Cellular model systems to study the tumor biological role of kallikrein-related peptidases in ovarian and prostate cancer

Since the identification of the gene family of kallikrein related peptidases (KLKs), their function has been robustly studied at the biochemical level. In vitro biochemical studies have shown that KLK proteases are involved in a number of extracellular processes that initiate intracellular signaling pathways by hydrolysis, as reviewed in Chapters 8, 9, and 15, Volume 1. These events have been associated with more invasive phenotypes of ovarian, prostate, and other cancers. Concomitantly, aberrant expression of KLKs has been associated with poor prognosis of patients with ovarian and prostate cancer (Borgoño and Diamandis, 2004; Clements et al., 2004; Yousef and Diamandis, 2009), with prostate-specific antigen (PSA, KLK3) being a long standing, clinically employed biomarker for prostate cancer (Lilja et al., 2008). Data generated from patient samples in clinical studies, alongwith biochemical activity, suggests that KLKs function in the development and progression of these diseases. To bridge the gap between their function at the molecular level and the clinical need for efficacious treatment and prognostic biomarkers, functional assessment at the in vitro cellular level, using various culture models, is increasing, particularly in a three-dimensional (3D) context (Abbott, 2003; Bissell and Radisky, 2001; Pampaloni et al., 2007; Yamada and Cukierman, 2007).

Markers of cutaneous human papillomavirus infection in individuals with tumor-free skin, actinic keratoses, and squamous cell carcinoma

Separately, actinic keratosis (AK) and cutaneous squamous cell carcinoma (SCC) have been associated with cutaneous human papillomavirus (HPV) infections. To further explore the association between HPV infection and SCC development, we determined markers of cutaneous HPV infection within a single population in persons with precursor lesions (AK), cancerous lesions (SCC), and without. Serum and plucked eyebrow hairs were collected from 57 tumor-free controls, 126 AK, and 64 SCC cases. Presence of HPV L1 and E6 seroreactivity and viral DNA were determined for HPV types 5, 8, 15, 16, 20, 24, and 38. Significant positive associations with increasing severity of the lesions (controls, AK, and SCC, respectively) were observed for overall HPV L1 seropositivity (13%, 26%, and 37%) and for HPV8 (4%, 17%, and 30%). In parallel, the proportion of L1 seropositive individuals against multiple HPV types increased from 14% to 39% and 45%. The overall E6 seroreactivity, however, tended to decline with AK and SCC, especially for HPV8 (21%, 11%, and 2%). HPV DNA positivity was most prevalent in the AK cases (54%) compared with the SCC cases (44%) and the tumor-free controls (40%). Among all participants, there was a positive trend between overall HPV DNA positivity and L1 seropositivity, but not E6 seropositivity. Taken together, our data suggest that cutaneous HPV infections accompanied by detectable HPV DNA in eyebrow hairs and HPV L1 seropositivity, but not E6 seropositivity, are associated with an increased risk of AK and SCC.

Multifocal urothelial cancers with the mutator phenotype are of monoclonal origin and require panurothelial treatment for tumor clearance

Purpose: UC is a disease of the entire urothelium, characterized by multiplicity and multifocality. The clonal relationship among multiple UCs has implications regarding adjuvant chemotherapy. It has been investigated in studies of chromosomal alteration and single gene mutation. However, these genetic changes can occur in unrelated tumors under similar carcinogenic selection pressures. Tumors with high MSI have numerous DNA mutations, of which many provide no selection benefit. While these tumors represent an ideal model for studying UC clonality, their low frequency has prevented their previous investigation. Materials and Methods: We investigated 32 upper and lower urinary tract UCs with high MSI and 4 nonUC primary cancers in 9 patients. We used the high frequency and specificity of individual DNA mutations in these tumors (MSI at 17 loci) and the early timing of epigenetic events (methylation of 7 gene promoters) to investigate tumor clonality. Results: Molecular alterations varied among tumors from different primary organs but they appeared related in the UCs of all 9 patients. While 7 patients had a high degree of concordance among UCs, in 2 the UCs shared only a few similar alterations. Genetic and epigenetic abnormalities were frequently found in normal urothelial samples. Conclusions: Multiple UCs in each patient appeared to arise from a single clone. The molecular order of tumor development varied from the timing of clinical presentation and suggested that residual malignant cells persist in the urinary tract despite apparent curative surgery. These cells lead to subsequent tumor relapse and new methods are required to detect and eradicate them.

Humanised xenograft models of bone metastasis revisited : novel insights into species-specific mechanisms of cancer cell osteotropism

The determinants and key mechanisms of cancer cell osteotropism have not been identified, mainly due to the lack of reproducible animal models representing the biological, genetic and clinical features seen in humans. An ideal model should be capable of recapitulating as many steps of the metastatic cascade as possible, thus facilitating the development of prognostic markers and novel therapeutic strategies. Most animal models of bone metastasis still have to be derived experimentally as most syngeneic and transgeneic approaches do not provide a robust skeletal phenotype and do not recapitulate the biological processes seen in humans. The xenotransplantation of human cancer cells or tumour tissue into immunocompromised murine hosts provides the possibility to simulate early and late stages of the human disease. Human bone or tissue-engineered human bone constructs can be implanted into the animal to recapitulate more subtle, species-specific aspects of the mutual interaction between human cancer cells and the human bone microenvironment. Moreover, the replication of the entire "organ" bone makes it possible to analyse the interaction between cancer cells and the haematopoietic niche and to confer at least a partial human immunity to the murine host. This process of humanisation is facilitated by novel immunocompromised mouse strains that allow a high engraftment rate of human cells or tissue. These humanised xenograft models provide an important research tool to study human biological processes of bone metastasis.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines: targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

CD4+ tumor infiltrating lymphocytes are prognostic and independent of R-IPI in patients with DLBCL receiving R-CHOP chemo-immunotherapy

Despite the Revised International Prognostic Index's (R-IPI) undoubted utility in diffuse large B-cell lymphoma (DLBCL), significant clinical heterogeneity within R-IPI categories persists. Emerging evidence indicates that circulating host immunity is a robust and R-IPI independent prognosticator, most likely reflecting the immune status of the intratumoral microenvironment. We hypothesized that direct quantification of immunity within lymphomatous tissue would better permit stratification within R-IPI categories. We analyzed 122 newly diagnosed consecutive DLBCL patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) chemo-immunotherapy. Median follow-up was 4 years. As expected, the R-IPI was a significant predictor of outcome with 5-year overall survival (OS) 87% for very good, 87% for good, and 51% for poor-risk R-IPI scores (P < 0.001). Consistent with previous reports, systemic immunity also predicted outcome (86% OS for high lymphocyte to monocyte ratio [LMR], versus 63% with low LMR, P = 0.01). Multivariate analysis confirmed LMR as independently prognostic. Flow cytometry on fresh diagnostic lymphoma tissue, identified CD4+ T-cell infiltration as the most significant predictor of outcome with ≥23% infiltration dividing the cohort into high and low risk groups with regard to event-free survival (EFS, P = 0.007) and OS (P = 0.003). EFS and OS were independent of the R-IPI and LMR. Importantly, within very good/good R-IPI patients, CD4+ T-cells still distinguished patients with different 5 year OS (high 96% versus low 63%, P = 0.02). These results illustrate the importance of circulating and local intratumoral immunity in DLBCL treated with R-CHOP.

High-resolution loss of heterozygosity screening implicates PTPRJ as a potential tumor suppressor gene that affects susceptibility to non-Hodgkin's Lymphoma

We employed a Hidden-Markov-Model (HMM) algorithm in loss of heterozygosity (LOH) analysis of high-density single nucleotide polymorphism (SNP) array data from Non-Hodgkin’s lymphoma (NHL) entities, follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). This revealed a high frequency of LOH over the chromosomal region 11p11.2, containing the gene encoding the protein tyrosine phosphatase receptor type J (PTPRJ). Although PTPRJ regulates components of key survival pathways in B-cells (i.e., BCR, MAPK, and PI3K signaling), its role in B-cell development is poorly understood. LOH of PTPRJ has been described in several types of cancer but not in any hematological malignancy. Interestingly, FL cases with LOH exhibited down-regulation of PTPRJ, in contrast no significant variation of expression was shown in DLBCLs. In addition, sequence screening in Exons 5 and 13 of PTPRJ identified the G973A (rs2270993), T1054C (rs2270992), A1182C (rs1566734), and G2971C (rs4752904) coding SNPs (cSNPs). The A1182 allele was significantly more frequent in FLs and in NHLs with LOH. Significant over-representation of the C1054 (rs2270992) and the C2971 (rs4752904) alleles were also observed in LOH cases. A haplotype analysis also revealed a significant lower frequency of haplotype GTCG in NHL cases, but it was only detected in cases with retention. Conversely, haplotype GCAC was over-representated in cases with LOH. Altogether, these results indicate that the inactivation of PTPRJ may be a common lymphomagenic mechanism in these NHL subtypes and that haplotypes in PTPRJ gene may play a role in susceptibility to NHL, by affecting activation of PTPRJ in these B-cell lymphomas.

Differential gene expression in breast cancer cell lines and stroma-tumor differences in microdissected breast cancer biopsies revealed by display array analysis

To examine gene-expression patterning in late-stage breast cancer biopsies, we used a microdissection technique to separate tumor from the surrounding breast tissue or stroma. A DD-PCR protocol was then used to amplify expressed products, which were resolved using PAGE and used as probe to hybridize with representative human arrays and cDNA libraries. The probe derived from the tumor–stroma comparison was hybridized with a gene array and an arrayed cDNA library derived from a GCT of bone; 21 known genes or expressed sequence tags were detected, of which 17 showed differential expression. These included factors associated with epithelial to mesenchymal transition (vimentin), the cargo selection protein (TIP47) and the signal transducer and activator of transcription (STAT3). Northern blot analysis was used to confirm those genes also expressed by representative breast cancer cell lines. Notably, 6 genes of unknown function were restricted to tumor while the majority of stroma-associated genes were known. When applied to transformed breast cancer cell lines (MDA-MB-435 and T47D) that are known to have different metastatic potential, DD array analysis revealed a further 20 genes; 17 of these genes showed differential expression. Use of microdissection and the DD-PCR array protocol allowed us to identify factors whose localized expression within the breast may play a role in abnormal breast development or breast carcinogenesis.

Current status of pharmacogenomics testing for anti-tumor drug therapies : approaches to non-melanoma skin cancer

Skin cancer is one of the most commonly occurring cancer types, with substantial social, physical, and financial burdens on both individuals and societies. Although the role of UV light in initiating skin cancer development has been well characterized, genetic studies continue to show that predisposing factors can influence an individual's susceptibility to skin cancer and response to treatment. In the future, it is hoped that genetic profiles, comprising a number of genetic markers collectively involved in skin cancer susceptibility and response to treatment or prognosis, will aid in more accurately informing practitioners' choices of treatment. Individualized treatment based on these profiles has the potential to increase the efficacy of treatments, saving both time and money for the patient by avoiding the need for extensive or repeated treatment. Increased treatment responses may in turn prevent recurrence of skin cancers, reducing the burden of this disease on society. Currently existing pharmacogenomic tests, such as those that assess variation in the metabolism of the anticancer drug fluorouracil, have the potential to reduce the toxic effects of anti-tumor drugs used in the treatment of non-melanoma skin cancer (NMSC) by determining individualized appropriate dosage. If the savings generated by reducing adverse events negate the costs of developing these tests, pharmacogenomic testing may increasingly inform personalized NMSC treatment.

Identification of TFG (TRK-fused gene) as a putative metastatic melanoma tumor suppressor gene

High density SNP arrays can be used to identify DNA copy number changes in tumors such as homozygous deletions of tumor suppressor genes and focal amplifications of oncogenes. Illumina Human CNV370 Bead chip arrays were used to assess the genome for unbalanced chromosomal events occurring in 39 cell lines derived from stage III metastatic melanomas. A number of genes previously recognized to have an important role in the development and progression of melanoma were identified including homozygous deletions of CDKN2A (13 of 39 samples), CDKN2B (10 of 39), PTEN (3 of 39), PTPRD (3 of 39), TP53 (1 of 39), and amplifications of CCND1 (2 of 39), MITF (2 of 39), MDM2 (1 of 39), and NRAS (1 of 39). In addition, a number of focal homozygous deletions potentially targeting novel melanoma tumor suppressor genes were identified. Because of their likely functional significance for melanoma progression, FAS, CH25H, BMPR1A, ACTA2, and TFG were investigated in a larger cohort of melanomas through sequencing. Nonsynonymous mutations were identified in BMPR1A (1 of 43), ACTA2 (3 of 43), and TFG (5 of 103). A number of potentially important mutation events occurred in TFG including the identification of a mini mutation ‘‘hotspot’’ at amino acid residue 380 (P380S and P380L) and the presence of multiple mutations in two melanomas. Mutations in TFG may have important clinical relevance for current therapeutic strategies to treat metastatic melanoma.

Phase III study of matrix metalloproteinase inhibitor prinomastat in non-small-cell lung cancer

Purpose: Matrix metalloproteinases (MMPs) degrade extracellular proteins and facilitate tumor growth, invasion, metastasis, and angiogenesis. This trial was undertaken to determine the effect of prinomastat, an inhibitor of selected MMPs, on the survival of patients with advanced non-small-cell lung cancer (NSCLC), when given in combination with gemcitabine-cisplatin chemotherapy. Patients and Methods: Chemotherapy-naive patients were randomly assigned to receive prinomastat 15 mg or placebo twice daily orally continuously, in combination with gemcitabine 1,250 mg/m2 days 1 and 8 plus cisplatin 75 mg/m2 day 1, every 21 days for up to six cycles. The planned sample size was 420 patients. Results: Study results at an interim analysis and lack of efficacy in another phase III trial prompted early closure of this study. There were 362 patients randomized (181 on prinomastat and 181 on placebo). One hundred thirty-four patients had stage IIIB disease with T4 primary tumor, 193 had stage IV disease, and 34 had recurrent disease (one enrolled patient was ineligible with stage IIIA disease). Overall response rates for the two treatment arms were similar (27% for prinomastat v 26% for placebo; P = .81). There was no difference in overall survival or time to progression; for prinomastat versus placebo patients, the median overall survival times were 11.5 versus 10.8 months (P = .82), 1-year survival rates were 43% v 38% (P = .45), and progression-free survival times were 6.1 v 5.5 months (P = .11), respectively. The toxicities of prinomastat were arthralgia, stiffness, and joint swelling. Treatment interruption was required in 38% of prinomastat patients and 12% of placebo patients. Conclusion: Prinomastat does not improve the outcome of chemotherapy in advanced NSCLC. © 2005 by American Society of Clinical Oncology.

Bone matters in lung cancer

Background: Bone metastases are a significant and undertreated clinical problem in patients with advanced lung cancer. Design: We reviewed the incidence of bone metastases and skeletal-related events (SREs) in patients with lung cancer and examined the burden on patients' lives and on health care systems. Available therapies to improve survival and lessen the impact of SREs on quality of life (QoL) were also investigated. Results: Bone metastases are common in lung cancer; however, owing to short survival times, data on the incidences of SREs are limited. As with other cancers, the costs associated with treating SREs in lung cancer are substantial. Bisphosphonates reduce the frequency of SREs and improve measures of pain and QoL in patients with lung cancer; however, nephrotoxicity is a common complication of therapy. Denosumab, a recently approved bone-targeted therapy, is superior to zoledronic acid in increasing the time to first on-study SRE in patients with solid tumours, including lung cancer. Additional roles of bone-targeted therapies beyond the prevention of SREs are under investigation. Conclusions: With increasing awareness of the consequences of SREs, bone-targeted therapies may play a greater role in the management of patients with lung cancer, with the aim of delaying disease progression and preserving QoL. © The Author 2012. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

The role of prostacyclin synthase and thromboxane synthase signaling in the development and progression of cancer

Prostacyclin synthase and thromboxane synthase signaling via arachidonic acid metabolism affects a number of tumor cell survival pathways such as cell proliferation, apoptosis, tumor cell invasion and metastasis, and angiogenesis. However, the effects of these respective synthases differ considerably with respect to the pathways described. While prostacyclin synthase is generally believed to be anti-tumor, a pro-carcinogenic role for thromboxane synthase has been demonstrated in a variety of cancers. The balance of oppositely-acting COX-derived prostanoids influences many processes throughout the body, such as blood pressure regulation, clotting, and inflammation. The PGI2/TXA2 ratio is of particular interest in-vivo, with the corresponding synthases shown to be differentially regulated in a variety of disease states. Pharmacological inhibition of thromboxane synthase has been shown to significantly inhibit tumor cell growth, invasion, metastasis and angiogenesis in a range of experimental models. In direct contrast, prostacyclin synthase overexpression has been shown to be chemopreventive in a murine model of the disease, suggesting that the expression and activity of this enzyme may protect against tumor development. In this review, we discuss the aberrant expression and known functions of both prostacyclin synthase and thromboxane synthase in cancer. We discuss the effects of these enzymes on a range of tumor cell survival pathways, such as tumor cell proliferation, induction of apoptosis, invasion and metastasis, and tumor cell angiogenesis. As downstream signaling pathways of these enzymes have also been implicated in cancer states, we examine the role of downstream effectors of PGIS and TXS activity in tumor growth and progression. Finally, we discuss current therapeutic strategies aimed at targeting these enzymes for the prevention/treatment of cancer. © 2010 Elsevier B.V. All rights reserved.

Matrix metalloproteinases 2 and 9 (gelatinases A and B) expression in malignant mesothelioma and benign pleura

Matrix metalloproteinases (MMPs), in particular the gelatinases (MMP-2 and -9), play a significant role in tumour invasion and angiogenesis. The expression and activities of MMPs have not been characterised in malignant mesothelioma (MM) tumour samples. In a prospective study, gelatinase activity was evaluated in homogenised supernatants of snap frozen MM (n = 35), inflamed pleura (IP, n = 12) and uninflammed pleura (UP, n = 14) tissue specimens by semiquantitative gelatin zymography. Matrix metalloproteinases were correlated with clinicopathological factors and with survival using Kaplan-Meier and Cox proportional hazard models. In MM, pro- and active MMP-2 levels were significantly greater than for MMP-9 (P = 0.006, P<0.001). Active MMP-2 was significantly greater in MM than in UP (P=0.04). MMP-2 activity was equivalent between IP and MM, but both pro- and active MMP-9 activities were greater in IP (P=0.02, P=0.009). While there were trends towards poor survival with increasing total and pro-MMP-2 activity (P=0.08) in univariate analysis, they were both independent poor prognostic factors in multivariate analysis in conjunction with weight loss (pro-MMP-2 P = 0.03, total MMP-2 P = 0.04). Total and pro-MMP-2 also contributed to the Cancer and Leukemia Group B prognostic groups. MMP-9 activities were not prognostic. Matrix metalloproteinases, and in particular MMP-2, the most abundant gelatinase, may play an important role in MM tumour growth and metastasis. Agents that reduce MMP synthesis and/or activity may have a role to play in the management of MM. © 2003 Cancer Research UK.

Tumor necrosis correlates with angiogenesis and is a predictor of poor prognosis in malignant mesothelioma

Objectives: Malignant mesothelioma (MM) is a fatal tumor of increasing incidence related to asbestos exposure. Microscopic tumor necrosis (TN) is a poor prognostic factor in solid tumors, but it has not been characterized in MM. We wished to evaluate the incidence of TN in MM and its correlations with clinicopathologic factors, angiogenesis, and survival. Methods: TN was graded in 171 routine formalin-fixed, paraffin-embedded hematoxylin-eosinstained tumor sections by two independent observers. Angiogenesis was assessed by the microvessel count (MVC) of CD34 immunostained sections. TN was correlated with survival by Kaplan-Meier and log-rank analysis, and stepwise, multivariate Cox models were used to compare TN with angiogenesis and established prognostic factors and prognostic scoring systems. Results: TN was identified in 39 cases (22.8%) and correlated with low hemoglobin (p = 0.01), thrombocytosis (p = 0.04), and high MVC (p = 0.02). TN was a poor prognostic factor in univariate analysis (p = 0.008). Patients with TN had a median survival of 5.3 months vs 8.3 months in negative cases. Independent indicators of poor prognosis in multivariate analysis were nonepithelioid cell type (p = 0.0001), performance status > 0 (p = 0.007), and increasing MVC (p = 0.004) but not TN. TN contributed independently to the European Organisation for Research and Treatment of Cancer (EORTC) [p = 0.03] and to the Cancer and Leukemia Group B (CALGB) [p = 0.03] prognostic groups in respective multivariate Cox analyses. Conclusions: TN correlates with angiogenesis and is a poor prognostic factor in MM. TN contributes to the EORTC and CALGB prognostic scoring systems.