Characterization of a novel radiation-induced sarcoma cell line.

BACKGROUND: Radiation-induced sarcoma (RIS) is a potential complication of cancer treatment. No widely available cell line models exist to facilitate studies of RIS. METHODS: We derived a spontaneously immortalized primary human cell line, UACC-SARC1, from a RIS. RESULTS: Short tandem repeat (STR) profiling of UACC-SARC1 was virtually identical to its parental tumor. Immunohistochemistry (IHC) analysis of the tumor and immunocytochemistry (ICC) analysis of UACC-SARC1 revealed shared expression of vimentin, osteonectin, CD68, Ki67 and PTEN but tumor-restricted expression of the histiocyte markers α1-antitrypsin and α1-antichymotrypsin. Karyotyping of the tumor demonstrated aneuploidy. Comparative genomic hybridization (CGH) provided direct genetic comparison between the tumor and UACC-SARC1. Sequencing of 740 mutation hotspots revealed no mutations in UACC-SARC1 nor in the tumor. NOD/SCID gamma mouse xenografts demonstrated tumor formation and metastasis. Clonogenicity assays demonstrated that 90% of single cells produced viable colonies. NOD/SCID gamma mice produced useful patient-derived xenografts for orthotopic or metastatic models. CONCLUSION: Our novel RIS strain constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. UACC-SARC1 is an aneuploid cell line with complex genomics lacking common oncogenes or tumor suppressor genes as drivers of its biology. The UACC-SARC1 cell line will enable further studies of the drivers of RIS.

Randomized, double-blind, placebo-controlled trial of sulindac in individuals at risk for melanoma: evaluation of potential chemopreventive activity.

BACKGROUND: Reduced melanoma risk has been reported with regular use of nonsteroidal anti-inflammatory drugs (NSAIDs). However, the ability of NSAIDs to reach melanocytes in vivo and modulate key biomarkers in preneoplastic lesions such as atypical nevi has not been evaluated. METHODS: This randomized, double-blind, placebo-controlled trial of sulindac was conducted in individuals with atypical nevi (AN) to determine bioavailability of sulindac and metabolites in nevi and effect on apoptosis and vascular endothelial growth factor A (VEGFA) expression in AN. Fifty subjects with AN ≥ 4 mm in size and 1 benign nevus (BN) were randomized to sulindac (150 mg twice a day) or placebo for 8 weeks. Two AN were randomized for baseline excision, and 2 AN and BN were excised after intervention. RESULTS: Postintervention sulindac, sulindac sulfone, and sulindac sulfide concentrations were 0.31 ± 0.36, 1.56 ± 1.35, and 2.25 ± 2.24 µg/mL in plasma, and 0.51 ± 1.05, 1.38 ± 2.86, and 0.12 ± 0.12 µg/g in BN, respectively. Sulindac intervention did not significantly change VEGFA expression but did increase expression of the apoptotic marker cleaved caspase-3 in AN (increase of 3 ± 33 in sulindac vs decrease of 25 ± 45 in the placebo arm, P = .0056), although significance was attenuated (P = .1103) after adjusting for baseline expression. CONCLUSIONS: Eight weeks of sulindac intervention resulted in high concentrations of sulindac sulfone, a proapoptotic metabolite, in BN but did not effectively modulate VEGFA and cleaved caspase-3 expression. Study limitations included limited exposure time to sulindac and the need to optimize a panel of biomarkers for NSAID intervention studies.

Integrins and Epithelial-Mesenchymal Cooperation in the Tumor Microenvironment of Muscle-Invasive Lethal Cancers.

Muscle-invasive lethal carcinomas traverse into and through this specialized biophysical and growth factor enriched microenvironment. We will highlight cancers that originate in organs surrounded by smooth muscle, which presents a barrier to dissemination, including prostate, bladder, esophageal, gastric, and colorectal cancers. We propose that the heterogeneity of cell-cell and cell-ECM adhesion receptors is an important driver of aggressive tumor networks with functional consequences for progression. Phenotype heterogeneity of the tumor provides a biophysical advantage for tumor network invasion through the tensile muscle and survival of the tumor network. We hypothesize that a functional epithelial-mesenchymal cooperation (EMC)exists within the tumor invasive network to facilitate tumor escape from the primary organ, invasion and traversing of muscle, and navigation to metastatic sites. Cooperation between specific epithelial cells within the tumor and stromal (mesenchymal) cells interacting with the tumor is illustrated using the examples of laminin-binding adhesion molecules-especially integrins-and their response to growth and inflammatory factors in the tumor microenvironment. The cooperation between cell-cell (E-cadherin, CDH1) and cell-ECM (α6 integrin, CD49f) expression and growth factor receptors is highlighted within poorly differentiated human tumors associated with aggressive disease. Cancer-associated fibroblasts are examined for their role in the tumor microenvironment in generating and organizing various growth factors. Cellular structural proteins are potential utility markers for future spatial profiling studies. We also examine the special characteristics of the smooth muscle microenvironment and how invasion by a primary tumor can alter this environment and contribute to tumor escape via cooperation between epithelial and stromal cells. This cooperative state allows the heterogenous tumor clusters to be shaped by various growth factors, co-opt or evade immune system response, adapt from hypoxic to normoxic conditions, adjust to varying energy sources, and survive radiation and chemotherapeutic interventions. Understanding the epithelial-mesenchymal cooperation in early tumor invasive networks holds potential for both identifying early biomarkers of the aggressive transition and identification of novel agents to prevent the epithelial-mesenchymal cooperation phenotype. Epithelial-mesenchymal cooperation is likely to unveil new tumor subtypes to aid in selection of appropriate therapeutic strategies.

Association of long-term administration of the survivin mRNA-targeted antisense oligonucleotide LY2181308 with reversible kidney injury in a patient with metastatic melanoma.

A 57-year-old man with metastatic melanoma was treated with the survivin inhibitor and antisense oligonucleotide LY2181308 as part of a First-in-Human Dose trial. After 18 months of treatment, he developed kidney injury and the treatment was discontinued. At 9 months and before the development of kidney injury, LY2181308 concentrations were 8- to 10-fold higher relative to median predicted values, but within the targeted exposure considered to be safe. However, at 17 months, 28 days after stopping LY2181308 therapy, LY2181308 concentration exceeded the predicted range by 38-fold. His decreased kidney function was slow to improve after stopping treatment. A kidney biopsy showed signs of acute tubular injury with regeneration. Complete recovery of kidney function occurred 6 months after treatment was stopped. The relationship between high exposures and slow LY2181308 clearance with the gradual improvement in kidney function after stopping the antisense treatment suggests that the oligonucleotide was related to the kidney injury. Based on this case report, kidney function should be monitored frequently in patients receiving long-term treatment with antisense oligonucleotides that specifically target survivin, particularly when they receive concomitant angiotensin-converting enzyme inhibitors or nonsteroidal anti-inflammatory drugs.

Glyoxalase 1 Expression as a Novel Diagnostic Marker of High-Grade Prostatic Intraepithelial Neoplasia in Prostate Cancer.

Glyoxalase 1 (GLO1) is an enzyme involved in the detoxification of methylglyoxal (MG), a reactive oncometabolite formed in the context of energy metabolism as a result of high glycolytic flux. Prior clinical evidence has documented GLO1 upregulation in various tumor types including prostate cancer (PCa). However, GLO1 expression has not been explored in the context of PCa progression with a focus on high-grade prostatic intraepithelial neoplasia (HGPIN), a frequent precursor to invasive cancer. Here, we have evaluated GLO1 expression by immunohistochemistry in archival tumor samples from 187 PCa patients (stage 2 and 3). Immunohistochemical analysis revealed GLO1 upregulation during tumor progression, observable in HGPIN and PCa versus normal prostatic tissue. GLO1 upregulation was identified as a novel hallmark of HGPIN lesions, displaying the highest staining intensity in all clinical patient specimens. GLO1 expression correlated with intermediate-high risk Gleason grade but not with patient age, biochemical recurrence, or pathological stage. Our data identify upregulated GLO1 expression as a molecular hallmark of HGPIN lesions detectable by immunohistochemical analysis. Since current pathological assessment of HGPIN status solely depends on morphological features, GLO1 may serve as a novel diagnostic marker that identifies this precancerous lesion.

The effect of difluoromethylornithine on decreasing prostate size and polyamines in men: results of a year-long phase IIb randomized placebo-controlled chemoprevention trial.

BACKGROUND: Prostate cancer is a major health issue, and prevention of prostate cancer and/or its progression will yield benefits for men. Difluoromethylornithine (DFMO) is an antiproliferative agent, inhibiting ornithine decarboxylase, the first enzyme in the polyamine pathway, and has been studied as a therapeutic and chemopreventive agent. The prostate has high levels of tissue polyamines and has shown sensitivity to DFMO both in vitro and in vivo. METHODS: Eighty-one men participated in a 1-year randomized trial of placebo or DFMO. Prostate volume determination and biopsy of the prostate for histology and polyamine content were done at baseline and after 12 months. Other biomarker variables were assessed, including total and free prostate-specific antigen and prostate-specific antigen doubling time. RESULTS: Compared with baseline, men receiving DFMO had a smaller increase in prostate volume (0.14 cm(3)) than those on placebo (2.95 cm(3); P = 0.0301) at 1 year. In addition, DFMO caused a 60.8% reduction of prostate putrescine levels compared with a 139.5% increase in the placebo arm (P = 0.0014). Stratification by ornithine decarboxylase genotype showed that DFMO reduced prostate volume (P = 0.029) and putrescine levels (P = 0.0053) in the AA + GA group but not in the GG group. There were no grade 3 or 4 toxicities. There was no clinical ototoxicity, with one subclinical grade 2 hearing decline on audiogram. CONCLUSION: In this randomized placebo-controlled trial, DFMO induced a decrease of prostate putrescine levels and rate of prostate growth. The potential of this compound for prostate cancer or hyperplasia should be further studied.

Breast cancer among Hispanic and non-Hispanic White women in Arizona.

BACKGROUND: Breast cancer in Hispanic women is poorly understood and data on tumor hormone receptor status in this population are limited. METHODS: Using data from the Arizona Cancer Registry, we assessed differences in tumor characteristics between Hispanic and non-Hispanic White (NHW) women using logistic regression modeling. 25,494 invasive breast cancer cases (23,657 NHWs and 1,837 Hispanics) reported to the cancer registry in 1995 to 2003 were included in the analysis. RESULTS: In age-adjusted models, compared with NHW women, Hispanics were more likely to have high-grade cancers, larger tumors, a greater number of positive lymph nodes, and advanced stage at diagnosis. Hispanic women were less likely to have tumors that are both estrogen and progesterone receptor positive (ER+/PR+), particularly those under age 60. CONCLUSIONS: The profile of tumor presentation in Hispanic women in Arizona is consistent with a more aggressive disease pattern and less favorable prognosis than that of NHWs.

Inhibition of Ciliogenesis Promotes Hedgehog Signaling, Tumorigenesis, and Metastasis in Breast Cancer.

Primary cilia are chemosensors that play a dual role to either activate or repress Hedgehog signaling, depending on presence or absence of ligand, respectively. While inhibition of ciliogenesis has been shown to be characteristic of breast cancers, the functional consequence is unknown. Here, for the first time, inhibition of ciliogenesis led to earlier tumor formation, faster tumor growth rate, higher grade tumor formation, and increased metastasis in the polyoma middle T (PyMT) mouse model of breast cancer. In in vitro model systems, inhibition of ciliogenesis resulted in increased expression of Hedgehog-target genes through a mechanism involving loss of the repressor form of the GLI transcription factor (GLIR) and activation of Hedgehog target gene expression through cross-talk with TGF-alpha (TGFA) signaling. Bioinformatics analysis revealed that increased Hedgehog signaling is frequently associated with increased TGFA; signaling in patients with triple-negative breast cancers (TNBC), a particularly aggressive breast cancer subtype. These results identify a previously unrecognized role for inhibition of ciliogenesis in breast cancer progression. This study identifies inhibition of ciliogenesis as an important event for activation of Hedgehog signaling and progression of breast cancer to a more aggressive, metastatic disease.Implications: These findings change the way we understand how cancer cells turn on a critical signaling pathways and a provide rationale for developing novel therapeutic approaches to target noncanonical Hedgehog signaling for the treatment of breast cancer. Mol Cancer Res; 15(10); 1421-30. ©2017 AACR.

A pilot study exploring the molecular architecture of the tumor microenvironment in human prostate cancer using laser capture microdissection and reverse phase protein microarray.

The cross-talk between tumor epithelium and surrounding stromal/immune microenvironment is essential to sustain tumor growth and progression and provides new opportunities for the development of targeted treatments focused on disrupting the tumor ecology. Identification of novel approaches to study these interactions is of primary importance. Using laser capture microdissection (LCM) coupled with reverse phase protein microarray (RPPA) based protein signaling activation mapping we explored the molecular interconnection between tumor epithelium and surrounding stromal microenvironment in 18 prostate cancer (PCa) specimens. Four specimen-matched cellular compartments (normal-appearing epithelium and its adjacent stroma, and malignant epithelium and its adjacent stroma) were isolated for each case. The signaling network analysis of the four compartments unraveled a number of molecular mechanisms underlying the communication between tumor cells and stroma in the context of the tumor microenvironment. In particular, differential expression of inflammatory mediators like IL-8 and IL-10 by the stroma cells appeared to modulate specific cross-talks between the tumor cells and surrounding microenvironment.

Laminin-5 beta3A expression in LNCaP human prostate carcinoma cells increases cell migration and tumorigenicity.

Interactions between extracellular matrix proteins and prostate carcinoma cells change dramatically during prostate tumor progression. We have concentrated on two key modifications that occur in the hemidesmosome in prostate carcinoma: loss of laminin-5 protein expression and altered basal cell polarity of the alpha6beta4 integrin. We previously demonstrated two cell line-specific isoforms (beta3A and beta3B) of the LAMB3 message. Cells expressing only the beta3B isoform did not translate the beta3 protein and were unable to assemble the laminin-5 trimer. One such cell line, LNCaP, was selected to determine whether restoration of the laminin-5 beta3A isoform would cause expression of a functional laminin-5 beta3 chain, assembly and secretion of the laminin-5 trimer, and reversion to a non-neoplastic phenotype. Laminin-5 beta3A cDNA was cloned and stably transfected into LNCaP cells. We observed the restoration of the beta3 protein, but a laminin-5 trimer was not secreted. Moreover, increased cell migration was demonstrated, and tumorigenicity was increased in SCID mice. A microarray analysis, performed between transfected and nontransfected LNCaP cells, showed most changing genes to be associated with signal transduction. The beta3 chain of laminin-5 may thus play an important role in signal transduction, which may enhance cell motility and tumorigenesis.

Cell line-specific translation of two laminin 5 beta3 chain isoforms.

In sequencing the beta3 chain of laminin 5 mRNA from LNCaP cells, we observed three different human cDNA clones (XM_001716, NM_000228 and L25541) in the GenBank that identified different sequences in the untranslated regions (UTR). XM_001716 and NM_000228 are almost identical cDNA clones with approximately 99% homology. However, they are quite different from L25541 in both the 5' UTR and the 3' UTR. Development of a PCR assay to specifically detect two of these different forms of the message led to the observation that they were differentially expressed in various cell lines. The message designated B3A (NM_000228, and XM_001716) was absent in LNCaP and MCF7 and greatly reduced in PC3-N, but was present in eight other epithelial cell lines. B3B (L25541) was present in all cell lines studied. The cell lines that failed to express the B3A form also failed to express the protein based on both immunoblotting and immunohistochemical analysis. It appears from this data that there are two isoforms of the beta3 mRNA, and that the 5' UTRs of the mRNAs play an important role in regulating translation of the beta3 protein. Since laminin 5 is lost in prostate carcinoma, the mechanism of control that results in the translation of the two forms of message may be important in tumorigenesis.

Loss of primary cilia occurs early in breast cancer development.

BACKGROUND: Primary cilia are microtubule-based organelles that protrude from the cell surface. Primary cilia play a critical role in development and disease through regulation of signaling pathways including the Hedgehog pathway. Recent mouse models have also linked ciliary dysfunction to cancer. However, little is known about the role of primary cilia in breast cancer development. Primary cilia expression was characterized in cancer cells as well as their surrounding stromal cells from 86 breast cancer patients by counting cilia and measuring cilia length. In addition, we examined cilia expression in normal epithelial and stromal cells from reduction mammoplasties as well as histologically normal adjacent tissue for comparison. RESULTS: We observed a statistically significant decrease in the percentage of ciliated cells on both premalignant lesions as well as in invasive cancers. This loss of cilia does not correlate with increased proliferative index (Ki67-positive cells). However, we did detect rare ciliated cancer cells present in patients with invasive breast cancer and found that these express a marker of basaloid cancers that is associated with poor prognosis (Cytokeratin 5). Interestingly, the percentage of ciliated stromal cells associated with both premalignant and invasive cancers decreased when compared to stromal cells associated with normal tissue. To understand how cilia may be lost during cancer development we analyzed the expression of genes required for ciliogenesis and/or ciliary function and compared their expression in normal versus breast cancer samples. We found that expression of ciliary genes were frequently downregulated in human breast cancers. CONCLUSIONS: These data suggest that primary cilia are lost early in breast cancer development on both the cancer cells and their surrounding stromal cells.

Immunohistochemical quantification of the vitamin B12 transport protein (TCII), cell surface receptor (TCII-R) and Ki-67 in human tumor xenografts.

BACKGROUND/AIM: Cancer cells have an essential demand for vitamin B12 (cobalamin) to enable cellular replication. The present pilot study quantified the immunohistochemical expression of vitamin B12 transport protein (Transcobalamin II; TCII), cell surface receptor (Transcobalamin II-R; TCII-R) and proliferation protein (Ki-67) in human tumor xenografts. MATERIALS AND METHODS: Tissue microarray slides containing 34 xenograft tumor tissues were immunohistochemically stained using TCN2 (anti-TCII), CD320 (anti-TCII-R) and MIB-1 (anti-Ki-67) antibodies. Representatively stained areas of all slides were digitally imaged and protein expression was quantified using ImageJ software plugins. RESULTS: All xenograft tumor tissues stained positively for TCII, TCII-R and Ki-67 proteins; expression varied both within and between tumor types. Correlation between TCII/TCII-R and Ki-67 expression was not significant in xenograft tissues. CONCLUSION: Proliferating cancer cells express measurable levels of TCII and TCII-R. Immunohistochemical quantification of these markers may be useful as a tool for detection of tumors, tailored selection of anti-tumor therapies and surveillance for evidence of recurrent disease.

Primary cilia are lost in preinvasive and invasive prostate cancer.

Prostate cancer is the second most commonly diagnosed cancer in men worldwide. Little is known about the role of primary cilia in preinvasive and invasive prostate cancer. However, reduced cilia expression has been observed in human cancers including pancreatic cancer, renal cell carcinoma, breast cancer, cholangiocarcinoma, and melanoma. The aim of this study was to characterize primary cilia expression in preinvasive and invasive human prostate cancer, and to investigate the correlation between primary cilia and the Wnt signaling pathway. Human prostate tissues representative of stages of prostate cancer formation (normal prostate, prostatic intraepithelial neoplasia (PIN), and invasive prostate cancer (including perineural invasion)) were stained for ciliary proteins. The frequency of primary cilia was determined. A decrease in the percentage of ciliated cells in PIN, invasive cancer and perineural invasion lesions was observed when compared to normal. Cilia lengths were also measured to indirectly test functionality. Cilia were shorter in PIN, cancer, and perineural invasion lesions, suggesting dysfunction. Primary cilia have been shown to suppress the Wnt pathway. Increased Wnt signaling has been implicated in prostate cancer. Therefore, we investigated a correlation between loss of primary cilia and increased Wnt signaling in normal prostate and in preinvasive and invasive prostate cancer. To investigate Wnt signaling in our cohort, serial tissue sections were stained for ß-catenin as a measure of Wnt signaling. Nuclear ß-catenin was analyzed and Wnt signaling was found to be higher in un-ciliated cells in the normal prostate, PIN, a subset of invasive cancers, and perineural invasion. Our results suggest that cilia normally function to suppress the Wnt signaling pathway in epithelial cells and that cilia loss may play a role in increased Wnt signaling in some prostate cancers. These results suggest that cilia are dysfunctional in human prostate cancer, and increase Wnt signaling occurs in a subset of cancers.

Human breast tissue disposition and bioactivity of limonene in women with early-stage breast cancer.

Limonene is a bioactive food component found in citrus peel oil that has shown chemopreventive and chemotherapeutic activities in preclinical studies. We conducted an open-label pilot clinical study to determine the human breast tissue disposition of limonene and its associated bioactivity. We recruited 43 women with newly diagnosed operable breast cancer electing to undergo surgical excision to take 2 grams of limonene daily for two to six weeks before surgery. Blood and breast tissue were collected to determine drug/metabolite concentrations and limonene-induced changes in systemic and tissue biomarkers of breast cancer risk or carcinogenesis. Limonene was found to preferentially concentrate in the breast tissue, reaching high tissue concentration (mean = 41.3 µg/g tissue), whereas the major active circulating metabolite, perillic acid, did not concentrate in the breast tissue. Limonene intervention resulted in a 22% reduction in cyclin D1 expression (P = 0.002) in tumor tissue but minimal changes in tissue Ki67 and cleaved caspase-3 expression. No significant changes in serum leptin, adiponectin, TGF-ß1, insulin-like growth factor binding protein-3 (IGFBP-3), and interleukin-6 (IL-6) levels were observed following limonene intervention. There was a small but statistically significant postintervention increase in insulin-like growth factor I (IGF-I) levels. We conclude that limonene distributed extensively to human breast tissue and reduced breast tumor cyclin D1 expression that may lead to cell-cycle arrest and reduced cell proliferation. Furthermore, placebo-controlled clinical trials and translational research are warranted to establish limonene's role for breast cancer prevention or treatment.

CD44 Promotes Epithelial Mammary Gland Development and Exhibits Altered Localization during Cancer Progression.

The basal cell layer has emerged as a critical player in cancer progression, and understanding the molecular contribution of specific cell types is important in treatment and prevention. The adhesion receptor CD44, which mediates epithelial-stromal and cell-cell interactions, has been shown to both promote and suppress tumor progression. To better understand the normal function of CD44, we have investigated its role in mouse mammary gland development and its expression in human breast and prostate cancer. We have found that CD44 is expressed in the myoepithelium of the developing mammary gland and modulates ductal development of FVB/N mice. The loss of CD44 results in defective luminal-myoepithelial cell-cell adhesion and promotes the mixing of luminal and myoepithelial layers, disrupting epithelial bilayer organization, and CD44-null mice experience delayed ductal outgrowth and impaired terminal end bud formation. The myoepithelial expression of CD44 is also relevant to its expression in cancer, as CD44 is expressed in the basal cells of early-stage breast and prostate cancer but exhibits altered localization with increasing tumorigenicity and is strongly expressed by tumor epithelium.

Silver in situ hybridization (SISH) for determination of HER2 gene status in breast carcinoma: comparison with FISH and assessment of interobserver reproducibility.

The importance of HER2 status in breast cancer management has focused attention on the ability of clinical assays to correctly assign HER2 amplification status. There is no consensus as to the best method for assessing HER2 status. Disadvantages of fluorescence in situ hybridization (FISH) testing include longer time required for staining and scoring slides, requirements for specialized training and fluorescence microscopy, and loss of the signal due to quenching of the fluorescent dye. Silver-enhanced in situ hybridization (SISH) is a rapid fully automated assay providing permanently stained slides that are interpreted by conventional bright field microscopy which enables pathologists to evaluate slides within the context of tissue morphology. This study evaluates the concordance between SISH and FISH assays in determining the status of HER2 gene amplification in a cohort of 298 primary invasive breast carcinomas. Furthermore, we assessed in detail the variables contributing to interobserver interpretive reproducibility of HER2 SISH among 10 pathologists. HER2 was quantified using the ratio of HER2 to CHR17 signals using the conventional historical interpretation scale and also by the American Society of Clinical Oncology/College of American Pathologists reporting scheme. For SISH status determined by consensus among 10 pathologists, overall concordance between SISH and FISH was identified in 288 of 298 cases (96.6%) using the conventional Food and Drug Administration approved criteria. Overall agreement was observed in 282 of 285 cases (98.9%) using the American Society of Clinical Oncology/College of American Pathologists result reporting scheme (with equivocal cases removed). In conclusion, SISH represents a novel approach for the determination of HER2 status in breast cancer. The overall concordance between SISH and FISH is excellent, and the interpretation of SISH results by pathologists is most reproducible using the HER2/CHR17 ratio.

Extracellular engagement of alpha6 integrin inhibited urokinase-type plasminogen activator-mediated cleavage and delayed human prostate bone metastasis.

Expression of alpha(6) integrin, a laminin receptor, on tumor cell surfaces is associated with reduced patient survival and increased metastasis in a variety of tumors. In prostate cancer, tumor extracapsular escape occurs in part via laminin-coated nerves and vascular dissemination, resulting in clinically significant bone metastases. We previously identified a novel form of alpha(6) integrin, called alpha(6)p, generated by urokinase-type plasminogen activator-dependent cleavage of the laminin-binding domain from the tumor cell surface. Cleavage increased laminin-dependent migration. Currently, we used the known conformation sensitivity of integrin function to determine if engagement of the extracellular domain inhibited integrin cleavage and the extravasation step of metastasis. We show that alpha(6) integrin was present on prostate carcinoma escaping the gland via nerves. Both endogenous and inducible levels of alpha(6)p were inhibited by engaging the extracellular domain of alpha(6) with monoclonal antibody J8H. J8H inhibited tumor cell invasion through Matrigel. A severe combined immunodeficient mouse model of extravasation and bone metastasis produced detectable, progressive osteolytic lesions within 3 weeks of intracardiac injections. Injection of tumor cells, pretreated with J8H, delayed the appearance of metastases. Validation of the alpha(6) cleavage effect on extravasation was confirmed through a genetic approach using tumor cells transfected with uncleavable alpha(6) integrin. Uncleavable alpha(6) integrin significantly delayed the onset and progression of osseous metastases out to six weeks post-injection. The results suggest that alpha(6) integrin cleavage permits extravasation of human prostate cancer cells from circulation to bone and can be manipulated to prevent metastasis.

Morphine treatment accelerates sarcoma-induced bone pain, bone loss, and spontaneous fracture in a murine model of bone cancer.

Metastatic bone cancer causes severe pain that is primarily treated with opioids. A model of bone cancer pain in which the progression of cancer pain and bone destruction is tightly controlled was used to evaluate the effects of sustained morphine treatment. In cancer-treated mice, morphine enhanced, rather than diminished, spontaneous, and evoked pain; these effects were dose-dependent and naloxone-sensitive. SP and CGRP positive DRG cells did not differ between sarcoma or control mice, but were increased following morphine in both groups. Morphine increased ATF-3 expression only in DRG cells of sarcoma mice. Morphine did not alter tumor growth in vitro or tumor burden in vivo but accelerated sarcoma-induced bone destruction and doubled the incidence of spontaneous fracture in a dose- and naloxone-sensitive manner. Morphine increased osteoclast activity and upregulated IL-1 beta within the femurs of sarcoma-treated mice suggesting enhancement of sarcoma-induced osteolysis. These results indicate that sustained morphine increases pain, osteolysis, bone loss, and spontaneous fracture, as well as markers of neuronal damage in DRG cells and expression of pro-inflammatory cytokines. Morphine treatment may result in "add-on" mechanisms of pain beyond those engaged by sarcoma alone. While it is not known whether the present findings in this model of osteolytic sarcoma will generalize to other cancers or opioids, the data suggest a need for increased understanding of neurobiological consequences of prolonged opioid exposure which may allow improvements in the use of opiates in the effective management of cancer pain.

Role of the extracellular matrix in prostate carcinogenesis.

This review summarizes the current state of knowledge regarding the proteins composing the extracellular matrix in the human prostate. The normal expression as well as the changes which occur in PIN and carcinoma are described for the lamins, collagens, and glycosaminoglycans.