Harold L. Asch

Downregulation of Gelsolin Correlates with the Progression to Breast Carcinoma

The actin cytoskeleton underlies several normal cellular functions and is deranged during carcinogenesis. Gelsolin, a multifunctional actin-binding protein, is downregulated in several types of tumors and its abnormal expression is one of the most common defects noted in invasive breast carcinoma (ICA). This study utilizes immunohistochemistry to examine the expression of gelsolin in 95 ICA, 59 ductal carcinoma in situ (DCIS) and 36 benign lesions, including 17 atypical ductal hyperplasia (ADH). Cytoplasmic staining was scored as positive, reduced or negative. Gelsolin expression was then correlated with patient’s age, tumor size, histologic grade and lymph node status. All unremarkable breast biopsies, 88% of ADH, 44% of DCIS and 28% of ICA were positive for gelsolin. This represents a significant difference among the groups (p=<0.0001) and the trend towards reduced gelsolin with the progression to ICA is significantly linear (p=<0.0001). For invasive carcinoma, patients older than 44 years were significantly more likely to have decreased expression of gelsolin than patients 44 years old and younger (p=0.007). Bivariate analysis showed no correlation of gelsolin expression with lymph node status (p=0.62), tumor size (p=0.10), histologic grade (p=0.42), estrogen receptor status (p=1.0) or other clinicopathologic parameters. In clinical follow-up, there were 18 breast tumor related deaths within a median follow-up time of 4.2 years. Survival analysis indicated that the level of gelsolin expression may be associated with survival (p=0.06). In summary, the frequency of gelsolin deficiency increases significantly with progression from ADH to DCIS to ICA. Additionally, gelsolin expression may be an independent marker of prognosis.

Downregulated gelsolin expression in hyperplastic and neoplastic lesions of the prostate.

Because of its role in cell motility and growth regulation, gelsolin, an actin‐binding protein, has been considered a tumor suppressor and a potential prognostic marker in some neoplasias, such as breast and bladder cancer. Little is known about its immunoexpression in prostatic adenocarcinoma (PCA).

Prognostic significance of MCM2, Ki-67 and gelsolin in non-small cell lung cancer.

BackgroundUncontrolled proliferation and increased motility are hallmarks of neoplastic cells, therefore markers of proliferation and motility may be valuable in assessing tumor progression and prognosis. MCM2 is a member of the minichromosome maintenance (MCM) protein family. It plays critical roles in the initiation of DNA replication and in replication fork movement, and is intimately related to cell proliferation. Ki-67 is a proliferation antigen that is expressed during all but G0 phases of the cell cycle. Gelsolin is an actin-binding protein that regulates the integrity of the actin cytoskeletal structure and facilitates cell motility. In this study, we assessed the prognostic significance of MCM2 and Ki-67, two markers of proliferation, and gelsolin, a marker of motility, in non-small cell lung cancer (NSCLC).Methods128 patients with pathologically confirmed, resectable NSCLC (stage I-IIIA) were included. Immunohistochemistry was utilized to measure the expressions of these markers in formalin-fixed, paraffin-embedded tumor tissues. Staining and scoring of MCM2, Ki-67 and gelsolin was independently performed. Analyses were performed to evaluate the prognostic significance of single expression of each marker, as well as the prognostic significance of composite expressions of MCM2 and gelsolin. Cox regression and Kaplan-Meier survival analysis were used for statistical analysis.ResultsOf the three markers, higher levels of gelsolin were significantly associated with an increased risk of death (adjusted RR = 1.89, 95% CI = 1.17–3.05, p = 0.01), and higher levels of MCM2 were associated with a non-significant increased risk of death (adjusted RR = 1.36, 95% CI = 0.84–2.20, p = 0.22). Combined, adjusted analyses revealed a significantly poor prognostic effect for higher expression of MCM2 and gelsolin compared to low expression of both biomarkers (RR = 2.32, 95% CI = 1.21–4.45, p = 0.01). Ki-67 did not display apparent prognostic effect in this study sample.ConclusionThe results suggest that higher tumor proliferation and motility may be important in the prognosis of NSCLC, and composite application of biomarkers might be of greater value than single marker application in assessing tumor prognosis.

Down-regulation of gelsolin expression in human breast ductal carcinoma in situ with and without invasion

Expression of gelsolin, an actin filament regulatory protein, in human breast ductal carcinoma in situ (DCIS) was analyzed by immunohistochemistry using a monoclonal antibody. Formalin‐fixed paraffin‐embedded tissues from 59 pure DCIS specimens and 33 DCIS specimens with associated invasive components were evaluated for gelsolin reactivity and compared to eight normal breast cases and 76 invasive breast cancers. The proportion of cases exhibiting negative/low expression of gelsolin in the epithelium was as follows – normal, 0%; pure DCIS, 56%; DCIS associated with invasion, 58% in the DCIS component and 66% in the invasive component; invasive carcinoma, 70%. These data demonstrate that down‐regulation of gelsolin expression in breast epithelium frequently parallels progression to malignancy. Testing gelsolin expression (normal vs. negative/low levels) in the DCIS lesions for associations with patient age or any of the following histopathologic parameters revealed no significant (95% probability level) correlations – tumor size; pathologic (Van Nuys system) grade; nuclear grade; necrosis; presence of histologic calcifications; presence or type of adjacent benign lesions; architectural histologic pattern; and mammographic extent. Gelsolin loss was more commonly associated with mammographic soft tissue lesions as compared to calcified lesions (P = 0.009). A positive trend of borderline significance (P = 0.06) found in the DCIS with invasion group was a correlation between down‐regulated gelsolin expression in the DCIS component and size (< versus ≥ 15 mm) of the invasive tumor. In conclusion, reduced gelsolin protein is detectable in at least half of breast lesions which have progressed to DCIS. The trend between increasing gelsolin loss and malignant progression from normal epithelium to DCIS to invasive breast cancer (P < 0.0001) suggests additional investigation is needed to determine the potential of altered gelsolin expression as a marker for prognosis and for therapeutic interventions in breast cancer.

Genes, Chromatin, and Breast Cancer: An Epigenetic Tale

The production of heritable changes in gene expression is the driving force in the development and progression of breast cancer. Such changes can result from mutations or from epigenetic events such as hypermethylation of DNA and hypoacetylation of histones. Histone acetylation and DNA methylation are major determinants of chromatin structure, and chromatin structure is a primary regulator of gene transcription. Cancer cells frequently contain both mutated genes and genes with altered expression due to one or more epigenetic mechanisms. This review describes the epigenetic changes that disrupt normal chromatin architecture and modify the expression of key genes in breast cancer cells. The structural integrity of the latter genes is usually intact, but their expression has been substantially altered due to methylation in their promoter region or deacetylation of histones that interact with their promoter region or both mechanisms. Genes affected by epigenetic changes in breast cancers include HoxA5, p21WAF, gelsolin, BRCA1, BRCA2, E-cadherin, steroid hormone receptors, and retinoic acid receptor II. Because these epigenetic modifications are usually reversible by treatment with certain drugs, they represent vulnerabilities in the cancer cell that can be exploited as novel targets for new prevention and therapeutic strategies.

Expression of keratins and other cytoskeletal proteins in mouse mammary epithelium during the normal developmental cycle and primary culture

Mammary epithelium is composed of ductal, alveolar, and myoepithelial cells, and undergoes dramatic responses in growth, differentiation, and function to hormonal stimuli during the four stages of the mammary developmental cycle represented in virgin, pregnant, lactating, and involuting animals. To determine if progression of the epithelium through the cycle is accompanied by changes in cytoskeletal composition, particularly the keratins, the polypeptides in cytoskeletal extracts from BALB/c mouse mammary tissues were analyzed by one- and two-dimensional gel electrophoresis combined with immunoblots using polyclonal and monoclonal antikeratin antibodies. The major polypeptides in cytoskeletal fractions enriched in intermediate filaments included seven acidic and three basic components ranging in molecular weight from 40,000 to 90,000. Two major polypeptides of Mr 50,000 and 40,000, along with two minor components of Mr 57,000 and 55,000 were identified as keratins. The polypeptide profiles of mammary glands from virgin, pregnant, lactating, and involuting mice were very similar, indicating a remarkable stability of cytoskeletal composition during hormonal shifts and periods of minimal or maximal cell growth and differentiated function. The data also suggest that ductal and alveolar cells express the same set of cytoskeletal polypeptides, including keratins. Mammary cells grown in primary culture exhibited a loss or reduction in most of the basic polypeptides, a large increase in an acidic Mr 55,000 keratin, and the appearance of a prominent acidic polypeptide of Mr 46,000. The latter results demonstrate that keratin expression in mouse mammary epithelial cells is subject to regulation by certain environmental factors.

Altered expression and localization of PKC eta in human breast tumors

Protein kinase C (PKC) eta is a PKC isoform whose upregulation is associated with differentiation in many epithelial tissues, including the rat mammary gland. The purpose of this study was to examine whether PKC eta is altered, in expression or localization, in human breast cancer. Paraffin sections of 49 in situ breast lesions, 29 invasive breast tumors, and nine normal breast biopsies were examined for PKC eta expression by immunohisto chemistry. Adjacent regions of normal epithelium, and in situ lesions that were present adjacent to invasive lesions were also analyzed. In normal epithelium, regardlessof the presence of adjacent in situ or invasive lesions, PKC eta was present in the cytoplasm of the luminal epithelium, and increased inareas of normal lobular development, similar to normal rat mammary gland. PKC eta staining intensity was homogeneous in normal lobules, but heterogeneous in in situ and invasive lesions, being focally increased in cells with aberrant nuclear morphology. In situ lesions were similar to adjacent normal epithelium in average staining intensity, regardless of whether invasion was also present. However, the invasive lesions themselves were significantly decreased in staining intensity compared to adjacent in situ lesions. In addition, 75% of invasive breast cancer lesions showed decreased staining relative to adjacent normal epithelium, compared to 37% of in situ lesions. The invasive tumors which possessed high PKC eta staining were associated with positive lymph node status. These results demonstrate that quantitative and qualitative alterations in PKC eta occur in human breast cancers.

Concurrent deregulation of gelsolin and cyclin D1 in the majority of human and rodent breast cancers.

Decreased gelsolin and increased cyclin D1 are among the most common defects found in human and rodent breast cancers. Our purpose was to determine the frequency of concurrence of these 2 alterations in this malignancy. Our results demonstrate that gelsolin protein and mRNA were significantly reduced in 80–100% of rodent mammary carcinomas that developed spontaneously, following oncogene introduction, or after treatment with viral, chemical or hormonal agents. The reduction in gelsolin most likely occurs during the transition from preneoplasia to carcinoma because hyperplasias had normal levels of gelsolin whereas microtumors had reduced expression. Southern analysis revealed no major mutations in the gelsolin gene of tumors with low expression. Cyclin D1 mRNA was increased in 50–100% of these rodent mammary tumors, although the cyclin D1 gene was not amplified. By nuclear runon assay, downregulation of gelsolin in both human and mouse mammary cancer cells involved diminished transcription and, conversely, human breast cancer cells expressing high levels of cyclin D1 had increased initiation of cyclin D1 transcription compared with cyclin D1 low expressors. Thus, alteration in the rate of transcription appears to be an important factor underlying the dysfunction of these genes. According to our data, concurrent deregulation of gelsolin and cyclin D1 is highly prevalent among breast cancers of humans and rodents, with both defects present in 89% of the neoplasms analyzed in this study. In fact, most tumors in every rodent model of mammary tumorigenesis examined had the 2 alterations. Int. J. Cancer 81:930–938, 1999.

Lipids noncovalently associated with keratins and other cytoskeletal proteins of mouse mammary epithelial cells in primary culture

Lipids noncovalently associated with cytoskeletal (CS) proteins of mouse mammary epithelial cells (MMEC) grown in primary culture were analyzed. A CS fraction, prepared by subjecting MMEC to 1.5 M KCl and 1% Triton X-100 in phosphate buffered saline (pH 7.4), was extracted 4-6 times with chloroform/methanol. Thin-layer chromatography (TLC) indicated that in comparison to whole cell lipid extracts, CS lipids consisted mostly of neutral lipids, especially triacylglycerols and, possibly cholesteryl esters. TLC analysis of chloroform/methanol CS extracts prepared from MMEC that had been incubated 4 h in [3H]palmitate revealed similar results, with the majority of label appearing in triacylglycerols and other neutral lipids. By autoradiography of sodium dodecyl sulfate polyacrylamide gels, all of the major CS proteins appeared labelled. The major regions of autoradiographic density of the gel were excised, the protein solubilized, and the lipids extracted and subjected to TLC. Most of the radiolabel appeared at the origin and ion front and resolved as neutral lipids. In contrast, keratins of 54-55 kDa and 46 kDa appeared to be associated noncovalently with a higher ratio of polar lipids (possibly phospholipids) to nonpolar (neutral lipids). Very little radioactivity, mostly neutral lipid, was associated with actin. A previously unidentified CS component of 30 kDa had primarily noncovalently bound neutral lipid. The results are discussed in terms of the apparent interactions of keratin filaments with the plasma membrane, nuclear envelope and cytoplasmic organelles.

Structural Components as Markers of Differentiation and Neoplastic Progression in Mammary Epithelial Cells

One of the primary aims in breast cancer research has been the development of molecular markers that can a) distinguish among the different cell types (epithelial, myoepithelial, stem cell, stromal, etc.) present in normal and abnormal mammary tissues and in cultures derived from these tissues, and b) identify a cell as normal, hyperplastic/preneoplastic, or malignant. Components of the cell surface and the cytoskeleton are especially promising candidates for such markers. A variety of surface antigens, usually defined by monoclonal antibodies, have now been described and were the subject of a number of recent reviews (1–5). The focus of the present chapter will therefore be on the cytoskeleton and in particular on intermediate filaments and their constituent proteins.