Higher human kallikrein gene 4 (KLK4) expression indicates poor prognosis of ovarian cancer patients.

PURPOSE: Kallikrein gene 4 (KLK4, also known as prostase/KLK-L1), located on chromosome 19q13.4, is one of the newly discovered members of the human KLK-like gene family. This gene is up-regulated by androgens in the LNCaP prostatic carcinoma cell line and by androgens and progestins in the BT-474 breast cancer cell line. On the basis of its apparent association with hormonally regulated tissues, we have undertaken to examine the prognostic value of KLK4 expression in 147 malignant ovarian tissues. EXPERIMENTAL DESIGN: Tumors were pulverized, total RNA was extracted, and cDNA was prepared by reverse transcription. KLK4 was amplified by PCR using gene-specific primers, and its identity was verified by sequencing. Ovarian tissues were then classified as KLK4-positive or -negative, based on ethidium bromide visualization of the PCR product on agarose gels. RESULTS: KLK4 was found to be expressed in 69 (55%) of 147 of ovarian cancer samples. We found a strong positive association between KLK4 expression and tumor grade (P = 0.02) and clinical stage (P < 0.001). Univariate survival analysis revealed that patients with ovarian tumors positive for KLK4 expression had an increased risk for relapse and death (P = 0.003 and 0.001, respectively). Whereas knowledge of KLK4 status did not significantly increase the prognostic power of the multivariate models, additional analyses did determine that KLK4 was an independent unfavorable prognostic factor in patients with grade 1 and 2 tumors. CONCLUSIONS: Our findings indicate that KLK4 expression is associated with more aggressive forms of ovarian cancer.

Prostate-specific antigen and human glandular kallikrein 2 are markedly elevated in urine of patients with polycystic ovary syndrome.

Prostate-specific antigen (PSA) is a well-established tumor marker of prostatic adenocarcinoma. Human glandular kallikrein 2 (hK2), another serine protease closely related to PSA, is also gaining ground as a promising diagnostic tool in prostate cancer. The expression of these 2 proteases is known to be regulated by androgens and progestins in hormonally responsive tissues, such as the male prostate and the female breast. Previously, we have shown that serum PSA levels in normal women are very low but still detectable by ultrasensitive PSA immunoassays. We have also demonstrated that some women with hyperandrogenic syndromes have elevated serum PSA levels. In this study, we have measured urinary PSA and urinary hK2 levels in 35 polycystic ovary syndrome (PCOS) patients and compared them to those of 41 age-matched controls. We found that urinary PSA levels were significantly higher (P < 0.0001) in PCOS patients (mean +/- SE = 820 +/- 344 ng/L) than in the controls (mean +/- SE = 4.3 +/- 1.8 ng/L). Similarly, the difference between urinary hK2 of patients (mean +/- SE = 8.2 +/- 3.1 ng/L) and controls (0.5 +/- 0.3 ng/L) was also significant (P < 0.001). A weak correlation was observed between urinary PSA and serum 3 alpha-androstanediol glucuronide (r(s) = 0.42, P = 0.03) as well as between urinary PSA and serum testosterone (r(s) = 0.40, P = 0.04). The results of this study indicate that urinary PSA, and possibly urinary hK2, are promising markers of hyperandrogenism in females suffering from PCOS.

Serum and urinary prostate-specific antigen and urinary human glandular kallikrein concentrations are significantly increased after testosterone administration in female-to-male transsexuals.

BACKGROUND: The genes that encode prostate-specific antigen (PSA) and human glandular kallikrein (hK2) are up-regulated by androgens and progestins in cultured cells, but no published studies have described the effect of androgen administration in women on serum and urinary PSA or hK2. METHODS: We measured serum and urinary PSA and hK2 before, and 4 and 12 months post testosterone treatment by immunofluorometric methods in 32 female-to-male transsexuals. RESULTS: Mean serum PSA increased from 1.1 ng/L to 11.1 ng/L and then to 22 ng/L by 4 and 12 months post treatment, respectively; the corresponding mean values in urine were 17, 1420, and 18 130 ng/L, respectively. Serum hK2, another kallikrein closely related to PSA, remained undetectable at the three time points. However, urinary hK2 concentration rose from below the detection limit (<6 ng/L) before treatment to 18 and 179 ng/L by the 4th and the 12th month of treatment, respectively. All changes were statistically significant (P <0.001) at 4 months. CONCLUSIONS: Testosterone administration increases serum and urinary PSA and urinary hK2 in women. These measurements may be useful as indicators of androgenic stimulation in women.

The new human kallikrein gene family: implications in carcinogenesis.

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.

Dramatic suppression of plasma and urinary prostate specific antigen and human glandular kallikrein by antiandrogens in male-to-female transsexuals.

PURPOSE: Prostate specific antigen (PSA) and human glandular kallikrein (hK2) are mainly produced by the prostate and their genes are regulated by androgens through the androgen receptor. We determine whether PSA and hK2 change significantly in plasma and urine after antiandrogen treatment in male-to-female transsexuals. MATERIALS AND METHODS: Plasma and urine PSA and hK2 were measured with highly sensitive immunofluorometric procedures capable of detecting within 1 or 6 ng./l. PSA or hK2, respectively. Study groups consisted of 10 men treated with cyproterone acetate only (group 1), 15 transdermal estradiol plus cyproterone acetate (group 2) and 31 ethinyl estradiol plus cyproterone acetate (group 3). Plasma and urine samples were collected before initiation of treatment as well as after 4 months of hormonal therapy. For a subset of group 3 patients blood and urine samples were also obtained after 12 months of treatment. RESULTS: Cyproterone acetate, a steroidal antiandrogen, alone or with estradiol was able to suppress greater than 90% of plasma and urinary PSA and hK2 concentration after 4 or 12 months of therapy. CONCLUSIONS: Cyproterone acetate therapy causes dramatic suppression of plasma and urinary PSA and hK2 in men without prostate cancer. Since cyproterone acetate is used for prostate cancer treatment, suppression of PSA after hormonal therapy may not accurately reflect therapy success in reducing tumor burden.

Expression of a prostate-associated protein, human glandular kallikrein (hK2), in breast tumours and in normal breast secretions.

The recent demonstration of human glandular kallikrein (hK2) expression in a breast carcinoma cell line has suggested that this putatively prostate-restricted, steroid hormone-regulated protease may also be expressed in breast epithelium in vivo and secreted into the mammary duct system. Given that the only substrate yet identified for hK2 activity is the precursor of prostate-specific antigen (PSA), the expression of which in breast carcinomas may be associated with favourable prognosis, our purpose was to examine the expression pattern of both hK2 and PSA in breast tumour tissues. Cytosolic extracts of 336 primary breast carcinomas prepared for routine oestrogen receptor (ER) and progesterone receptor (PR) analysis, as well as 31 nipple aspirates from six women with non-diseased mammary glands, were assayed for hK2 and PSA using immunofluorometric assays developed by the authors. In the tumour extracts, measurable hK2 and PSA concentrations were detected in 53% and 73% of cases respectively, and were positively correlated to each other (r = 0.59, P = 0.0001). Higher concentrations of PSA and hK2 were found in tumours expressing steroid hormone receptors (P = 0.0001 for PSA and P = 0.0001 for hK2, by Wilcoxon tests for both ER and PR), and both PSA (r = 0.25, P = 0.0001) and hK2 (r = 0.22, P = 0.0001) correlated directly with PR levels. A negative correlation between patient age and PSA (r = -0.12, P = 0.03) was also found. Both proteins were present in nipple aspirate fluid at relatively high concentrations which were positively correlated (r = 0.53, P = 0.002). The molecular weights of the immunoreactive species quantified by the hK2 and PSA assays were established by high-performance liquid chromatography (HPLC) and were consistent with the known molecular weights of hK2 and PSA. Together these data provide the first evidence, to our knowledge, that both malignant breast tissue and normal breast secretion contain measurable quantities of hK2, and that the degree of hK2 expression or secretion is directly proportional to the expression of PSA and steroid hormone receptors. hK2 expression may therefore be a marker of steroid hormone action in breast tissue.

Prostase/KLK-L1 is a new member of the human kallikrein gene family, is expressed in prostate and breast tissues, and is hormonally regulated.

By using the positional candidate gene approach, we were able to identify a novel serine protease gene that maps to chromosome 19q13.3-q13.4. Screening of expressed sequence tags allowed us to establish the expression of the gene and delineate its genomic organization (GenBank accession no. AF135023). We named this gene KLK-L1. Another group, by using a subtraction hybridization method, cloned the same gene and named it prostase (GenBank accession nos. AF113140 and AF113141). Here, we describe the precise mapping and localization of the prostase/KLK-L1 gene between the known genes KLK2 (human glandular kallikrein) and zyme (also known as protease M/neurosin). The direction of transcription of prostase/KLK-L1 is the same as that of zyme but opposite to that of KLK2 and prostate-specific antigen genes. Contrary to the initial impression, prostase/KLK-L1 is expressed at high levels not only in prostate tissue but also in testis, mammary gland, adrenals, uterus, thyroid, and salivary glands. We have further demonstrated with in vitro experiments with the breast carcinoma cell line BT-474 that this gene is expressed and that its expression is up-regulated by androgens and progestins. On the basis of information on other genes that are localized in the same region (prostate-specific antigen, KLK2, zyme, and normal epithelial cell specific-1 gene), we speculate that prostase/KLK-L1 may be involved in the pathogenesis and/or progression of prostate, breast, and possibly other malignancies.

Development of an ultrasensitive immunoassay for human glandular kallikrein with no cross-reactivity from prostate-specific antigen.

BACKGROUND: Studies demonstrating that human glandular kallikrein (hK2) is increased in prostate cancer patients have prompted speculation that this marker may of use in addition to prostate-specific antigen (PSA). METHODS: An ultrasensitive hK2 sandwich immunoassay was developed, and its detection limit, cross-reactivity, analytical recovery, precision, and linearity of dilution were evaluated. hK2 was measured in seminal plasma and sera from healthy males, females, and prostatectomized patients. RESULTS: Our assay has an excellent detection limit (6 ng/L) and precision (>90%). Recovery studies indicated that hK2 binds to serum protease inhibitors. All sera from healthy males had measurable hK2 concentrations (median, 402 ng/L). Almost all female sera had undetectable hK2. Serum hK2 and PSA in males correlated positively (r = 0.44), but hK2 was present at concentrations approximately 2. 5-fold lower than PSA. The PSA/hK2 ratio in male sera was 0.1-34, with a median of 2.6. In seminal plasma, this ratio was 100-500. More than 94% of immunoreactive hK2 in serum was in the free form ( approximately 30 kDa); traces of hK2 complexed to alpha1-antichymotrypsin were present. CONCLUSIONS: The limit of detection of the method for hK2 measurement described here ( approximately 20-fold lower than any other reported assay for hK2) allows the generation of new clinical information. When combined with a previously described method for PSA measurement that has no cross-reactivity from hK2, this methods allows the relative proportions of hK2 and PSA in biological fluids to be measured.