Pubmed #18515984 No free full text.

Abstract: OBJECTIVES: Our aim was to examine how certain signal transduction pathways influence the regulation of hormone-dependent kallikrein (KLK) gene expression in androgen-sensitive breast cancer cell lines. METHODS: We used the breast cancer cell lines T47D and BT474, treated with steroid hormones or various pathway inhibitors. KLKs were quantified by ELISA. RT-PCR, Western blots and immunoprecipitations were used to assess transcript and protein levels. RESULTS: PSA, KLK10, KLK11, KLK13 and KLK14 are upregulated upon androgen stimulation in the T47D cell line. The expression of PSA, KLK10 and KLK11 was repressed by the MEK1/2 inhibitor U0126 and the PI3K inhibitor Wortmannin in the presence of the hormone, thus implicating the RAS/MEK/ERK and PI3K/AKT signaling pathways in regulating hormone-dependent KLK gene activation. Analysis of inhibitor-treated cells revealed changes in c-MYC expression with a pattern parallel to KLK gene expression. Chromatin immunoprecipitations identified androgen-dependent recruitment of specific transcription factors to the KLK proximal promoters, including c-MYC binding to PSA and KLK11. CONCLUSION: The hormone-specific upregulation of PSA, KLK10 and KLK11 in the breast cancer cell line T47D is dependent on major intracellular signaling pathways. This work provides a new dimension to the regulation of these cancer-related genes and the potential for new therapeutic targeting strategies.

Kulasingam V, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, University of Toronto, and Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto M5G 1X5, ON, Canada. · Biol Chem. · Pubmed #17937626 No free full text.

Abstract: Using the breast cell lines MCF-10A, MDA-MB-468 and T-47D, we investigated the role of various glucocorticoids in regulating human kallikrein 10 expression. We found that increased concentrations of glucocorticoids decreased KLK10 expression in MCF-10A and increased KLK10 expression in MDA-MB-468 and T-47D cells. Stimulation of the cell lines using other steroid hormones did not yield any difference in KLK10 expression in MCF-10A and MDA-MB-468 cells, suggesting that regulation of KLK10 occurs primarily through glucocorticoids. However, T-47D cells expressed higher levels of KLK10 upon dihydrotestosterone stimulation. Blocking the glucocorticoid receptor (GR) demonstrated that the mechanisms of induction and repression are different in the three cell lines studied. Taken together, our results suggest an alternative mode of KLK10 regulation - by glucocorticoids via GR-dependent mechanisms.

Kulasingam V, Diamandis EP. · Department of Laboratory Medicine and Pathobiology, Mount Sinai Hospital, University of Toronto, Toronto, Ontario M5G 1X5, Canada. · Mol Cell Proteomics. · Pubmed #17656355 links to  free full text

Abstract: A "bottom-up" proteomics approach and a two-dimensional (strong cation exchange followed by reversed-phase) LC-MS/MS strategy on a linear ion trap (LTQ) were utilized to identify and compare expressions of extracellular and membrane-bound proteins in the conditioned media of three breast cell lines (MCF-10A, BT474, and MDA-MB-468). Proteomics analysis of the media identified in excess of 600, 500, and 700 proteins in MCF-10A, BT474, and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6, and 10 (ranging in concentration from 2 to 50 microg/liter) in MDA-MB-468 conditioned medium as validated by ELISA and confidently identified Her-2/neu in BT474 cells. Subcellular localization was determined based on Genome Ontology terms for all the 1,139 proteins of which 34% were classified as extracellular and membrane-bound. Proteomics analysis of MDA-MB-468 cell lysate demonstrated that only 5% of all identified proteins were extracellular. This confirmed our hypothesis that examining the CM of cell lines, as opposed to the cell lysates, leads to a significant enrichment in secreted proteins. Tissue specificity, functional classifications, and spectral counting were performed. Elafin, a protease inhibitor, identified in the conditioned media of BT474 and MDA-MB-468 and the three kallikreins (KLK5, KLK6, and KLK10) were validated using an immunoassay on various serum and biological samples. Some of the secreted proteins identified have established roles in breast cancer development (cell growth, differentiation, and metastasis) and/or are linked to early onset breast cancer. Our approach to mining for low abundance molecules could identify proteins in various stages of breast cancer development. Many of the identified proteins are potentially useful to investigate as circulating serum breast cancer biomarkers.

Darling MR, Tsai S, Jackson-Boeters L, Daley TD, Diamandis EP. · Division of Oral Pathology, Department of Pathology, University of Western Ontario, London, Ontario N6A 5C1, Canada. · Int J Biol Markers. · Pubmed #17177156 No free full text.

Abstract: The human kallikrein 5 protein (hK5) is expressed in many normal tissues, most notably in skin, breast, salivary gland and esophagus. It has also been shown to be a potential biomarker for breast, ovarian and testicular cancer. Human kallikrein 3 (hK3; prostate-specific antigen) is the most useful marker for adenocarcinoma of the prostate gland. The aim of this study was to determine whether hK3 and hK5 are expressed in salivary gland tissues and salivary gland tumors (both benign and malignant), in order to compare normal with tumor tissues. Pleomorphic adenomas, adenoid cystic carcinomas, polymorphous low-grade adenocarcinomas, acinic cell carcinomas, mucoepidermoid carcinomas and adenocarcinomas not otherwise specified of both minor and major salivary glands were examined. The results of this study indicate that most salivary gland tumors do not show high levels of expression of hK5. Staining was most prominent in keratinizing epithelia in pleomorphic adenomas. hK3 is not expressed in salivary gland tumors.

Borgoño CA, Michael IP, Shaw JL, Luo LY, Ghosh MC, Soosaipillai A, Grass L, Katsaros D, Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada. · J Biol Chem. · Pubmed #17110383 links to  free full text

Abstract: Human tissue kallikrein 14 (KLK14) is a novel extracellular serine protease. Clinical data link KLK14 expression to several diseases, primarily cancer; however, little is known of its (patho)-physiological role. To functionally characterize KLK14, we expressed and purified recombinant KLK14 in mature and proenzyme forms and determined its expression pattern, specificity, regulation, and in vitro substrates. By using our novel immunoassay, the normal and/or diseased skin, breast, prostate, and ovary contained the highest concentration of KLK14. Serum KLK14 levels were significantly elevated in prostate cancer patients compared with healthy males. KLK14 displayed trypsin-like specificity with high selectivity for P1-Arg over Lys. KLK14 activity could be regulated as follows: 1) by autolytic cleavage leading to enzymatic inactivation; 2) by the inhibitory serpins alpha1-antitrypsin, alpha2-antiplasmin, antithrombin III, and alpha1-antichymotrypsin with second order rate constants (k(+2)/Ki) of 49.8, 23.8, 1.48, and 0.224 microM(-1) min(-1), respectively, as well as plasminogen activator inhibitor-1; and 3) by citrate and zinc ions, which exerted stimulatory and inhibitory effects on KLK14 activity, respectively. We also expanded the in vitro target repertoire of KLK14 to include collagens I-IV, fibronectin, laminin, kininogen, fibrinogen, plasminogen, vitronectin, and insulin-like growth factor-binding proteins 2 and 3. Our results indicate that KLK14 may be implicated in several facets of tumor progression, including growth, invasion, and angiogenesis, as well as in arthritic disease via deterioration of cartilage. These findings may have clinical implications for the management of cancer and other disorders in which KLK14 activity is elevated.

Paliouras M, Diamandis EP. · Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Ave., M5G 1X5 Toronto, ON, Canada. · Breast Cancer Res Treat. · Pubmed #16897430 No free full text.

Abstract: The regulation of gene expression by steroid hormones plays an important role in the normal development and function of many organs, as well in the pathogenesis of endocrine-related cancers. Previous experiments have shown that many kallikrein genes are under steroid hormone regulation in breast cancer cell lines. We here examine the coordinated expression of multiple kallikrein genes in several breast cancer cell lines after steroid hormone stimulation. Breast cancer cell lines were treated with various steroid hormones and kallikrein (KLK/hK) expression of hK3 (prostate-specific antigen, PSA), hK5, hK6, hK7, hK8, hK10, hK11, hK13, and hK14 was analyzed at the RNA level via RT-PCR and at the protein level by immunofluorometric ELISA assays. We identified several distinct hK hormone-dependent and hormone-independent expression patterns. Hormone-specific modulation of expression was seen for several kallikreins in BT-474, MCF-7, and T-47D cell lines. hK6 was specifically up-regulated upon estradiol treatment in all three cell lines whereas PSA expression was induced by dihydrotestosterone (DHT) and norgestrel stimulation in BT-474 and T-47D. hK10, hK11, hK13, and hK14 were specifically up-regulated by DHT in T-47D and by estradiol in BT-474 cells. Bioinformatic analysis of upstream proximal promoter sequences for these hKs did not identify any recognizable hormone-response elements (HREs), suggesting that the coordinated activation of these four hKs represents a unique expression "cassette", utilizing a common hormone-dependent mechanism. We conclude that groups of human hKs are coordinately expressed in a steroid hormone-dependent manner. Our data supports clinical observations linking expression of multiple hKs with breast cancer prognosis.