Grace Bradley

P-glycoprotein, multidrug resistance and tumor progression

P-glycoprotein (Pgp) is a plasma membrane protein that was first characterised in multidrug resistant cell lines. The occurrence of Pgp in clinical tumors has been widely studied. Recent investigations have begun to focus on the relationship between Pgp detection in tumors and treatment outcome. In several types of tumors, detection of Pgp correlates with poor response to chemotherapy and shorter survival. P-glycoprotein overexpression often occurs upon relapse from chemotherapy but may also occur at the time of diagnosis. Studies of experimental rat liver carcinogenesis have shown that Pgp expression increases in late stages of carcinogenesis, suggesting that Pgp may be involved in tumor progression. While some of the Pgp isoforms are known to transport hydrophobic chemotherapeutic drugs out of tumor cells, the biologic effects of Pgp overexpression in tumor cells are not fully understood, because the spectrum of substrates for Pgp-mediated transport has not been determined. In the rat liver carcinoma model, strong expression of Pgp is associated with a highly vascular stroma, suggesting that Pgp in tumor cells may affect the connective tissue stroma. The regulation of Pgp appears to be complex, and little is known about how it is up-regulated during carcinogenesis. Further studies of the role of Pgp in malignancy may contribute to our understanding of molecular mechanisms which underlie tumor progression.

Identification of a microRNA signature associated with progression of leukoplakia to oral carcinoma

MicroRNAs (miRs) are non-coding RNA molecules involved in cancer initiation and progression. Deregulated miR expression has been implicated in cancer; however, there are no studies implicating an miR signature associated with progression in oral squamous cell carcinoma (OSCC). Although OSCC may develop from oral leukoplakia, clinical and histological assessments have limited prognostic value in predicting which leukoplakic lesions will progress. Our aim was to quantify miR expression changes in leukoplakia and same-site OSCC and to identify an miR signature associated with progression. We examined miR expression changes in 43 sequential progressive samples from 12 patients and four non-progressive leukoplakias from four different patients, using TaqMan Low Density Arrays. The findings were validated using quantitative RT-PCR in an independent cohort of 52 progressive dysplasias and OSCCs, and five non-progressive dysplasias. Global miR expression profiles distinguished progressive leukoplakia/OSCC from non-progressive leukoplakias/normal tissues. One hundred and nine miRs were highly expressed exclusively in progressive leukoplakia and invasive OSCC. miR-21, miR-181b and miR-345 expressions were consistently increased and associated with increases in lesion severity during progression. Over-expression of miR-21, miR-181b and miR-345 may play an important role in malignant transformation. Our study provides the first evidence of an miR signature potentially useful for identifying leukoplakias at risk of malignant transformation.

EWSR1‐ATF1 fusion is a novel and consistent finding in hyalinizing clear‐cell carcinoma of salivary gland

Hyalinizing clear‐cell carcinoma (HCCC) is a rare, low‐grade salivary gland tumor with distinctive clear‐cell morphology and pattern of hyalinization as well as focal mucinous differentiation. However, histological overlap exists with other salivary gland tumors, such as epithelial–myoepithelial carcinoma (EMCa), salivary myoepithelial carcinoma, and mucoepidermoid carcinoma (MEC). The potential relationship between HCCC and its morphological mimics has not been yet investigated at the genetic level. In this study, we conducted a molecular analysis for the presence of rearrangements in MAML2, commonly seen in MECs, and EWSR1, involved in “soft tissue myoepithelial tumors” (SMET) by fusion with POU5F1, PBX1, or ZNF444. Fluorescence in situ hybridization (FISH) was performed on 23 HCCC cases for abnormalities in MAML2, EWSR1, FUS, POU5F1, PBX1, and ZNF444. FISH for MAML2 was negative in all cases (0 of 14), including those with mucinous differentiation (0 of 7). An EWSR1 rearrangement was identified in 18 of 22 HCCCs (82%), while no break‐apart signals were seen in FUS, POU5F1, PBX1, or ZNF444. 3′RACE on an EWSR1 rearranged HCCC identified an EWSR1‐ATF1 fusion, which was confirmed by RT‐PCR. ATF1 involvement was further confirmed by FISH analysis in 13 of 14 EWSR1‐rearranged HCCC cases (93%). In contrast, all control cases tested, including among others 5 EMCa and 3 MEC with clear cells, were negative for EWSR1 and ATF1 rearrangements. The presence of EWSR1‐ATF1 fusion in most HCCCs reliably separates these tumors from its histological mimics. The distinction from MEC is particularly important, as conventional MEC grading schemes overgrade these indolent HCCCs, potentially impacting on treatment.

Analysis of Genomic Integrity and p53-Dependent G1 Checkpoint in Telomerase-Induced Extended-Life-Span Human Fibroblasts

ABSTRACT Life span determination in normal human cells may be regulated by nucleoprotein structures called telomeres, the physical ends of eukaryotic chromosomes. Telomeres have been shown to be essential for chromosome stability and function and to shorten with each cell division in normal human cells in culture and with age in vivo. Reversal of telomere shortening by the forced expression of telomerase in normal cells has been shown to elongate telomeres and extend the replicative life span (H. Vaziri and S. Benchimol, Curr. Biol. 8:279–282, 1998; A. G. Bodnar et al., Science 279:349–352, 1998). Extension of the life span as a consequence of the functional inactivation of p53 is frequently associated with loss of genomic stability. Analysis of telomerase-induced extended-life-span fibroblast (TIELF) cells by G banding and spectral karyotyping indicated that forced extension of the life span by telomerase led to the transient formation of aberrant structures, which were subsequently resolved in higher passages. However, the p53-dependent G1 checkpoint was intact as assessed by functional activation of p53 protein in response to ionizing radiation and subsequent p53-mediated induction of p21Waf1/Cip1/Sdi1. TIELF cells were not tumorigenic and had a normal DNA strand break rejoining activity and normal radiosensitivity in response to ionizing radiation.

p53 gene mutations in sequential oral epithelial dysplasias and squamous cell carcinomas

Previous studies of oral cancer have suggested that alterations of the p53 tumour suppressor gene occur early in the precancerous stage of development. However, these observations have been based on cross‐sectional assessment of abnormal p53 protein staining by immunohistochemistry and may not necessarily reflect gene changes. The purpose of this longitudinal study was to examine the changes in the p53 gene in progressive, sequential epithelial dysplasias and carcinomas from the oral cavity. The study analysed 24 formalin‐fixed, paraffin‐embedded tissue biopsies from ten patients with two or more temporally distinct lesions from the same site in the oral cavity with the diagnosis of hyperkeratosis, epithelial dysplasia, carcinoma in situ or squamous cell carcinoma. Exons 5–8 of the p53 gene were amplified from genomic DNA using intronic primers and directly sequenced using fluorescent‐labelled primers. Standard immunohistochemistry with the DO7 monoclonal antibody was used to detect mutant and wild‐type p53 protein. Mutations of the p53 gene were identified in 9 of 24 samples. Eight were missense mutations and one occurred at a splice site. In six patients, mutations of the p53 gene occurred late after the transformation of epithelial dysplasia to carcinoma. In two patients with progressive dysplasia, but who had yet to develop invasive carcinoma, p53 missense mutations occurred at the carcinoma in situ stage in one case and in a moderate dysplasia in the other. There was an inconsistent relationship between gene mutations and the level of p53 protein staining by immunohistochemistry. It is concluded that during oral carcinogenesis, p53 gene mutations seem to occur relatively late and are associated with transformation to the invasive phenotype. Copyright

Patterns of p53 and Ki-67 protein expression in epithelial dysplasia from the floor of the mouth

Oral squamous cell carcinoma develops through a series of precancerous stages manifested at the microscopic level as epithelial dysplasia. Mutation of the p53 tumour suppressor gene is thought to be an important component of oral carcinogenesis. p53 regulates cell proliferation and DNA repair by inhibiting the cell cycle at G1/S; loss of p53 function may therefore lead to aberrant cell kinetics. To date, no studies have examined the relationship between p53 protein and alterations in cell kinetics in oral epithelial dysplasia from a single anatomical site. Serial sections were studied from 40 routinely processed biopsy specimens of epithelial dysplasia from the floor of the mouth. The expression of p53 protein was determined by immunohistochemistry and cell proliferation was studied by immunostaining for the cell cycle‐dependent protein Ki‐67. The number of positive cells per millimetre of basement membrane was determined using computer image analysis and compared with site‐matched normal controls. The mean p53 labelling index (LI) in normal mucosa was low, 3·48±0·92 [mean±95 per cent confidence interval (CI)], and increased sharply in the transition from mild (42·49±21·71) to moderate (104·86±51·39) epithelial dysplasia. The mean p53 LI for severe dysplasia was 119·09±56·50. Differences were also observed in the distribution of p53‐positive cells between grades of dysplasia, with the development of compact p53‐positive foci in severe dysplasia. Mean proliferative indices, as determined by Ki‐67 expression, were significantly associated with grade of epithelial dysplasia. Furthermore, there was a significant correlation between p53 LI and Ki‐67 score (r2=0·37, P=0·01). It is concluded that altered p53 protein expression is probably an early event in oral carcinogenesis in the floor of the mouth and is associated with dysregulation of cell proliferation at this site.

Abnormalities of the ARF-p53 pathway in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is associated with heavy smoking and drinking, but the molecular pathway of tumorigenesis is not understood. Inactivation of the p53 tumor suppressor gene is likely to play an important role since p53 mutation is frequently found. The p14ARF tumor suppressor gene is functionally linked to p53, because it is activated by oncogenes and causes p53-dependent growth arrest and apoptosis. The relationship between p14ARF and p53 inactivation has not been described for OSCC. We studied 25 cases of OSCC to determine if there is an inverse correlation between p53 mutation and p14ARF inactivation by homozygous deletion or mutation. p53 mutation was found in 16 of 25 cases (64%), including nine missense and seven truncating mutations. While all cases with missense mutations showed abnormal accumulation of p53 protein, there were also five carcinomas which showed increased p53 staining in the absence of mutation. p14ARF deletion or mutation was found in eight cases (32%), six of which also demonstrated p53 mutation. Our findings indicate that OSCC often involves loss of both p14ARF and p53 function and suggest that inactivation of these two tumor suppressor genes are not functionally equivalent during tumorigenesis.

INCREASED P-GLYCOPROTEIN MESSENGER RNA STABILITY IN RAT LIVER TUMORS IN VIVO

P‐glycoproteins (Pgp) are comprised of a small family of plasma membrane proteins whose abundance in cultured cells is often associated with the multidrug resistance phenotype. Overexpression of Pgp has been observed in many types of human cancers, but the molecular basis for this overexpression has not been established. We have used primary monolayer cultures of adult rat hepatocytes and a stepwise model of rat liver carcinogenesis to study the regulation of Pgp gene expression. We observed a marked overexpression of Pgp, specifically the class II Pgp, in both systems. In addition, we observed that a number of unrelated genes including α‐tubulin, β‐actin, γ‐actin, cytokeratin 8, cytokeratin 18, and c‐myc are overexpressed in cultured hepatocytes, and they are also overexpressed during liver carcinogenesis and in transplantable tumors. Nuclear run‐on assays showed no increase in the transcriptional activity of Pgp genes in transplantable liver tumors compared to normal liver. Studies of in vivo mRNA stability, however, revealed that all three Pgp mRNAs were relatively stable in transplantable liver tumors (t1/2 > 12 h), in contrast to what was found in normal liver (t1/2 < 2 h). In addition, mRNA for several other genes, including α‐tubulin, c‐myc, and cyclin D1, all appear to be stabilized in the tumors. These findings suggest that the overexpression of Pgp genes in rat liver tumors may be the result of a mechanism involving stabilization of a diverse group of mRNAs. J. Cell. Physiol. 177:1–12, 1998.

SNAI1 expression and the mesenchymal phenotype: an immunohistochemical study performed on 46 cases of oral squamous cell carcinoma

BackgroundSNAI1 can initiate epithelial-mesenchymal transition (EMT), leading to loss of epithelial characteristics and, in cancer, to invasion and metastasis. We hypothesized that SNAI1 reactivation occurs in oral squamous cell carcinoma (OSCC) where it might also be associated with focal adhesion kinase (FAK) expression and p63 loss.MethodsImmunohistochemistry was performed on 46 tumors and 26 corresponding lymph node metastases. Full tissue sections were examined to account for rare and focal expression. Clinical outcome data were collected and analyzed.ResultsSNAI1-positivity (nuclear, ≥ 5% tumor cells) was observed in 10 tumors and 5 metastases (n = 12 patients). Individual SNAI1(+) tumor cells were seen in primary tumors of 30 patients. High level SNAI1 expression (>10% tumor cells) was rare, but significantly associated with poor outcome. Two cases displayed a sarcomatoid component as part of the primary tumor with SNAI1(+)/FAK(+)/E-cadherin(-)/p63(-) phenotype, but disparate phenotypes in corresponding metastases. All cases had variable SNAI1(+) stroma. A mesenchymal-like immunoprofile in primary tumors characterized by E-cadherin loss (n = 29, 63%) or high cytoplasmic FAK expression (n = 10, 22%) was associated with N(+) status and tumor recurrence/new primary, respectively.ConclusionsSNAI1 is expressed, although at low levels, in a substantial proportion of OSCC. High levels of SNAI1 may herald a poor prognosis and circumscribed SNAI1 expression can indicate the presence of a sarcomatoid component. Absence of p63 in this context does not exclude squamous tumor origin. Additional EMT inducers may contribute to a mesenchymal-like phenotype and OSCC progression.

Cyclin Alterations in Giant Cell Tumor of Bone

Cyclins play an important role in regulating the passage of dividing cells through critical checkpoints in the cell cycle. Because alterations of several cyclins, especially cyclin D1, have been implicated in the development of many human neoplasms, we examined 32 cases of giant cell tumor of long bones for cyclin D1 gene amplification and protein overexpression using differential polymerase chain reaction and immunohistochemistry, respectively. In addition, the expression of cyclin D3, cyclin B1, and the proliferation-associated antigen Ki-67 (MIB-1) was assessed immunohistochemically. Low-level cyclin D1 gene amplification was detected in 61% of giant cell tumor cases. All tumors showed cyclin D1, cyclin D3, cyclin B1, and Ki-67 (MIB-1) staining; however, the distribution was very characteristic. Cyclin D1 protein expression was seen predominantly in the nuclei of the giant cells, with occasional mononuclear cells staining. There was no correlation between cyclin D1 gene amplification and protein overexpression. Cyclin D3 staining showed a similar distribution, with 88% of cases showing protein overexpression. Cyclin D1 and/or D3 staining in the giant cells was never associated with staining for either cyclin B1 or Ki-67 (MIB-1), as the expression of the latter two proteins was restricted to the mononuclear cells. Cyclin B1 overexpression was seen in 44% of cases. Ki-67 (MIB-1) staining was present in all cases, and between 10 to 50% of the mononuclear cells were positive. These results suggest that alterations in cyclin D1 and/or D3 might play a role in the pathogenesis of giant cell tumor of bone.