Stages Of Neoplastic Progression Research Articles

Multiple aspects of matrix stiffness in cancer progression.

The biophysical and biomechanical properties of the extracellular matrix (ECM) are crucial in the processes of cell differentiation and proliferation. However, it is unclear to what extent tumor cells are influenced by biomechanical and biophysical changes of the surrounding microenvironment and how this response varies between different tumor forms, and over the course of tumor progression. The entire ensemble of genes encoding the ECM associated proteins is called matrisome. In cancer, the ECM evolves to become highly dysregulated, rigid, and fibrotic, serving both pro-tumorigenic and anti-tumorigenic roles. Tumor desmoplasia is characterized by a dramatic increase of α-smooth muscle actin expressing fibroblast and the deposition of hard ECM containing collagen, fibronectin, proteoglycans, and hyaluronic acid and is common in many solid tumors. In this review, we described the role of inflammation and inflammatory cytokines, in desmoplastic matrix remodeling, tumor state transition driven by microenvironment forces and the signaling pathways in mechanotransduction as potential targeted therapies, focusing on the impact of qualitative and quantitative variations of the ECM on the regulation of tumor development, hypothesizing the presence of matrisome drivers, acting alongside the cell-intrinsic oncogenic drivers, in some stages of neoplastic progression and in some tumor contexts, such as pancreatic carcinoma, breast cancer, lung cancer and mesothelioma.

Some aspects of thyroid neoplasia after chernobyl

Significant increase in thyroid carcinoma (TC) incidence was registered after the Chernobyl accident among people exposed as children and adolescents. The quantity of radiogenic cases and dose-effect correlations have been overestimated due the screening-effect, improved medical surveillance after the accident, overdiagnosis, recall bias, dose-dependent selection and self-selection. Besides, some non-exposed patients were counted among Chernobyl victims. Before the accident, the registered incidence of paediatric TC was lower in the USSR compared to other industrialised countries. The screening found not only small nodules but also late-stage TC interpreted as rapidly growing radiogenic cancers developing after a short latent period. Some supposed markers of radiogenic malignancy were in fact associated with a later stage of neoplastic progression. In conclusion, the overestimation of Chernobyl consequences may lead to a biased concept about carcinogenicity of low-dose low-rate exposures, especially from radioiodine.

A Possible Role of Human Herpes Viruses Belonging to the Subfamily Alphaherpesvirinae in the Development of Some Cancers.

Seroepidemiological studies suggest that human herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) are linked with several types of cancer; however, they do not appear to play a direct role and are considered to be cofactors. The abilities of HSV-1 and -2 to transform cells in vitro can be demonstrated by suppressing their lytic ability via irradiation with a specific dose of ultraviolet light, photoinactivation in the presence of photosensitizers (e.g., neutral red or methylene blue), and culture under specific conditions. Several mechanisms have been proposed to explain the actions of these viruses. According to the hit-and-run mechanism, viral DNA initiates transformation by interacting with cellular DNA and thereby inducing mutations and epigenetic changes, but is not involved in other stages of neoplastic progression. By contrast, according to the hijacking mechanism, viral products in infected cells can activate signaling pathways and thereby cause uncontrolled proliferation. Such products include RR1PK, an oncoprotein that activates the Ras pathway and is encoded by the HSV-2 gene ICP10. Virus-encoded microRNAs may act as cofactors in tumorigenesis of serous ovarian carcinoma and some prostate tumors. Herpes virus-associated growth factors that facilitate or suppress transformation may play important roles in tumor formation. Finally, there is much evidence that HSV-2 increases the risk of cervical cancer after infection of human papilloma viruses. Key words: HSV-1 - HSV-2 - cancer - mechanisms of transformation This work was supported by APVV 0621-12. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 29. 11. 2016 Accepted: 20. 3. 2018.

Vimentin, colon cancer progression and resistance to butyrate and other HDACis.

Dietary fibre protects against colorectal cancer (CRC) most likely through the activity of its fermentation product, butyrate. Butyrate functions as a histone deacetylase inhibitor (HDACi) that hyperactivates Wnt signalling and induces apoptosis of CRC cells. However, individuals who consume a high‐fibre diet may still develop CRC; therefore, butyrate resistance may develop over time. Furthermore, CRC cells that are resistant to butyrate are cross‐resistant to clinically relevant therapeutic HDACis, suggesting that the development of butyrate resistance in vivo can result in HDACi‐resistant CRCs. Butyrate/HDACi‐resistant CRC cells differ from their butyrate/HDACi‐sensitive counterparts in the expression of many genes, including the gene encoding vimentin (VIM) that is usually expressed in normal mesenchymal cells and is involved in cancer metastasis. Interestingly, vimentin is overexpressed in butyrate/HDACi‐resistant CRC cells although Wnt signalling is suppressed in such cells and that VIM is a Wnt activity‐targeted gene. The expression of vimentin in colonic neoplastic cells could be correlated with the stage of neoplastic progression. For example, comparative analyses of LT97 microadenoma cells and SW620 colon carcinoma cells revealed that although vimentin is not detectable in LT97 cells, it is highly expressed in SW620 cells. Based upon these observations, we propose that the differential expression of vimentin contributes to the phenotypic differences between butyrate‐resistant and butyrate‐sensitive CRC cells, as well as to the differences between early‐stage and metastatic colorectal neoplastic cells. We discuss the hypothesis that vimentin is a key factor integrating epithelial to mesenchymal transition, colonic neoplastic progression and resistance to HDACis.

Molecular pathways: myeloid complicity in cancer.

Cancer-induced inflammation results in accumulation of myeloid cells. These myeloid cells include progenitors and progeny of monocytes, granulocytes, macrophages, and dendritic cells. It has become increasingly evident that tumor-dependent factors can condition myeloid cells toward an immunosuppressive and protumorigenic phenotype. Thus, myeloid cells are not simply bystanders in malignancy or barometers of disease burden. Reflecting their dynamic and plastic nature, myeloid cells manifest a continuum of cellular differentiation and are intimately involved at all stages of neoplastic progression. They can promote tumorigenesis through both immune-dependent and -independent mechanisms and can dictate response to therapies. A greater understanding of the inherent plasticity and relationships among myeloid subsets is needed to inform therapeutic targeting. New clinical trials are being designed to modulate the activities of myeloid cells in cancer, which may be essential to maximize the efficacy of both conventional cytotoxic and immune-based therapies for solid tumors. Clin Cancer Res; 20(20); 5157-70. ©2014 AACR.

Morphological and quantitative analysis of BCL6 expression in human colorectal carcinogenesis

The aim of the present study was to determine whether BCL6 is expressed during malignant transformation of the large bowel and to assess whether, and to what extent, immunoreactivity is related to the different stages of neoplastic progression. Samples of normal colorectal mucosa (n=22), microadenomas (n=22) and colorectal cancer (n=22), were analyzed by immunohistochemistry, immunofluorescence coupled with confocal microscopy and western blotting. Our results clearly outlined the marked increase occurring in both intensity and density of BCL6 protein expression in the normal mucosa-microadenoma-carcinoma sequence. Immunohistochemistry and immunofluorescence analyses showed that BCL6 is expressed at low levels in normal mucosa and increases in microadenoma and in cancer with statistical significance. These results were confirmed by western blotting data. The increasing expression of BCL6 in human colorectal cancer development suggests the involvement of BCL6 in tumor progression, from the earliest stages of carcinogenesis with significant increase in cancer. The enhanced understanding of the biological role of BCL6, previously shown to exert a key role in lymphomagenesis, may lead to a re-evaluation of this protein and may highlight the importance of performing further studies in order to identify novel therapeutic targets for colorectal cancer.

Abstract 3915: Insulin-like growth factor 1 promotes cancer cell growth via RAC1 activation in ovarian epithelial cancer cells

Abstract Ovarian cancer is the most lethal of all gynecological malignancies, due in part to the diagnosis at an advanced stage caused by the lack of specific signs and symptoms and the absence of reliable tests for screening and early detection. Furthermore, survival is reduced by recurrence of chemo-resistant tumors. The insulin-like growth factor 1 (IGF1)/IGF1 receptor (IGFR) signaling system exerts a broad anti-apoptotic function and plays a crucial role in human cancer progression, including ovarian cancers. The small GTP-binding protein RAC1 regulates a diverse array of cellular events, including the control of cell growth, cytoskeleton reorganization for motility, and the activation of protein kinases. Although RAC1 is expressed in most tissues, it can be elevated in tumors at various stages of neoplastic progression. This study was conducted to explore the role of RAC1 in IGF1-stimulated ovarian cancer cell growth. While IGFR was expressed in normal human ovarian surface epithelial (HOSE) cells and all OSE cancer cell lines examined, the IGF1R was over expressed in IGROV1 ovarian cancer cells. Exposure of IGROV1 cells to IGF1 rapidly (<0.5 min) induced IGFR phosphorylation which triggered the phosphorylation of AKT (S473) (≥0.5 min), ERK1[[Unsupported Character - ⁄]] 2 (>1.0 min) and p70S6K (T389) (≥1.0 min); indicating that IGF1 actives PI3K and MAPK signaling pathways that are known to be important in cell growth. We observed a maximal effect of IGF1 on IGROV1 cell growth at 50 ng/ml (2.2 fold increase, p<0.001) after 24 h. In contrast, exposure of IGROV1 cells to the PI3K inhibitor LY294002 or the MEK1/2 inhibitor UO126 decreased cell viability. Pull-down assays revealed that IGF1 stimulated GTP loading of RAC1 in less than 1.0 min with maximal increases observed after 5 min (2.6 fold, p<0.05). Treatment with the PI3K inhibitor LY294002 significantly reduced RAC1 activation in response to IGF1. Pre-treatment of IGROV1 cells with the RAC1 inhibitor (NSC23766) for 1 h inhibited IGF1-induced activation of RAC1; and decreased cell survival in a concentration-dependent manner (5- 50 µM NSC23766, p<0.05). The reduction in viability after 48 hr was accompanied by an increase in apoptotic cells, cleaved PARP and elevated cytosolic cytochrome C. In contrast, treatment of HOSE cells with NSC23766 did not alter cell viability. Of interest, following 6 h treatment of IGROV1 cells with 20-50 µM NSC23766 we observed increases in the phosphorylation of Akt (S473), GSK3β (S9), and p70S6K (T389), suggesting the activation of PI3K/AKT pathway was not sufficient to rescue cells from death. Furthermore, treatment with NSC23766 (50 µM) and cisplatin (20 µM) reduced cell viability to an extent greater than achieved by either agent alone (p<0.05). Thus, disruption of RAC1 activation reduces IGF1-stimulated cell growth, induces apoptosis, and enhances cell death by anti-cancer agents in ovarian cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3915. doi:1538-7445.AM2012-3915

Accurate Reconstruction of the Temporal Order of Mutations in Neoplastic Progression

The canonical route from normal tissue to cancer occurs through sequential acquisition of somatic mutations. Many studies have constructed a linear genetic model for tumorigenesis using the genetic alterations associated with samples at different stages of neoplastic progression from cross-sectional data. The common interpretation of these models is that they reflect the temporal order within any given tumor. Linear genetic methods implicitly neglect genetic heterogeneity within a neoplasm; each neoplasm is assumed to consist of one dominant clone. We modeled neoplastic progression of colorectal cancer using an agent-based model of a colon crypt and found clonal heterogeneity within our simulated neoplasms, as observed in vivo. Just 7.3% of cells within neoplasms acquired mutations in the same order as the linear model. In 41% of the simulated neoplasms, no cells acquired mutations in the same order as the linear model. We obtained similarly poor results when comparing the temporal order with oncogenetic tree models inferred from cross-sectional data. However, when we reconstructed the cell lineage of mutations within a neoplasm using several biopsies, we found that 99.7% cells within neoplasms acquired their mutations in an order consistent with the cell lineage mutational order. Thus, we find that using cross-sectional data to infer mutational order is misleading, whereas phylogenetic methods based on sampling intratumor heterogeneity accurately reconstructs the evolutionary history of tumors. In addition, we find evidence that disruption of differentiation is likely the first lesion in progression for most cancers and should be one of the few regularities of neoplastic progression across cancers.

Malignant Transformation of Non-Neoplastic Barrett's Epithelial Cells through Well-Defined Genetic Manipulations

BackgroundHuman Barrett's cancer cell lines have numerous, poorly-characterized genetic abnormalities and, consequently, those lines have limited utility as models for studying the early molecular events in carcinogenesis. Cell lines with well-defined genetic lesions that recapitulate various stages of neoplastic progression in Barrett's esophagus would be most useful for such studies.Methodology/Principal FindingsTo develop such model cell lines, we started with telomerase-immortalized, non-neoplastic Barrett's epithelial (BAR-T) cells, which are spontaneously deficient in p16, and proceeded to knock down p53 using RNAi, to activate Ras by introducing oncogenic H-RasG12V, or both. BAR-T cells infected with either p53 RNAi or oncogenic H-RasG12V alone maintained cell-to-cell contact inhibition and did not exhibit anchorage-independent growth in soft agar. In contrast, the combination of p53 RNAi knockdown with expression of oncogenic H-RasG12V transformed the p16-deficient BAR-T cells, as evidenced by their loss of contact inhibition, by their formation of colonies in soft agar, and by their generation of tumors in immunodeficient mice.Conclusions/SignificanceThrough these experiments, we have generated a number of transformed and non-transformed cell lines with well-characterized genetic abnormalities recapitulating various stages of carcinogenesis in Barrett's esophagus. These lines should be useful models for the study of carcinogenesis in Barrett's esophagus, and for testing the efficacy of chemopreventive and chemotherapeutic agents.

Immunohistochemical Evaluation of a Panel of Tumor Cell Markers During Malignant Progression in Barrett Esophagus

Histopathologic grading of dysplasia in Barrett esophagus (BE) shows substantial interobserver and intraobserver variation. We used immunohistochemical analysis with a set of tumor cell markers, ie, epidermal growth factor receptor (EGFR), ERBB2 (HER2/neu), MYC, CDKN2A (p16), SMAD4, MET, CCND1 (cyclin D1), CTNNB1 (beta-catenin), and TP53 (p53), in histologic sections of endoscopic biopsies of 86 patients with BE in various stages of neoplastic progression. The markers, except SMAD4, were scored as 0 (<1% of cells stained), 1 (1%-25%), 2 (26%-50%), or 3 (>50%). All markers, except EGFR, showed a significant trend for immunohistochemical protein overexpression during malignant progression in BE (P <.01). When the successive stages along the metaplasia-low-grade dysplasia (LGD)-high-grade dysplasia (HGD)-adenocarcinoma axis were compared, protein overexpression of beta-catenin separated LGD from metaplasia, whereas protein overexpression of cyclin D1 and p53 discriminated HGD from LGD (all P <.001). beta-Catenin can be helpful for a diagnosis of LGD in BE, although it stains positively in a subset only, whereas p53 remains an appropriate marker to define HGD. In case of doubt, cyclin D1 can be added to separate LGD from HGD in BE.

Induction of secreted matrix metalloproteinase-9 activity in human melanoma cells by extracellular matrix proteins and cytokines

Degradation of matrix proteins that constitute the dermal-epidermal junction and dermis by proteolytic enzymes is an essential step of melanoma invasion and metastasis, and this is primarily achieved by the matrix metalloproteinases. In this report, using zymography, we compared the basal secretion levels of active matrix metalloproteinase-2 and matrix metalloproteinase-9 to levels in response to various extracellular matrix proteins, cytokines, and growth factors in normal human melanocyte cells and melanoma cell lines from different stages of neoplastic progression. Basal matrix metalloproteinase-9 activity was only detected in vertical growth phase and metastatic melanoma cell lines, suggesting that matrix metalloproteinase-9 is a candidate biomarker for identifying vertical growth phase and metastatic melanomas. Most melanoma cell lines and cultured normal melanocytes produced high levels of matrix metalloproteinase-2. In addition, both tumor necrosis factor-alpha and interleukin-1beta are strong inducers of active matrix metalloproteinase-9 in vertical growth phase melanoma cell lines, indicating a possible role of these cytokines in the switch from radial growth phase to vertical growth phase. We propose that these proinflammatory cytokines promote melanoma invasion in part through upregulating matrix metalloproteinase-9. Both these cytokines are released from keratinocytes in the epidermis by ultraviolet radiation. Thus, our study suggests that the microenvironment of melanoma cells is an important feature in melanoma progression, and ultraviolet-radiation-induced cytokines might promote the progression of melanoma through the release or activation of matrix metalloproteinases.

Progressive changes in Met-dependent signaling in a human ovarian surface epithelial model of malignant transformation

We used an experimental in vitro model of human ovarian surface epithelium (OSE), the tissue of origin of >90% of ovarian cancers, to more precisely define the contribution of hepatocyte growth factor (HGF) to various OSE phenotypes at different stages of neoplastic progression. Neoplastic transformation of OSE in cultures was achieved by multiple genetic manipulations, resulting in the nontumorigenic line IOSE-29, the tumorigenic IOSE-Ov29, and the tumor-derived, more highly malignant IOSE-Ov29/T4. We demonstrate here that, compared to IOSE-29, IOSE-Ov29 and IOSE-Ov29/T4 exhibited higher levels of the HGF receptor Met and an increasing duration of ERK1/2 activation with malignant progression, in conjunction with other neoplastic properties. HGF activated Met signaling in all lines but elicited different responses: HGF induced cell dispersion (scattering) and collagen gel invasion in IOSE-Ov29 and IOSE-Ov29/T4 but did not alter the growth pattern of IOSE-29. Inhibition with PD98059 and LY294002 independently prevented HGF-induced invasive growth. Furthermore, our results show that HGF-induced invasion can be mediated through a rapamycin-sensitive p70 S6K cascade, which demonstrates that p70S6K can regulate cell motility in addition to its well-established role in protein synthesis. Taken together, our data correlate specific responses to HGF-mediated signaling with specific signaling pathways and with progressive neoplastic changes.

Involvement of Arp2/3 complex in the process of colorectal carcinogenesis

Increased motility is one of the characteristics of cancer cells, and actin polymerization and disassembly are essential for cellular motility. Since actin-related protein (Arp) 2/3 complex acts as a nucleus for actin polymerization, in this study, we immunohistochemically investigated the expression of Arp2 and Arp3 in 175 colorectal tumors in various stages of neoplastic progression. Arp2 and Arp3 showed identical expression patterns, and both were expressed in the stromal cells around neoplastic tubules or glands and in the tumor cells themselves. The frequency of expression of Arp2 and Arp3 (Arp2&3) by the stromal cells increased with the atypia of the colorectal neoplasms, from 5.5% (3/55) in adenoma with mild or moderate atypia, to 11.8% (2/17) in adenoma with severe atypia, 53.3% (16/30) in intramucosal carcinoma, and 91.8% (67/73) in invasive carcinoma (P<0.0001). The frequency of expression of Arp2&3 in the tumor cells was similar and was 1.8% (1/55) in adenoma with mild or moderate atypia, 23.5% (4/17) in adenoma with severe atypia, 23.5% (7/30) in intramucosal carcinoma, and 32.9% (24/73) in invasive carcinoma. Expression of Arp2&3 by the stromal cells was significantly correlated with nuclear accumulation of p53 in the tumor cells and stromal expression of CD10. These results suggest that formation of Arp2/3 complex by both neoplastic and stromal cells contributes to the increased motility of both cell types and thus provides suitable conditions for invasion.

Profiling the progression of cancer: separation of microsomal proteins in MCF10 breast epithelial cell lines using nonporous chromatophoresis.

The heterogeneity of cellular protein expression has stimulated development of separations targeting smaller groups of related proteins rather than entire proteomes. The following work describes the development of a technique for the characterization of membrane subproteomes from five different breast epithelial cell lines. Intact membrane proteins are separated by hydrophobicity in the first dimension using nonporous reversed-phase high-performance liquid chromatography (RP-HPLC) to generate unique chromatographic profiles. Fractions of eluent are further separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to create distinct banding patterns. This hybrid liquid phase/gel phase method circumvents issues of membrane protein precipitation and provides a simple strategy aimed at isolating and characterizing a traditionally underrepresented protein class. Membrane protein profiles are created that discriminate between microsomal fractions of breast epithelial cells in different stages of neoplastic progression. Proteins are subsequently identified using matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) mass fingerprinting and MALDI-quadrupole time of flight - tandem mass spectrometry (QTOF-MS/MS) peptide sequencing. Furthermore, as this strategy preserves intact protein structure, further characterization can be performed on proteins producing mass fingerprint spectra and fragmentation spectra that did not result in database protein identifications. The coupling of nonporous RP-HPLC with SDS-PAGE provides a useful alternative to two-dimensional PAGE (2-D-PAGE) for membrane protein analysis.

Differential in vivo modifications of the HMGI(Y) nonhistone chromatin proteins modulate nucleosome and DNA interactions.

The HMGI(Y) family of "high mobility group" nonhistone proteins are architectural transcription factors whose overexpression is highly correlated with both cancerous transformation and increased malignancy and metastatic potential of tumors in vivo. Here we report on the types of posttranslational modifications found in vivo on the HMG-I and HMG-Y proteins isolated from two human breast epithelial cell lines, MCF-7 and MCF-7/PKC-alpha, that represent different stages of neoplastic progression. The MCF-7 cell line exhibits many characteristics of normal breast epithelial cells and does not form tumors when injected into nude mice, whereas the MCF-7/PKC-alpha cell line, a derivative of MCF-7 that expresses a transgene coding for the enzyme protein kinase C-alpha (PKC-alpha), is both malignant and highly metastatic. Using MALDI mass spectrometry, we show that the HMG-Y protein is more highly modified than the HMG-I protein in both the MCF-7 and the MCF-7/PKC-alpha cells. Significantly, the HMG-Y protein isolated from the highly metastatic MCF-7/PKC-alpha cells possesses a unique constellation of phosphorylations, methylations, and acetylations not found on the HMG-I protein isolated from either the MCF-7 or MCF-7/PKC-alpha cells. We further demonstrate that some of the same amino acid residues phosphorylated on recombinant HMGI(Y) proteins by purified PKC in vitro are also phosphorylated on the HMG-I(Y) proteins isolated from MCF-7/PKC-alpha cells, suggesting that PKC phosphorylates these proteins in vivo. Quantitative substrate binding analyses indicate that the biochemical modifications present on the HMG-I and HMG-Y proteins differentially influence the ability of these proteins to interact with both A.T-rich DNA substrates and nucleosome core particles in vitro, suggesting a similar modulation of such binding affinities in vivo. To our knowledge, this is the first demonstration of differences in the types of in vivo biochemical modifications found on the HMG-I and HMG-Y proteins in cells and also the first experimental evidence suggesting a possible linkage between such posttranslational modifications and the neoplastic potential of cells.

Cloning of a novel human Rac1b splice variant with increased expression in colorectal tumors.

Rac1 is a member of the Ras superfamily of small GTPases involved in signal transduction pathways that induce the formation of lamellipodia, stimulate cell proliferation and activate the JNK/SAPK protein kinase cascade. Here we describe that amplification by RT-PCR of the entire Rac1 coding sequence from a series of human adult and fetal tissues revealed beside the expected Rac1 cDNA, a variant product which contained additional 57 nucleotides between codons 75 and 76. This variant resulted in an in-frame insertion of 19 new amino acids immediately behind the switch II region, including two potential threonine phosphorylation sites for casein kinase II and protein kinase C. Primers designed within and downstream of the inserted nucleotide sequence allowed isolation of a genomic clone with intronic consensus sequences demonstrating that the insertion corresponds to a novel, yet undescribed exon 3b. This Rac1 splice variant, designated Rac1b, was predominantly identified in skin and epithelial tissues from the intestinal tract. Most notably, the expression of rac1b versus rac1 was found to be elevated in colorectal tumors at various stages of neoplastic progression, as compared to their respective adjacent tissues. We suggest that the 19 amino acid-insertion following the switch II region may create a novel effector binding site in rac1b, and thus participate in signaling pathways related to the normal or neoplastic growth of the intestinal mucosa.

Constitutive and conditional cadherin expression in cultured human ovarian surface epithelium: Influence of family history of ovarian cancer

Epithelial ovarian carcinomas arise in a simple mesothelium (ovarian surface epithelium, OSE) but exhibit properties of oviductal and endometrial epithelia. Thus, during malignant progression, their differentiation proceeds from simple to complex, in contrast to carcinomas in other tissues. Related changes in OSE of women with a history of familial ovarian cancer indicate that this aberrant differentiation is initiated very early in neoplastic progression. The mechanisms underlying this process are not understood. Because cadherins are known regulators of differentiation, we investigated the relationship of the cadherins E, N and P to OSE morphology, growth patterns and differentiation in cultures of normal and metaplastic OSE from women with (FH-OSE) and without (NFH-OSE) a family history of ovarian cancer and in the ovarian carcinoma lines OVCAR-3 and CaOV3. We used immunofluorescence, RT-PCR, in situ hybridization and Western blotting. Our results define N-cadherin as the constitutively expressed cadherin of normal and metaplastic OSE and indicate that P-cadherin is undetectable while E-cadherin expression is conditional and related to genotype, stage of neoplastic progression and growth pattern. The altered expression of E-cadherin in apparently normal OSE of women with hereditary ovarian cancer syndromes in conjunction with the known capacity of E-cadherin to induce epithelial characteristics implicates this adhesion molecule as a possible inducer of the aberrant Mullerian differentiation which characterizes epithelial ovarian carcinomas. Abnormal differentiation in such (pre)-neoplastic tissues may represent an early, irreversible, non-mutational step in ovarian epithelial neoplastic progression. Int. J. Cancer 81:180–188, 1999. © 1999 Wiley-Liss, Inc.

Expression of cytokeratin-19 as a marker of neoplastic progression of human prostate epithelial cells.

Our earlier studies demonstrated neoplastic transformation of SV40-immortalized neonatal human prostate epithelial cells (267B1) by fractionated doses of ionizing radiation or by introduction of v-ki-ras oncogene. X-ray-treated 267B1 cells represent three different stages of neoplastic progression: nontumorigenic F3-SAC cells that acquired morphological changes and anchorage independence when treated with 2 x 2 Gy of X-rays; malignantly transformed 267B1-XR and 267B1-SXR cells that received 2-Gy doses to a total of 30 Gy. We also reported alterations in cell size, morphology, actin stress fibers, and levels of actin-binding proteins in these transformed human prostate cells. We analyzed intermediate filament-nuclear matrix (IF-NM) protein expression in the various 267B1 cells as a consequence of neoplastic progression by two-dimensional gel electrophoresis and immunofluorescence. Our present study revealed that the 267B1 cells experienced progressive changes in their intermediate filament protein composition during the process of neoplastic conversion, achieved either by X-rays or by ras-oncogene. In particular, we observed a stepwise downregulation of cytokeratin-19 in these in vitro transformed 267B1 cells. Our data suggest that loss of expression of cytokeratin-19 accompanied the morphological alterations associated with in vitro neoplastic transformation of SV40-immortalized prostate epithelial cells.

P21WAF1/Cip1 is associated with cyclin D1CCND1 expression and tubular differentiation but is independent of p53 overexpression in human breast carcinoma

p21WAF1/Cip1 is an inhibitor of cdk/cyclin complexes, and thus regulates the cell cycle. p21 is also related to cell differentiation and is regulated by wild-type p53, although p53-independent regulatory pathways have been proposed. In order to analyse p21 expression as well as its relationship with p53 in human breast cancer, an immunohistochemical analysis was undertaken of 77 breast carcinomas, 16 of them with an in situ component; 30 adjacent normal tissue samples; and five non-neoplastic specimens. Forty-four infiltrating carcinomas (57 per cent) were p21-positive. Expression of p21 was also observed in pre-invasive lesions, whereas normal ducts were negative or focally and weakly positive. p21 expression was associated with high histological grade (II + III) (P = 0.017) and poor tubule formation (P = 0.002), and was significantly less frequent in lobular carcinomas (P = 0.0001). p21 positivity also correlated with increased proliferation, but this seemed to be dependent on the histological grade. Twenty carcinomas (26 per cent) showed p53 overexpression, but this was not associated with p21 negativity, suggesting the existence of p53-independent mechanisms for p21 regulation in vivo. Cyclin D1CCND1 expression was analysed in the same series and an association between p21 and cyclin D1 expression was found, since 23 of 26 cyclin D1-positive carcinomas were p21-positive (P < 0.001 ...). In conclusion, p21 is frequently overexpressed in breast carcinomas and this occurs in the early stages of neoplastic progression. This overexpression seems to be independent of p53 status and might be involved in cyclin D1 modulation.

Physiologic and Pathologic Drug Resistance in Ovarian Carcinoma: A Hypothesis Based on a Clonal Progression Model

Conventional models for successful chemotherapy suggest that early-stage ovarian cancer should be more responsive to cytotoxic drugs than advanced disease, that multiple drugs will prove more efficacious than single agents and that more intensive chemotherapy will prove superior to conventional doses. The purpose of numerous clinical studies has been to test these predictions and the results often fall short of the expectations. In trials to date, adjuvant chemotherapy for high-risk, early-stage ovarian cancer has provided only a modest, equivocal increase in disease-free survival. Combination chemotherapy produces higher response rates but this has not consistently resulted in prolonged survival. Dose intensification with high-dose chemotherapy increases rate of response further, but most responses are of short duration. The objective of this review is to integrate clinical and molecular biological observations into a novel model of drug resistance. We hypothesize that drug sensitivity is an acquired characteristic of neoplastic cells, and that there are two broad cellular forms of resistance to cytotoxic drugs. 'Physiological drug resistance' is a cellular state when drug sensitivity of cancer cells is similar to that of the corresponding normal tissue. This form of drug resistance can precede the acquisition of drug sensitivity and may be predominant in the early stages of neoplastic progression. A distinct, 'pathological drug resistance' can reappear later during tumor progression or during chemotherapy as a result of increased detoxification, upregulation of repair pathways or defective apoptosis. Tumors are formed of heterogeneous cell populations and in terms of drug sensitivity three distinct cell types may be present: drug-sensitive, physiologically drug-resistant and pathologically drug-resistant. Clinical response to chemotherapy is determined by the relative contribution of these different cell populations to the total cellular mass. Depending on the predominant type of surviving population, distinct patterns of clinical failure may develop that require different treatment strategies for optimal management. For chemosensitive cells that survived insufficient chemotherapy, consolidation with further, perhaps, high-dose chemotherapy is a rational option. For pathologically drug-resistant cells, pharmacological manipulation of drug resistance, and signaling pathways in combination with chemotherapy could be exploited. For physiologically drug-resistant cells, non-chemotherapy-based, 'chemopreventive' strategies to arrest tumor progression may prove beneficial.