The Use of Smoothelin and Other Antibodies in the Diagnosis of Uterine and Soft Tissue Smooth Muscle Tumors.

Smoothelin is a cytoplasmic protein expressed in differentiated smooth muscle cells. Immunohistochemical evaluation of smoothelin has previously been reported in gastrointestinal (GI) smooth muscle tumors, but has yet to be studied in smooth muscle tumors of uterine and other soft tissue origin. DOG1 expression is reported to be specific for GI stromal tumors; however, variable expression has been reported in leiomyosarcomas (LMS) depending on site of origin. Overexpression of p16 is common in LMS of uterine and other sites of origin, but has not been correlated with tumor grade. This study explores the differential expression of these markers, as well as caldesmon, in LMS cases to assess diagnostic utility. Using tissue microarrays and cases from Tulane Medical Center and Medical College of Wisconsin, expression of smoothelin, DOG1, caldesmon, and p16 was evaluated by immunohistochemistry in 87 cases of LMS. The cases were subdivided by location of origin into uterine (N=31) and nonuterine (N=56) with 10 of the nonuterine of GI origin, as well as by grade into low grade (N=27) and intermediate and high grade (N=60). Differential expression among different grades and locations was evaluated. The same markers were evaluated in atypical leiomyoma cases (N=4) and 1 smooth muscle tumor of uncertain malignant potential case (N=1). Smoothelin expression was also assessed in 20 benign uterine leiomyomas. Weak DOG1 expression is rare but possible in extrauterine LMS. Expression of p16 is common in both uterine and extrauterine LMS, and more frequent in higher grades. Expression of smoothelin in this study differed depending on tumor type, grade, and site of origin. All leiomyomas and most atypical leiomyomas showed cytoplasmic positivity for smoothelin, whereas only 5% of LMS had cytoplasmic expression. The study suggests smoothelin may be downregulated in the cytoplasm of malignant smooth muscle tumor cells and may serve as a supportive aid in the distinction of LMS from benign smooth muscle tumors in cases where it is difficult by morphology alone.

Phospho-Akt overexpression in non-small cell lung cancer confers significant stage-independent survival disadvantage.

PURPOSE: Akt is a signal transduction protein that plays a central role in inhibiting apoptosis in a variety of cell types including human cancer cells. In cell lines derived from human non-small cell lung cancers (NSCLCs), Akt has been shown to confer chemoresistance by inhibition of apoptosis in response to different chemotherapeutic agents including platinum-based agents, which are often the first-line therapy for NSCLCs. Only 20% to 30% of patients with NSCLC treated with chemotherapy have clinical evidence of response. The purpose of this study is to determine whether or not overexpression of activated Akt [i.e., phosphorylated Akt (pAkt)] is correlated with survival. EXPERIMENTAL DESIGN: We studied tumors from 61 patients with NSCLC in three tissue microarrays. All patients were followed for a period of 10 years or until death. The arrays were studied immunohistochemically with antibodies against pAkt, p53, and Ki-67. RESULTS: There was a statistically significant difference in survival between the 14 patients with strong pAkt staining and the 47 patients with weak to absent pAkt staining both by log-rank (P = 0.0416) and Breslow analysis (P = 0.0446). Difference in survival time with respect to pAkt status was also statistically significant even after accounting for stage at diagnosis (P = 0.004). Neither p53 nor Ki-67 was a statistically significant prognostic factor. CONCLUSIONS: Overexpression of pAkt is an independent prognostic factor. Additional studies of human NSCLCs are warranted to drive the development of targeted tumor-specific antineoplastic therapies.

Spatial composition of prostate cancer spheroids in mixed and static cultures.

Aggregation of neoplastic cells produces multicellular spheroids resembling micrometastases. The objective of this study was to investigate the effects of mixing culture medium on the spatial composition of spheroids prepared from well (LNCaP) and poorly (DU 145) differentiated human prostate cancer cells. Spheroids were cultured in a mixed suspension within a high-aspect rotating wall vessel and static liquid-overlay plate. Results from this study demonstrate that mixed cultures consistently manifested differences in morphology and composition between DU 145 and LNCaP spheroids. For example, 40 +/- 12% of DU 145 cells were Ki-67 positive 100 microm from the surface within mixed spheroids versus 0% for LNCaP cells; there was no significant difference in this spatial profile for static cultures. The results suggest that poorly differentiated spheroids may be more likely to experience a change in composition from mixing culture medium than well-differentiated spheroids, due to low tissue density. Immunostaining for P-glycoprotein is representative of this trend; average staining intensity increased 50% for DU 145 spheroids on mixing but was unchanged for LNCaP spheroids. The effects of mixing on spheroid composition were attributed to faster interstitial mass transport. Applications include drug development and delivery, as well as basic research on drug action and resistance.

AKT regulation of estrogen receptor beta transcriptional activity in breast cancer.

Growth factor activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway has been shown to activate the estrogen receptor (ER) alpha and to mediate tamoxifen resistance in breast cancer. Here, we investigated the regulation of the transcriptional activity of the newer ER beta by PI3K-AKT signaling. Tissue arrays of breast cancer specimens showed a positive association between the expressions of AKT and ER beta in the clinical setting. Reporter gene assays using pharmacologic and molecular inhibitors of AKT and constitutively active AKT revealed for the first time the ability of AKT to (a) potentiate ER beta activity and (b) target predominantly the activation function-2 (AF2) domain of the receptor, with a requirement for residue K269. Given the importance of coactivators in ER transcriptional activity, we further investigated the possible involvement of steroid receptor coactivator 1 (SRC1) and glucocorticoid receptor-interacting protein 1 (GRIP1) in AKT regulation of ER beta. Mammalian two-hybrid assays revealed that AKT enhanced both SRC1 and GRIP1 recruitment to the ER beta-AF2 domain, and reporter gene analyses revealed that AKT and GRIP1 cooperatively potentiated ER beta-mediated transcription to a level much greater than either factor alone. Investigations into AKT regulation of GRIP with mammalian one-hybrid assays showed that AKT potentiated the activation domains of GRIP1 itself, and in vitro kinase assays revealed that AKT directly phosphorylated GRIP1. The cross-talk between the PI3K-AKT and ER beta pathways, as revealed by the ability of AKT to regulate several components of ER beta-mediated transcription, may represent an important aspect that may influence breast cancer response to endocrine therapy.

Immunohistochemical analysis of differentiation in static and mixed prostate cancer spheroids.

Neoplastic multicellular spheroids are in vitro models of solid tumors employed in drug testing and basic research. This study compares differentiation in static and mixed prostate cancer spheroids. Staining intensity of prostate specific antigen (PSA) was down-regulated upon mixing, from 0.21 +/- 0.03 to 0.13 +/- 0.03 in LNCaP multicellular spheroids, and from 0.13 +/- 0.04 to 0.03 +/- 0.02 in DU 145 multicellular spheroids. This was accompanied by 65% increase in the expression of cytokeratins 8 and 18 in DU 145 spheroids. PSA expression extended 60 micro m within static spheroids and was disrupted in mixed culture. Diminished PSA expression and spatial organization suggests a more aggressive cancer. Higher cytokeratin expression could result from either differentiation towards a luminal phenotype or activation of the Ras pathway during dedifferentiation. Thus, the existing paradigm of differentiation established for normal tissue does not apply for our neoplastic spheroids. Cell dedifferentiation is attributed to improved interstitial transport and synthesis of extracellular matrix.