Inhibition of androgen receptor activity by histone deacetylase 4 through receptor SUMOylation.

The transcriptional activity of the androgen receptor (AR) is regulated by both ligand binding and post-translational modifications, including acetylation and small ubiquitin-like modifier (SUMO)ylation. Histone deacetylases (HDACs) are known to catalyze the removal of acetyl groups from both histones and non-histone proteins. In this study, we report that HDAC4 binds to and inhibits the activity of the AR. This inhibition was found to depend on the SUMOylation, instead of deacetylation, of the AR. Consistently, HDAC4 increases the level of AR SUMOylation in both whole-cell and cell-free assay systems, raising the possibility that the deacetylase may act as an E3 ligase for AR SUMOylation. Knock down of HDAC4 increases the activity of endogenous AR and androgen induction of prostate-specific antigen expression and prostate cancer cell growth, which is associated with decreased SUMOylation of the receptor. Overall, the studies identify HDAC4 as a positive regulator for AR SUMOylation, revealing a deacetylase-independent mechanism of HDAC action in prostate cancer cells.

Deacetylation of cortactin by SIRT1 promotes cell migration.

Cortactin binds F-actin and promotes cell migration. We showed earlier that cortactin is acetylated. Here, we identify SIRT1 (a class III histone deacetylase) as a cortactin deacetylase and p300 as a cortactin acetylase. We show that SIRT1 deacetylates cortactin in vivo and in vitro and that the SIRT1 inhibitor EX-527 increases amounts of acetylated cortactin in ovarian cancer cells. We also show that p300 acetylates cortactin in vivo and that cells lacking or depleted of p300 express less-acetylated cortactin than do control cells. Deletion analysis mapped the SIRT1-binding domain of cortactin to its repeat region, which also binds F-actin. Mouse embryo fibroblasts (MEFs) lacking sir2alpha (the mouse homolog of SIRT1) migrated more slowly than did wild-type cells. The expression of SIRT1 in sir2alpha-null cells restored migratory capacity, as did expression of a deacetylation-mimetic mutant of cortactin. SIRT1 and cortactin were more abundant in breast tumor tissue than in their normal counterparts, whereas SIRT1 expression inversely correlates with the ratio of acetylation cortactin versus total cortactin. These data suggest that deacetylation of cortactin is associated with high levels of SIRT1 and tumorigenesis. Finally, breast and ovarian cancer cell lines expressing an acetylation mimetic mutant of cortactin are less motile than that of control cells, whereas cells expressing the deacetylation mimetic mutant of cortactin migrate faster than that of control cells in Transwell migration assays. In summary, our results suggest that cortactin is a novel substrate for SIRT1 and p300 and, for the first time, a possible role for SIRT1 in cell motility through deacetylation of cortactin.

Phosphatidylinositol-3-OH Kinase (PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen receptor alpha (ERalpha) via interaction between ERalpha and PI3K.

We have shown previously that the AKT2 pathway is essential for cell survival and important in malignant transformation. In this study, we demonstrate elevated kinase levels of AKT2 and phosphatidylinositol-3-OH kinase (PI3K) in 32 of 80 primary breast carcinomas. The majority of the cases with the activation are estrogen receptor alpha (ERalpha) positive, which prompted us to examine whether AKT2 regulates ERalpha activity. We found that constitutively activated AKT2 or AKT2 activated by epidermal growth factor or insulin-like growth factor-1 promotes the transcriptional activity of ERalpha. This effect occurred in the absence or presence of estrogen. Activated AKT2 phosphorylates ERalpha in vitro and in vivo, but it does not phosphorylate a mutant ERalpha in which ser-167 was replaced by Ala. The PI3K inhibitor, wortmannin, abolishes both the phosphorylation and transcriptional activity of ERalpha induced by AKT2. However, AKT2-induced ERalpha activity was not inhibited by tamoxifen but was completely abolished by ICI 164,384, implicating that AKT2-activated ERalpha contributes to tamoxifen resistance. Moreover, we found that ERalpha binds to the p85alpha regulatory subunit of PI3K in the absence or presence of estradiol in epithelial cells and subsequently activates PI3K/AKT2, suggesting ERalpha regulation of PI3K/AKT2 through a nontranscriptional and ligand-independent mechanism. These data indicate that regulation between the ERalpha and PI3K/AKT2 pathway (ERalpha-PI3K/AKT2-ERalpha) may play an important role in pathogenesis of human breast cancer and could contribute to ligand-independent breast cancer cell growth.

AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells.

Extensive studies have demonstrated that the Akt/AKT1 pathway is essential for cell survival and inhibition of apoptosis; however, alterations of Akt/AKT1 in human primary tumors have not been well documented. In this report, significantly increased AKT1 kinase activity was detected in primary carcinomas of prostate (16 of 30), breast (19 of 50), and ovary (11 of 28). The results were confirmed by Western blot and immunohistochemical staining analyses with phospho-Ser473 Akt antibody. The majority of AKT1-activated tumors are high grade and stage III/lV (13 of 16 prostate, 15 of 19 breast, and 8 of 11 ovarian carcinomas). Previous studies showed that wild-type AKT1 was unable to transform NIH3T3 cells. To demonstrate the biological significance of AKT1 activation in human cancer, constitutively activated AKT1 (Myr-Akt) was introduced into NIH3T3 cells. Overexpression of Myr-Akt in the stably transfected cells resulted in malignant phenotype, as determined by growth in soft agar and tumor formation in nude mice. These data indicate that AKT1 kinase, which is frequently activated in human cancer, is a determinant in oncogenesis and a potential target for cancer intervention.

Antagonism between PTEN/MMAC1/TEP-1 and androgen receptor in growth and apoptosis of prostatic cancer cells.

PTEN/MMAC1/TEP-1 (PTEN) tumor suppressor and androgen receptor play important roles in prostatic tumorigenesis by exerting opposite effects on homeostasis of prostatic epithelium. Here, we describe a mutual repression and selective dominance between PTEN and the androgen receptor (AR) in the growth and the apoptosis of prostatic cancer cells. On the one hand, PTEN and an inhibitor of phosphoinositide 3-kinase repressed the transcriptional activity of the AR as well as androgen-induced cell proliferation and production of prostate-specific antigen. On the other hand, androgens protected prostate cancer cells from PTEN-induced apoptosis in an AR-dependent manner. Whereas the repression of the transcriptional activity of the AR by PTEN is likely to involve the down-regulation of AKT, androgens protected prostate cancer cells from PTEN-induced apoptosis without an effect on AKT activity, demonstrating a differential involvement of AKT in the interaction between PTEN and the AR. Our data suggest that the loss of PTEN function may induce tumorigenesis through unopposed activity of the AR as well as contribute to the resistance of prostate cancers to androgen ablation therapy.

Antiproliferative and apoptotic effect of ascorbyl stearate in human glioblastoma multiforme cells: modulation of insulin-like growth factor-I receptor (IGF-IR) expression.

Human glioblastomas (gliomas) are characterized as highly invasive and rapidly growing brain tumors. In this study, we present data on in vitro effect of ascorbyl stearate (Asc-S), a liphophilic derivative of ascorbic acid on cell proliferation, transformation, apoptosis and modulation of expression of insulin-like growth factor-I receptor (IGF-IR) in human glioblastoma multiforme (T98G) cells. Asc-S showed significant inhibition of fetal bovine serum and human recombinant insulin-like growth factor-I (IGF-I) dependent cell proliferation in a dose dependent manner. Treatment of T98G cells with 0, 50, 100 and 150 microM Asc-S for 24h slowed down the cell multiplication cycle with significant accumulation of cells at late S/G2-M phase of cycle. Asc-S treatment (100 microM) reversed the transformed phenotype as determined by clonogenecity in soft agar and also induced apoptosis of T98G. These changes were found to be associated with significant decrease in IGF-IR expression in dose and time dependent manner compared to untreated controls. The data clearly demonstrate that Asc-S has antiproliferative and apoptotic effect on T98G cells probably through modulation of IGF-IR expression and consequent facilitation of programmed cell death.

Loss of p21WAF1/CIP1 accelerates Ras oncogenesis in a transgenic/knockout mammary cancer model.

Upregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and subsequent cell growth arrest or senescence is one mechanism by which normal cells are believed to respond to stress induced by the constitutively activated GTPase Ras. We hypothesize that in the absence of p21, the onset of Ras-dependent oncogenesis is accelerated. To test this hypothesis, we crossed MMTV/v-Ha-ras transgenic mice into a p21-deficient background. By 63 days of age, all 8 ras/p21-/- mice developed either malignant (mammary and/or salivary adenocarcinomas) or benign (Harderian hyperplasia) tumors. In contrast, by the same age, only one out of nine of the ras/p21+/+ mice developed a tumor. Furthermore, by 94 days of age, half of the ras/p21-/- mice, but none of the ras/p21+/+ mice, developed mammary tumors. p21-deficiency also accelerated the development of salivary (T50=66 days for ras/p21-/- vs T50=136 days for ras/p21+/+) and Harderian (T50=52 days for ras/p21-/- vs T50>221 days for ras/p21+/+) tumors. Furthermore, two out of the eight ras/p21-/- mice had metastatic lesions, one in its lungs, the other in its abdomen. None of the nine ras/p21+/+ mice had metastatic lesions. By 4 months of age, the mammary tumor multiplicity was 10-fold greater in ras/p21-/- (average 3.40 tumors/mouse) than in ras/p21+/+ (average 0.33 tumor/mouse) mice. However, once the tumors appeared, their growth rate, apoptosis level, and mitotic index were not affected by the loss of p21, suggesting that loss of p21 is critical in early but not late events of Ras oncogenesis. Altogether, the results show that tumor onset in MMTV/v-Ha-ras mice is p21-dependent with loss of p21 associated with earlier tumor appearance and increased tumor multiplicity and aggressiveness.

MEKK1 activation of human estrogen receptor alpha and stimulation of the agonistic activity of 4-hydroxytamoxifen in endometrial and ovarian cancer cells.

Estrogens are mitogens that stimulate the growth of both normal and transformed epithelial cells of the female reproductive system. The effect of estrogens is mediated through the estrogen receptors, which are ligand-regulated transcription factors. Tamoxifen, a selective estrogen receptor modulator, functions as an estrogen receptor antagonist in breast but an agonist in uterus. In the current study, we show that coexpression of a constitutively active MEKK1, but not RAF or MEKK2, significantly increases the transcriptional activity of the receptor in endometrial and ovarian cancer cells. The expression of wild-type MEKK1 and an active Rac1, which functions upstream of MEKK1, also increased the activity of the receptor while coexpression of dominant negative MEKK1 blocked the Rac1 induction, indicating that endogenous MEKK1 is capable of activating the receptor. Additional experiments demonstrated that the MEKK1-induced activation was mediated through both Jun N-terminal kinases and p38/Hog1 and was independent of the known phosphorylation sites on the receptor. p38, but not Jun N-terminal kinases, efficiently phosphorylated the receptor in immunocomplex kinase assays, suggesting a differential involvement of the two kinases in the receptor activation. More importantly, the expression of the constitutively active MEKK1 increased the agonistic activity of 4-hydroxytamoxifen to a level comparable to that of 17beta-estradiol and fully blocked its antagonistic activity. These findings suggest that the uterine-specific agonistic activity of the tamoxifen compound may be determined by the status of kinases acting downstream of MEKK1.

Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer.

We previously demonstrated that AKT2, a member of protein kinase B family, is activated by a number of growth factors via Ras and PI 3-kinase signaling pathways. Here, we report the frequent activation of AKT2 in human primary ovarian cancer and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase (PI 3-kinase)/Akt pathway. In vitro AKT2 kinase assay analyses in 91 ovarian cancer specimens revealed elevated levels of AKT2 activity (>3-fold) in 33 cases (36.3%). The majority of tumors displaying activated AKT2 were high grade and stages III and IV. Immunostaining and Western blot analyses using a phospho-ser-473 Akt antibody that detects the activated form of AKT2 (AKT2 phosphorylated at serine-474) confirmed the frequent activation of AKT2 in ovarian cancer specimens. Phosphorylated AKT2 in tumor specimens localized to the cell membrane and cytoplasm but not the nucleus. To address the mechanism of AKT2 activation, we measured in vitro PI 3-kinase activity in 43 ovarian cancer specimens, including the 33 cases displaying elevated AKT2 activation. High levels of PI 3-kinase activity were observed in 20 cases, 15 of which also exhibited AKT2 activation. The remaining five cases displayed elevated AKT1 activation. Among the cases with elevated AKT2, but not PI 3-kinase activity (18 cases), three showed down-regulation of PTEN protein expression. Inhibition of PI 3-kinase/AKT2 by wortmannin or LY294002 induces apoptosis in ovarian cancer cells exhibiting activation of the PI 3-kinase/AKT2 pathway. These findings demonstrate for the first time that activation of AKT2 is a common occurrence in human ovarian cancer and that PI 3-kinase/Akt pathway may be an important target for ovarian cancer intervention.

Estrogen stimulation of ovarian surface epithelial cell proliferation.

Ovarian cancer is the leading cause of gynecological cancer mortality, and 85-90% of this malignancy originates from the ovarian surface epithelium (OSE). The etiology of ovarian epithelial cancer is unknown, but a role for estrogens has been suspected. However, the effect of estrogens on OSE cell proliferation remains to be determined. Using the rabbit model, our studies have demonstrated that 17beta-estradiol stimulates OSE cell proliferation and the formation of a papillary ovarian surface morphology similar to that seen in human ovarian serous neoplasms of low malignant potential. Immunohistochemical staining of ovarian tissue sections with an antibody to the estrogen receptor alpha demonstrates its expression in both OSE cells and stromal interstitial cells. In primary ovarian cell cultures, the proliferative response of the epithelial cells to 17beta-estradiol depends on the expression of the estrogen receptor alpha in the epithelial cells. However, when the epithelial cells are grown together with ovarian stromal cells, their proliferative response to this hormone is greatly enhanced, suggesting the involvement of stromal-epithelial interactions. These studies suggest a role for estrogens and the estrogen receptor alpha in OSE growth.

The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis.

Farnesyltransferase inhibitors (FTIs) represent a novel class of anticancer drugs that exhibit a remarkable ability to inhibit malignant transformation without toxicity to normal cells. However, the mechanism by which FTIs inhibit tumor growth is not well understood. Here, we demonstrate that FTI-277 inhibits phosphatidylinositol 3-OH kinase (PI 3-kinase)/AKT2-mediated growth factor- and adhesion-dependent survival pathways and induces apoptosis in human cancer cells that overexpress AKT2. Furthermore, overexpression of AKT2, but not oncogenic H-Ras, sensitizes NIH 3T3 cells to FTI-277, and a high serum level prevents FTI-277-induced apoptosis in H-Ras- but not AKT2-transformed NIH 3T3 cells. A constitutively active form of AKT2 rescues human cancer cells from FTI-277-induced apoptosis. FTI-277 inhibits insulin-like growth factor 1-induced PI 3-kinase and AKT2 activation and subsequent phosphorylation of the proapoptotic protein BAD. Integrin-dependent activation of AKT2 is also blocked by FTI-277. Thus, a mechanism for FTI inhibition of human tumor growth is by inducing apoptosis through inhibition of PI 3-kinase/AKT2-mediated cell survival and adhesion pathway.

Differential diagnosis of endometrial hyperplasia and carcinoma by computerized image cytometry of cell proliferation, apoptosis and Bcl-2 expression.

BACKGROUND: The differential diagnosis between atypical endometrial hyperplasia and endometrial carcinoma is often difficult and based on controversial criteria. Cell kinetic parameters may be helpful. DESIGN: Cell proliferation, apoptosis and Bcl-2 expression were evaluated in benign endometrium, non atypical and atypical endometrial hyperplasia and endometrial carcinoma. The results were compared by one way analysis of variance and Bonferroni T tests. RESULTS: Cell proliferation was significantly higher (p < 0.01) in endometrial adenocarcinoma (25.6 percent) than in atypical hyperplasia (17.1 percent) and non-atypical hyperplasia (7.5 percent) of the endometrium. Apoptosis was observed in 12.3 percent of endometrial adenocarcinomas and less frequently in atypical hyperplasia (7.4 percent) and non-atypical hyperplasia of the endometrium (5.8 percent). Bcl-2 expression was significantly lower (p < 0.002) in endometrial adenocarcinoma (1.7 percent) than in atypical hyperplasia (4.2 percent) and non-atypical hyperplasia (5.3 percent) of the endometrium. In benign endometrium, cell proliferation and Bcl-2 expression were significantly higher during the proliferative phase while the rate of apoptosis was significantly higher during the secretory phase. CONCLUSIONS: Our data suggests that cell proliferation, apoptosis and Bcl-2 expression could be helpful when distinguishing endometrial carcinoma from non-atypical or atypical endometrial hyperplasia.

Expression of insulin-like growth factor-1 receptor in human colorectal cancer.

The activation of the insulinlike growth factor 1/IGF-1 receptor system (IGF1/IGF1-R) has recently emerged as critical event in transformation and tumorigenicity of several murine and human tumors. Expression of IGF1 and of IGF1-R has been demonstrated in normal and neoplastic intestinal cell lines of rats and humans. However, the modulation of IGF1-R expression during the progression from normal colonic mucosa to adenoma, to carcinoma, and to metastasis, has not been evaluated. In this retrospective study, we investigated the expression of IGF1-R in 12 colonic adenomas (AD), 36 primary colorectal adenocarcinomas (CA), and in 27 corresponding metastases (MT). Normal colonic mucosa (N) was adjacent to the CA in 34 cases. Formalin-fixed, paraffin-embedded tissues of each case were immunostained using the avidin-biotin-peroxidase method. We used an anti-IGF1-R rabbit polyclonal antibody (Santa Cruz Biotechnology, CA; dilution 1:100). Positive staining was quantitated by CAS-200. Moderate to strong cytoplasmic immunostaining was observed in 34 of 36 CA (96%), and in 25 of 27 MT (93%). In all of the positive MTs, the intensity of the staining was always strong. In 10 of 12 ADs (83%), only a faint cytoplasmic stain was identified. Normal mucosa when present was negative. Strong IGF1-R positivity correlated with higher grade and higher-stage tumors (P < .01). These data suggest a role of IGF1-R expression during the progression of colorectal adenoma to carcinoma. An increased number of IGF1-R receptors may favor the metastasis of colorectal cancer.

The insulin-like growth factor 1 receptor induces transformation and tumorigenicity of ovarian mesothelial cells and down-regulates their Fas-receptor expression.

Cell proliferation and papillogenesis are growth factor-sensitive events in the ovarian mesothelium, the tissue source of ovarian epithelial cancer. To further investigate the regulation of cell proliferation in this tissue, rabbit ovarian mesothelial cells (OMC) were transfected in vitro with a CVN expression vector carrying the human gene for insulin-like growth factor 1 receptor (IGF-1R). The growth characteristics of IGF-1R transfectants (OMIR) and their response to IGF-1 were then compared with those of OMC in serumless HL-1 cultures. OMIR cells formed epithelial-like colonies and, even when nonconfluent, produced tridimensional structures reminiscent of papillae seen in ovarian serous epithelial tumors. After 3 and 7 days of exposure to IGF-1, OMIR cells grew approximately 20-fold (P < 0.05), and papillogenesis was 15- to 25-fold over similar events in OMC, respectively. Exposure to treatment with antisense oligonucleotides against IGF-1R mRNA inhibited OMIR growth rate by 70%. Western immunoblotting and flow cytometry revealed higher expression of IGF-1R in OMIR cells than in OMC. The reverse was true when Fas-receptor expression was evaluated. OMIR cells were clonogenic in 15% serum-rich soft agar assay (OMIR:OMC colony-forming ratio 150-200:1), and tumorigenic in nude mice in which high-grade carcinomas with occasional lung metastases were observed. These data suggest that IGF-1R plays a role in ovarian epithelial carcinogenesis. The overexpression of this receptor induces transformation and morphogenesis of OMCs via an autocrine mechanism. IGF-1R may down-regulate the Fas expression rendering transformed ovarian mesothelial cells resistant to apoptosis.

Predictability of PSA failure in prostate cancer by computerized cytometric assessment of tumoral cell proliferation.

OBJECTIVES: To evaluate the relationship of DNA ploidy and cell proliferation (CP) with Gleason score (GS) and clinical outcome in prostate cancer. METHODS: Sixteen patients with benign prostatic hyperplasia (BPH) and 65 patients with prostate cancer classified by GS (four groups: 2 to 4, 5 to 6, 7, and 8 to 10) were studied. All patients with carcinoma underwent prostatectomy and were separated into prostate-specific antigen (PSA) failure and nonfailure groups (failure if PSA 0.1 ng/mL or more three times after surgery). Tumoral CP (Ki-67 inmunostaining and SG2M phase) and DNA ploidy were evaluated by computerized cytometry. RESULTS: BPH were diploid with low CP (8% SG2M cells or less). Carcinomas were either diploid with high CP (greater than 8% SG2M cells) or aneuploid. CP was significantly higher (P <0.001) in tumors with GS 7 or greater than in tumors with GS less than 7 (mean percent Ki-67 cells 18.3% versus 7.8%, respectively). PSA failure increased with GS (7.1% in GS 2 to 4, 21% in GS 5 to 6, 28.6% in GS 7, and 50% in GS 8 to 10), as well as with aneuploidy (18.5% in diploid tumors versus 72.7% in aneuploid tumors). Those experiencing PSA failure had significantly higher (P <0.001) CP than those not failing (mean percent Ki-67 cells 24% and mean percent SG2M 30.4% versus 8.7% and 13.5%, respectively). Cox regression analysis showed GS, DNA ploidy, Ki-67, and SG2M to each be univariately prognostic for time to PSA failure; however, Ki-67 and SG2M were more highly significant (P <0.0001 for both) than GS (P = 0.007) or DNA ploidy (P = 0.002). After adjusting for either SG2M or Ki-67 measures of CP, neither ploidy nor GS contained additional prognostic value. CONCLUSIONS: Tumor CP and DNA ploidy can be reliably determined in prostate cancer by computerized cytometry. On the basis of our preliminary results, CP correlates well with GS and predicts PSA failure better than DNA ploidy or GS.

Vascular endothelial growth factor levels in ovarian cyst fluid correlate with malignancy.

Ovarian cancer is a richly vascularized neoplasm with solid and cystic components. The purpose of this study was to determine whether cyst fluid could be used to quantitatively evaluate production of angiogenic factors in ovarian lesions. ELISA was used to measure vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the cyst fluid of patients with ovarian cancer (n = 13), benign cysts and cystadenomas (n = 23), borderline tumors (n = 5), and functional cysts (n = 8). VEGF levels were markedly elevated in the fluid of malignant cysts (38.5+/-8.2 ng/ml) as compared with benign (1.6+/-0.4 ng/ml; P < 0.001), borderline (5.7+/-1.5 ng/ml; P < 0.001), or functional cysts (3.8+/-2.0 ng/ml; P < 0.001). The presence of VEGF in cancer cells was confirmed by immunohistochemistry. Follow-up of patients with malignant and borderline lesions demonstrated a correlation between VEGF levels in cyst fluid and tumor recurrence (P = 0.03). bFGF in malignant cysts was either undetectable or very low (0.3+/-0.2 ng/ml), and no significant differences were found in bFGF levels among malignant, benign, borderline, and functional cysts. This study demonstrates that ovarian malignancy is associated with dramatic elevation of VEGF levels in ovarian cyst fluid. Conversely, there is no correlation between cyst fluid bFGF levels and malignant transformation. The high levels of VEGF in malignant cysts are consistent with the hypothesis that this growth factor plays an important role in ovarian cancer related-angiogenesis and tumor progression and represents a potentially important target of antiangiogenic therapy.

Effects of raloxifene in a guinea pig model for leiomyomas.

OBJECTIVE: Chronic exposure of oophorectomized guinea pigs to 17beta-estradiol causes leiomyoma formation. Our aims were to determine whether these leiomyomas can become estradiol independent after exposure to estradiol and if raloxifene inhibits leiomyoma growth when given concomitantly with estradiol. STUDY DESIGN: To induce leiomyoma development, 6 oophorectomized animals received two estradiol implants for 140 days. Next, the estradiol implants were replaced with empty implants in 3 animals, whereas the other 3 received 2 new estradiol implants and raloxifene given per os 10 mg/kg per day for 60 days. Tumor size was monitored biweekly by ultrasonography. RESULTS: On estradiol removal, abdominal wall leiomyomas regressed within 15 to 30 days; when estradiol implants were reintroduced, leiomyomas redeveloped. Within 30 days on raloxifene, all abdominal leiomyomas (n = 9) regressed as determined by ultrasonography and verified at laparotomy. Serum raloxifene and estradiol levels were 432 +/- 46 pg/mL and 78 +/- 13 pg/mL (mean +/- SEM, n = 3), respectively, after 60 days of treatment. CONCLUSIONS: Leiomyomas did not become estradiol independent, even after long exposure to estradiol; ultrasonography allowed frequent, noninvasive assessment of leiomyoma size, and raloxifene rapidly regressed leiomyomas in this animal model.

AKT2, a member of the protein kinase B family, is activated by growth factors, v-Ha-ras, and v-src through phosphatidylinositol 3-kinase in human ovarian epithelial cancer cells.

Three members have been identified in the protein kinase B (PKB) family, i.e., Akt/PKB alpha, AKT2/PKB beta, and AKT3/PKB gamma. Previous studies have demonstrated that only AKT2 is predominantly involved in human malignancies and has oncogenic activity. However, the mechanism of transforming activity of AKT2 is still not well understood. Here, we demonstrate the activation of AKT2 with several growth factors, including epidermal growth factor, insulin-like growth factor 1, insulin-like growth factor II, basic fibroblast growth factor, platelet-derived growth factor, and insulin, in human ovarian epithelial cancer cells. The kinase activity and the phosphorylation of AKT2 were induced by the growth factors and blocked by the phosphatidylinositol (PI) 3-kinase inhibitor, wortmannin, and dominant-negative Ras (N17Ras). Moreover, the activated Ras and v-Src, two proteins that transduce growth factor-generated signals, also activated AKT2, and this activation was not significantly enhanced by growth factor stimulation but was abrogated by wortmannin. These results indicate that AKT2 is a downstream target of PI 3-kinase and that Ras and Src function upstream of PI 3-kinase and mediate the activation of AKT2 by growth factors. The findings also provide further evidence that AKT2, in cooperation with Ras and Src, is important in the development of some human malignancies.

CD44V6 expression in human colorectal carcinoma.

CD44 is an adhesion molecule involved in cell-to-cell and cell-to-matrix interactions. This transmembrane glycoprotein exists in either standard or variant forms, originated by alternative splicing. One of the isoforms (CD44V6) has been shown, in some systems, to modify the metastatic potential of tumor cells. To investigate the role of this biomarker as possible prognostic antigen in colorectal cancer, we immunohistochemically analyzed the distribution of CD44V6 expression on formalin-fixed, paraffin-embedded tissues from resected colorectal cancers of 34 patients. The monoclonal antibody VFF7 against the amino acid sequence encoded by exon CD44V6 was applied using the avidin-biotin-peroxidase method. For each resected specimen, normal (N), adenomatous (AD), and carcinomatous (CA) colonic mucosa were tested. In 68% of the resected cases, these areas were present in the same slide, and in 76% of cases, nodal or liver metastases (MT) were available for evaluation. Adenomatous polyp biopsy specimens of 10 carcinoma-free patients were also tested. In selected cases, CD44V6 expression was also determined using the Western blot immunoprecipitation technique. CD44V6 immunoreactivity was detected in 100% of the ADs, and in 91% of CAs, but was mostly weak in only 38% of MTs (n=26). In 49% (n=35) of ADs, 11% (n=34) of CAs, and 4% of MTs (n=26), the stain was moderate to strong. CD44V6 immunoreactivity was predominantly membranous in ADs and cytoplasmic in MTs. In the CAs, both staining patterns were noted. Interestingly, the normal mucosa had a weak subnuclear localization of the stain. In the cases evaluated by Western blotting immunoprecipitation analysis, the level of CD44V6 protein expression was similar to that obtained by immunohistochemistry. No correlation was found with tumor type, stage, or patient survival. The predominant CD44V6 expression in ADs and CAs, but not in MTs, suggests that, in many cases, the expression of this adhesion molecule may be lost during the acquisition of migratory function by the tumor cells.

Analysis of p53, p21WAF1, and TGF-beta1 in human ductal adenocarcinoma of the pancreas: TGF-beta1 protein expression predicts longer survival.

Loss of p53 and p21WAF1 expression have previously been reported in pancreatic adenocarcinoma. Despite these findings in several reports of oncogene and tumor suppressor gene alterations in pancreatic cancer, the clinical significance of these changes is still poorly understood. In an attempt to detect molecular prognostic markers for pancreatic carcinoma, we studied the immunohistochemical expression of p53, p21WAF1, and TGF-beta1 proteins in 42 pancreatic adenocarcinomas of the ductal type. The results were correlated with clinicopathologic findings to identify the markers with prognostic significance. p53 nuclear immunoreactivity was seen in 20 (48%) of the cases, and it was strong to moderate in 14 (33%) of them. p21WAF1 cytoplasmic positivity was found in 16 (38%) of the tumors, with 72% staining strong to moderate. TGF-beta1 stained the cytoplasm of the tumor cells in 13 (31%). Of the p53-negative cases, 12 (54%) exhibited p21WAF1 expression. In 3 (30%) of cases, TGF-beta1 reactivity was seen in the absence of p53 and p21WAF1 p53 positivity identified tumors of higher grade, but did not correlate with stage or survival. TGF-beta1 expression, however, identified low-grade tumors and patients with longer survival. No correlation was found between the expression of any of these molecular markers and smoking history. We report a significant correlation between TGF-beta1 reactivity and low-grade tumors and between TGF-beta1 and better survival. This is a novel finding pointing to TGF-beta1 as a possible new stage-independent predictor of tumor survival in pancreatic ductal adenocarcinoma. In agreement with others, we also found p53 mutation in 20 (48%) of the tumors.