3-Phosphoinositide-dependent kinase-1 (PDK-1) was identified by its ability to phosphorylate and activate protein kinase B (PKB) in vitro [1,2] and can phosphorylate and activate additional protein kinases in the AGC family in vitro [3-6]. Its role in vivo has, however, only begun to be addressed. We used antisense oligonucleotides directed against PDK-1 expression to explore the role of PDK-1 in human glioblastoma cells (U87-MG), which express a mutant PTEN allele. Reduction in PDK-1 levels resulted in inhibition of PKB activity, and a reduction in phosphorylation on Thr308 and Ser473 of PKB. p70 S6 kinase (p70(S6K)) activity was also reduced. Cell proliferation was dramatically inhibited following treatment with PDK-1 antisense oligonucleotides, due to a combination of decreased cell doubling and an increase in apoptosis. This is in contrast to direct inhibition of phosphoinositide 3-OH kinase (PI 3-kinase), which results in G1 arrest with no effect on apoptosis. This study confirms both PKB and p70(S6K) as in vivo substrates for PDK-1. The effect of acute PDK-1 loss on cell proliferation and survival suggests the involvement of PI 3-kinase dependent and independent signaling events, and implicates PDK-1 as a potential therapeutic target for human neoplasms.

SUMMARY:Genetic heterogeneity in breast cancer has been observed both by cytogenetic and loss of heterozygosity (LOH) analyses; however, the frequency with which genetically heterogeneous clones arise is unknown. In this study, a panel of 115 breast carcinomas was analyzed to determine the extent of clonal divergence in tumor foci at progressive stages of tumor evolution. Intraductal, infiltrating, and metastatic tumor components were microdissected from each tumor and tested for LOH at 20 microsatellite markers on seven chromosomal arms. Of these cases, 24 (21%) demonstrated genetically divergent clones during tumor progression. Clonal divergence, inferred from discordant LOH patterns, was observed most commonly between intraductal and infiltrating tumor (18 cases), but was also demonstrated between infiltrating and metastatic tumor (11 cases). Discordant LOH was observed with markers on one chromosomal arm in 16 cases, on two in 7 cases, and on four in 1 case, and was observed most commonly with markers on 17p, 17q, and 16q. More detailed microdissection of four cases provided evidence for a specific chronology of genetic alterations occurring during the progression of each tumor. The results indicate that the different tumor components observed microscopically in breast cancer specimens often represent genetically divergent clones. (Lab Invest 2000, 80:291-301).E lucidation of the sequence of genetic events responsible for progression of breast cancer from in situ to infiltrating and metastatic carcinoma is an important goal of efforts to understand the biological basis of this common malignancy. Progression is believed to occur through the accumulation of genetic changes via a process of clonal evolution and clonal selection (Brenner and Aldaz, 1997;Nowell, 1976). Surgically resected breast carcinoma specimens provide a unique resource for analyzing the genetic changes that occur during progression, because specimens typically contain foci of tumor in various stages of progression, including in situ carcinoma, invasive tumor, and lymph node metastases. Morphologically normal epithelial constituents of the breast are usually represented in such specimens, and foci of benign proliferative epithelial lesions may also be present.Detailed studies of colon cancer have shown that adenomatous epithelium adjacent to carcinoma typically has some but not all of the genetic lesions present in the fully developed malignancy, consistent with direct progression from adenoma to carcinoma (Boland et al, 1995;Fearon and Vogelstein, 1990;Vogelstein et al, 1988). By analogy, it might be expected that a similar analysis of breast tumors in different stages of progression would likewise show the accumulation of genetic changes with progression. However, genetic analysis of breast cancer specimens has suggested that the individual foci of tumor identifiable by microscopic examination may not show the precursor-product relationship often observed in colon cancer. Cytogenetic studies in particular have revealed sufficient genetic...

Microdissection of histologically identifiable components from formalin-fixed, paraffin-embedded tissue sections allows molecular genetic analyses to be correlated directly with pathological findings. In this study, we have characterized loss of heterozygosity (LOH) at chromosome 11p15 at different stages of progression in microdissected tumor components from 115 ductal carcinomas of the breast. Microdissected foci of intraductal, infiltrating, and metastatic tumors were analyzed to determine the stage of progression at which LOH at 11p15 occurs. LOH was detected in 43 (37%) of 115 cases. Foci of intraductal carcinoma could be microdissected from 85 cases, of which 30 (35%) showed LOH at some stage of progression. LOH was detected in the intraductal component in 26 of these 30 cases. Interstitial deletions were characterized by using a panel of 10 highly polymorphic markers. The smallest region of overlap (SRO) for LOH at 11p15 was bounded by the markers D11S4046 and D11S1758. LOH at 11p15.5 showed no correlation with estrogen receptor status, the presence of positive lymph nodes, tumor size, histological grade, or long-term survival. We conclude that 11p15 LOH usually occurs early in breast cancer development but less frequently does not develop until the infiltrating or metastatic stages of tumor progression.