Loss of fibulin-2 expression is associated with breast cancer progression

Fibulin-2, an extracellular matrix protein expressed by normal epithelia, was found to be down-regulated in several breast cancer cell lines. Fibulin-2 protein expression was also decreased in breast cancer tissue samples as evaluated by immunohistochemistry. Reintroduction of Fibulin-2 into breast cancer cell lines that do not express Fibulin-2 reduced cancer cell motility and invasion in vitro but had no effect on cell growth and adhesion properties. Together with evidence that Fibulin-2 contributes to wound healing and inhibits smooth muscle cell migration, our findings suggest that loss of Fibulin-2 expression may facilitate migration and invasion in breast cancer.     

Loss of occludin leads to the progression of human breast cancer

Occludin is an integral membrane protein localised at tight junctions (TJs). It has become clear that the TJ is an important structure that cancer cells must overcome in order to metastasize successfully. The purpose of this study was to elucidate the importance of the expression of occludin in human breast cancer. Human tissues and breast cancer cell lines were amplified for functional regions of occludin. Tumour tissues showed truncated and/or variant signals. There was also considerable variation in the expression of occludin in the 10 human breast cancer cell lines investigated. Western blotting demonstrated that variants in the MDA-MB-231 and MCF-7 human breast cancer cell lines did not fit the expected occludin signals for changes in phosphorylation status. Immunostaining showed similarly disparate levels of expression. Ribozyme knockdown resulted in increased invasion, reduced adhesion and significantly reduced TJ functions. Q-RT-PCR analysis of 124 tumour and 33 background human breast tissues showed occludin to be significantly decreased in patients with metastatic disease. Immunohistochemical staining showed a decreased expression of occludin in the tumour sections. This study demonstrates for the first time that occludin is differentially expressed in human breast tumour tissues and cell lines. This loss of or aberrant expression has clear repercussions as to the importance of occludin in maintaining TJ integrity in breast tissues. Such inappropriate expression could play a part in breast cancer development.     

Loss of immunohistochemical E-cadherin expression in colon cancer is not due to structural gene alterations

 E-cadherin, a transmembrane cell adhesion molecule, has been observed to have an altered pattern of immunoreactivity in several types of carcinomas. In lobular breast cancer, loss of immunoreactivity has been shown to be due either to out-of-frame deletions or to nonsense mutations of the E-cadherin gene. We analysed 29 cases of completely resected colon carcinoma with immunohistochemistry using the HEC-D1 antibody. Normal protein expression similar to that in the adjacent nonmalignant mucosa was seen in 6 cases, whereas 23 tumours had reduced or absent E-cadherin expression. In the 8 cases with no expression of E-cadherin revealed by immunohistochemistry, the entire E-cadherin cDNA sequence was analysed. In these cases, sequence analysis failed to reveal any cDNA mutations despite the negative immunohistochemistry. Possible explanations for this discrepancy include regulatory defects in the E-cadherin promoter, abnormalities at the translation or protein processing levels and mutations in other parts of the gene that were not investigated by the cDNA analysis (e.g. intronic sequences), which could play a role in causing abnormal processing of the E-cadherin protein. Received: 14 September 1998 / Accepted: 23 February 1999     

Routine testing for PALB2 mutations in familial pancreatic cancer families and breast cancer families with pancreatic cancer is not indicated

PALB2-mutation carriers not only have an increased risk for breast cancer (BC) but also for pancreatic cancer (PC). Thus far, PALB2 mutations have been mainly found in PC patients from families affected by both PC and BC. As it is well known that the prevalence of gene mutations varies between different populations, we studied the prevalence of PALB2 mutations in a Dutch cohort of non-BRCA1/2 familial PC (FPC) families and in non-BRCA1/2 familial BC (FBC) families with at least one PC case. Mutation analysis included direct sequencing and multiplex ligation-dependent probe amplification (MLPA) and was performed in a total of 64 patients from 56 distinct families (28 FPC families, 28 FBC families). In total, 31 patients (48%) originated from FPC families; 24 were FPC patients (77%), 6 had a personal history of BC (19%) and 1 was a suspected carrier (3.2%). The remaining 33 patients (52%) were all female BC patients of whom 31 (94%) had a family history of PC and 2 (6.1%) had a personal history of PC. In none of these 64 patients a PALB2 mutation was found. Therefore, PALB2 does not have a major causal role in familial clustering of PC and BC in non-BRCA1/2 families in the Dutch population.     

Loss of phenotypic expression is related to tumour progression in early gastric differentiated adenocarcinoma

AIMS : To evaluate the relationship between phenotypic expression and tumour progression as represented by macroscopic features, submucosal invasion and lymph node metastasis in early differentiated gastric adenocarcinoma. METHODS : One hundred and fifty-five cases of early gastric differentiated adenocarcinoma without any poorly differentiated components were studied. The mucosal and submucosal components of carcinomas and lymph node metastatic lesions were classified into four categories, gastric type (G-type), incomplete intestinal type (I-type), complete intestinal type (C-type) and unclassified type (U-type), based on the combination of the phenotypic expression of HGM (gastric foveolar epithelium), MUC 6 (gastric pyloric glands), MUC 2 (intestinal goblet cells) and CD 10 (small intestinal brush border). In addition, a new classification representing a phenotypic shift from mucosa to submucosa or from primary lesion to lymph node metastasis was established with the categories of preserved group (P-group), loss group (L-group) and acquired group (A-group). RESULTS : (1) In submucosal carcinoma, U-type was more common in the submucosa (39%) than in the mucosa (9%). (2) U-type was more common in the metastatic lesions (42%) than in the primary lesions (5%). (3) The submucosal component and lymph node metastatic lesions were classified as P-group in 48% and 43% of cases, respectively, and as L-group in 50% and 52% of cases, respectively. (4) Lymph node metastatic lesions comprising undifferentiated carcinoma were classified as L-group in 100% of cases. CONCLUSION : During the course of tumour progression, early differentiated adenocarcinoma at first tends to lose its phenotypic expression despite preserving its morphology, but subsequently morphological dedifferentiation occurs.     

Loss of self-control in intertemporal choice may be attributable to logarithmic time-perception

Impulsivity and loss of self-control in drug-dependent patients have been associated with the manner in which they discount delayed rewards. Although drugs of abuse have been shown to modify perceived time-duration, little is known regarding the relationship between impulsive decision-making in intertemporal choice and estimation of time-duration. In classical economic theory, it has been hypothesized that people discount future reward value exponentially. In exponential discounting, a temporal discounting rate is constant over time, which has been referred to as dynamic consistency. However, accumulating empirical evidence in biology, psychopharmacology, behavioral neuroscience, and neuroeconomics does not support the hypothesis. Rather, dynamically inconsistent manners of discounting delayed rewards, e.g., hyperbolic discounting, have been repeatedly observed in humans and non-human animals. In spite of recent advances in neuroimaging and neuropsychopharmacological study, the reason why humans and animals discount delayed rewards hyperbolically is unknown. In this study, we hypothesized that empirically-observed dynamical inconsistency in intertemporal choice may result from errors in the perception of time-duration. It is proposed that perception of temporal duration following Weber's law might explain the dynamical inconsistency. Possible future study directions for elucidating neural mechanisms underlying inconsistent intertemporal choice are discussed.     

Oncogenic PIK3CA mutations in lobular breast cancer progression

Infiltrating lobular breast cancer (ILBC) is a tumor‐biologically distinct breast cancer subtype. A high frequency of oncogenic PIK3CA mutations has been reported in ILBC, which may allow for targeted therapy with newly developed PI3K inhibitors. This is of particular clinical relevance for ILBC patients, who have failed to respond to current treatment regimes and suffer from tumor recurrence or dissemination. In anticipation of this therapeutic strategy, we investigated PIK3CA mutations in ILBC with special reference to late stage tumor progression. A total of 88 ILBCs from 73 patients, including primary tumors (PTs, n = 43), ipsilateral locally recurrent tumors (LRTs, n = 15), and distant organ metastases (DOMs, n = 30), were compiled on tissue microarrays. Established ILBC marker proteins were evaluated by immunohistochemistry and PIK3CA hot spot mutations in exons 9 and 20 by direct sequencing. Matched PT/LRT, PT/DOM, and DOM/DOM cases were characterized on a patient‐by‐patient basis. Following correction for redundant patient representations, mutation frequencies were compared in PTs versus LRTs or DOMs. Nearly all specimens were E‐cadherin‐negative (99%), estrogen receptor (ER)‐positive (91%), and lacked basal epithelial markers (100%), demonstrating correct ILBC classification. PIK3CA mutations were detected in 32/88 (36%) specimens. The mutation rate was similar in PTs (33%) and DOMs (26%, P = 0.769), but approximately two‐fold increased in LRTs (69%, P = 0.022). Consistently, matched PT/LRT and LRT/DOM cases showed additional PIK3CA mutations in LRTs. Intriguingly, these findings imply that PIK3CA mutations are positively selected for during ILBC progression to local recurrence but not distant metastasis, which may have clinical implications for PI3K inhibitor‐based therapy. © 2012 Wiley Periodicals, Inc.     

Downregulation of GLTSCR2 expression is correlated with breast cancer progression

Glioma tumor-suppressor candidate region gene2 (GLTSCR2) is a recently identified nucleolus-localized protein participating in the regulation of cell cycle progression and apoptosis. Down-regulation of GLTSCR2 in several types of cancers and increased transforming activity in GLTSCR2-downregulated cancer cells indicated its tumor suppressive potential. The aim of this study was to evaluate GLTSCR2 expression in breast cancer and to investigate the question of whether reduced expression of GLTSCR2 may have any pathological significance in breast cancer development or progression. In this study, we performed quantitative RT-PCR and immunohistochemistry to evaluate the expression of GLTSCR2 and relevance with clinicopathological factors in the invasive ductal carcinoma (IDC). GLTSCR2 expression was reduced in 48% of IDC (n = 426) by a semi-quantitative scoring system using tissue microarray. GLTSCR2 mRNA was significantly reduced by 0.16 fold in 15 out of 17 (88%) cases of IDC. Reduction of GLTSCR2 was significantly correlated with increased histological grade (p < 0.005), increased tumor size (p < 0.001), axillary lymph node involvement (p < 0.001) and decreased disease free survival (p < 0.025). In addition, we show that upregulation of GLTSCR2 decreases the invasive potential of breast cancer cells. Taken together, our data suggest that GLTCR2 may play a role in the tumorigenesis, progression and biological behavior in breast cancer.     

Loss of heterozygosity of MCC is not associated with mutation of the retained allele in sporadic colorectal cancer

The adenomatous polyposis coli (APC) gene, which transmits familialadenomatous polyposis, is frequently mutated in sporadic colorectaltumours. Acquired somatic mutations have also been reportedin a second gene, mutated in colorectal cancer (MCC), whichlies within 500 kb of APC on chromosome 5q21 and has thus beenImplicated In tumour development. Further evidence for an oncosuppressorgene other than APC on chromosome 5q comes from recent studiesof lung, renal and hepatic cancers in which there Is loss ofheterozygosity of 5q21 but no somatic APC mutations. To investigatethe relative importance of APC and MCC In sporadic colorectalcancer, we have assessed the extent of 5q21 allellc loss in80 carcinomas. All informative tumours exhibiting allellc losshad deletions which included both APC and MCC. In 21 tumourswith loss of heterozygosity In MCC we have screened the entirecoding region of the gene for mutation of the retained alleleand found no evidence for mutation. The data Indicate that independentloss of MCC Is a rare event, and that in cases where alleleloss occurs mutation of the retained allele Is uncommon. Thissuggests that MCC does not function as an independent tumoursuppressor In the majority of colorectal cancers.     

Loss of microRNA-205 expression is associated with melanoma progression

In this study, we used formalin-fixed paraffin-embedded melanocytic tumors to demonstrate reproducible alterations in microRNA expression in nevi compared with melanomas using a microarray platform. We validated those results in an independent set of nevi and melanomas by quantitative RT-PCR. miR-205 demonstrated a statistically significant, progressive diminution in expression from nevi to primary melanomas to metastatic melanomas. Enforced miR-205 expression in melanoma cells profoundly impairs cell motility and migration along with significantly decreased F-actin polymerization with only a modest reduction in cell proliferation. Using a xenograft model, melanoma cells overexpressing miR-205 exhibit a reduced migratory capacity compared with control tumor cells. Mechanistically, miR-205 overexpression results in decreased expression of the zinc-finger E-box binding homeobox 2 (ZEB2) mRNA and protein. This coincides with increased expression of E-cadherin mRNA and protein. Furthermore, re-introduction of ZEB2 into melanoma cells overexpressing miR-205 rescues these phenotypic effects and results in a restoration of cell migration and F-actin polymerization with a concomitant reduction in E-cadherin expression. Together, these results provide in vitro and in vivo evidence for miR-205 as a critical suppressor of melanoma cell migration.     

Loss of raf-1 kinase inhibitor protein expression is associated with tumor progression and metastasis in colorectal cancer

Raf-1 kinase inhibitor protein (RKIP) is known as a critical down-regulator of the mitogen-activated protein kinase signaling pathway and a potential molecular determinant of malignant metastasis. The aim of this study was to determine the prognostic significance of RKIP expression in colorectal cancer (CRC).Immunohistochemical staining for RKIP was performed on a tissue microarray comprising 1,197 mismatch repair (MMR)-proficient and 141 MMRdeficient CRCs. The association of RKIP with clinicopathologic features was analyzed. Loss of cytoplasmic RKIP was associated with distant metastasis (P = .038), higher N stage (P = .032), vascular invasion (P = .01), and worse survival (P = .001) in the MMR-proficient group. In MMR-deficient CRCs, loss of cytoplasmic RKIP was associated with distant metastasis (P = .043) and independently predicted worse survival (P = .004). Methylation analysis of 28 cases showed that loss of RKIP expression is unlikely to be due to promoter methylation.Loss of RKIP expression is a marker of tumor progression and distant metastasis in MMR-proficient and MMR-deficient CRCs.     

Amplification of growth regulatory genes in intraductal breast cancer is associated with higher nuclear grade but not with the progression to invasiveness

Ductal carcinoma in situ (DCIS), as an identifiable progenitor lesion of invasive breast cancer, represents a morphologically, biologically, and prognostically heterogeneous disease. It is not clear which molecular mechanisms are involved in progression to infiltrative growth. In this study, 83 DCIS classified according to the Van Nuys grading scheme were examined for amplification of growth regulatory genes that have been found to be amplified in invasive breast cancer (c-erbB2, topoisomerase IIalpha, c-myc, and cyclinD1 genes). Exact quantification of gene amplification was enabled by a combination of laser microdissection of paraffin-embedded tissue with real-time PCR. In DCIS, gene amplifications of all tested genes were found. The most frequently amplified gene was c-erbB2 found in 21 of 83 (25%) cases. Amplification of the other genes under investigation was observed in 4% to 6% of cases, high-grade DCIS being predominantly affected. High-grade DCIS differed significantly from low- and intermediate-grade DCIS in frequency and level of c-erbB2 amplification. In addition, high-grade DCIS revealed an accumulation of genetic aberrations. Amplification status in pure in situ lesions did not differ from intraductal carcinoma with an infiltrative component, indicating that although associated with a higher nuclear grade gene amplification might not represent an independent prognostic marker of disease progression.     

Heterogeneity of rabbit endogenous pyrogens is not attributable to glycosylated variants of a single polypeptide chain.

Rabbit endogenous pyrogens were of about the same molecular size, but showed considerable heterogeneity of their isoelectric points. We attempted to show that this heterogeneity was attributable to variable glycosylation of a single polypeptide chain. When peritoneal exudate cells were stimulated to make pyrogens in the presence of 2-deoxy-D-glucose, there was a relatively trivial suppression of pyrogen release, and analysis by isoelectric focusing showed parallel inhibition of secretion of all the forms of endogenous pyrogen. When cells were stimulated in the presence of 3H-labeled amino acids and 14C-labeled glucosamine or glucose, the purified pyrogens were labeled with 3H but not with 14C. Macrophage membrane preparations were made which contained glycosyl transferases and could transfer sugar residues from sugar nucleotides to deglycosylated fetuin. These macrophage membrane preparations did not transfer sugars to the pI 7.3 endogenous pyrogen. Treatment of endogenous pyrogens with neuraminidase or with periodate produced no evidence suggesting that the pyrogens were glycosylated. Last, endogenous pyrogens did not bind to any of four lectins with different carbohydrate specificities. This evidence suggests that the heterogeneity of rabbit endogenous pyrogens is not attributable to glycosylation and must have some other cause.     

Loss of subcutaneous adipose tissue in HIV-associated lipodystrophy is not due to accelerated apoptosis

HIV-associated lipodystrophy is characterized by a loss of adipose tissue from the subcutaneous compartment. Previously reported data suggested that this loss of adipose tissue was the result of an increased rate of apoptosis in subcutaneous adipose tissue. The present study examined the rate of apoptosis in subcutaneous adipose tissue with a sensitive ligase-mediated polymerase chain reaction technique to amplify DNA ladders. Individuals with HIV lipodystrophy were compared with HIV-infected subjects without lipodystrophy and subjects without HIV disease. Although apoptosis was observed in subjects with HIV lipodystrophy, there was no difference in the frequency of individuals with apoptosis among those with HIV lipodystrophy (10/22), those with HIV but no lipodystrophy (13/25), and subjects without HIV disease (13/27).     

Hypogammaglobulinaemia in nephrotic rats is attributable to hypercatabolism of IgG

The effect of the nephrotic syndrome induced by puromycin aminonucleoside (PA) in rats on specific antibody responses to 2,4 dinitrophenyl (DNP) conjugated to either spider crab haemocyanin (MSH), a T cell-dependent antigen, or hydroxyethyl starch (HES), a T cell-independent type 2 antigen were studied. The serum IgG anti-DNP levels following immunization with both antigens were reduced in nephrotic animals compared with controls while IgM anti-DNP antibody titres were higher. The half-life of IgG anti-DNP antibodies passively transferred into non-immunized nephrotic rats was markedly reduced while the half-life of anti-DNP antibodies of the IgM class was comparable to that in controls. Low serum IgG and elevated IgM levels were seen in nephrotic animals compared to controls. Antibody-forming cells specific for DNP were demonstrated by immunohistology on rat spleens and the numbers of both IgG and IgM-producing cells were found to be significantly increased (P less than 0.05) in nephrotic animals in response to both DNP-HES and DNP-MSH. These data indicate that in nephrotic rats the alteration seen in the serum immunoglobulin levels is not attributable to reduced antibody production but increased catabolism of serum IgG antibodies.