Mislocalized cytoplasmic p27 activates PAK1-mediated metastasis and is a prognostic factor in osteosarcoma.

The development of pulmonary metastasis is the leading cause of death in osteosarcoma (OS), which is the most common malignant bone tumor in children. We have previously reported that the tumor suppressor p27 (KIP1, CDKN1B) is frequently mislocalized to the cytoplasm of OS. However, its prognostic significance and metastatic mechanism are still elusive. Here, we show that cytoplasmic p27 significantly correlated with a higher metastatic status and poorer survival of OS patients (n = 136, P < 0.05), highlighting the clinical significance of p27 mislocalization in OS. Mechanistically, cytoplasmic p27 is co-immunoprecipitated with p21-activated kinase 1 (PAK1), which resulted in higher PAK1 phosphorylations, actin polymerization, and cell motility in p27-mislocalized OS cells. Silencing PAK1 expression in different p27-mislocalized OS cell lines decreased the migratory and adhesion abilities in vitro, as well as the development of pulmonary metastases in vivo. Similar PAK1-dependent motility was also observed in other p27-mislocalized cancer cell lines. In summary, our study suggests that cytoplasmic p27-mediated PAK1 activation is crucial for OS metastasis. A biomarker-guided targeted therapeutic approach for metastatic OS and other cancers harboring p27 mislocalization can be developed, where cytoplasmic p27 is used for risk stratification and PAK1 can be exploited as a potential therapeutic target.

Clinical Impact of p27Kip1 and CaSR Expression on Primary Hyperparathyroidism.

We aimed to investigate the expressions of p27 kinase inhibitory protein 1 (p27Kip1) and calcium sensing receptor (CaSR) in adenomas and normal parathyroid tissue and to evaluate the relationship of these molecules with clinical and biochemical parameters in primary hyperparathyroidism (PHPT). Fifty-one patients with histopathologically confirmed parathyroid adenomas and 20 patients with normal parathyroid glands (which were removed incidentally during thyroid resection) were included. Immunohistochemical stainings of CaSR and p27Kip1 were performed in surgical specimens. Clinical features, biochemical parameters, and BMD measurements of patients with PHPT were evaluated retrospectively. Expressions of p27Kip1 and CaSR were decreased in parathyroid adenomas, compared to normal glands (p < 0.05). High intensity of CaSR staining (3+) was more frequent in normal parathyroid tissue (75%) than adenomas (12%) (p < 0.01). Hypertension was not observed in patients with high staining intensity of CaSR (p = 0.032). There was a negative association between CaSR expression and body mass index (BMI) (p = 0.027, r = - 0.313). There was no significant relationship between p27Kip1 and CaSR expressions, serum calcium, plasma parathormone, 25-hydroxy vitamin D levels, and bone density (p > 0.05). The expressions of p27Kip1 and CaSR were decreased in PHPT patients. This reduction may play an important role in the pathogenesis of PHPT. However, neither p27Kip1 nor CaSR expression was found to be useful in predicting prognosis or severity of disease.

The PI3K/Akt/FOXO3a pathway regulates regeneration following spinal cord injury in adult rats through TNF-α and p27kip1 expression.

The aim of the present study was to elucidate the expression and role of the phosphatidylinositol 3­kinase (PI3K)/Akt/forkhead box O3 (FOXO3a) pathway in the regeneration of the spinal cord following spinal cord injury (SCI), and its regulatory effect on tumor necrosis factor (TNF)-α and cyclin-dependent kinase inhibitor 1B (p27kip1) expression. Firstly, in a Sprague-Dawley rat model of SCI, western blot analysis revealed that the protein levels of PI3K, phosphorylated Akt and FOXO3a were markedly inhibited compared with those in the sham control group. In vitro experiments were also conducted, in which primary dissociated cultures of rat dorsal spinal cord cells were induced with lipopolysaccharide (LPS; 4 µg/ml). The downregulation of PI3K using LY294002 markedly suppressed cell viability, reduced the protein levels of FOXO3a and p27kip1, and increased TNF-α protein production in the LPS-induced spinal cord cells. In addition, when the LPS-induced spinal cord cells were infected with FOXO3a adenoviral vectors, the overexpression of FOXO3 markedly promoted cell proliferation, activated p27kip1 protein levels and inhibited TNF-α protein production in the spinal cord cells. These results suggest that the PI3K/Akt/FOXO3a pathway regulates regeneration following SCI in adult rats via its modulatory effects on TNF-α and p27kip1 expression.

Forkhead box protein O3 suppresses uveal melanoma development by increasing the expression of Bcl­2­like protein 11 and cyclin­dependent kinase inhibitor 1B.

Forkhead box protein O3 (FoxO3a) is a forkhead box family transcription factor which serves an important role in a number of biological functions, including tumor growth. A previous study indicated that FoxO3a serves a role in insulin like growth factor­induced growth, migration and invasion of uveal melanoma (UM) cells; however, whether FoxO3a is associated with the development and formation of UM remains unknown. In the present study, the role of FoxO3a in UM development and formation was investigated by modulating the expression of FoxO3a in a human UM cell line. The results of the present study demonstrated that FoxO3a overexpression in UM cells inhibited cell proliferation and promoted cellular apoptosis, leading to an accumulation of cells at the G1 cell cycle phase. Western blot analysis demonstrated that FoxO3a overexpression increased the transcription and protein expression of Bcl­2­like protein 11 and cyclin­dependent kinase inhibitor 1B, and inhibited cyclin D1 transcription and expression. The opposite effects were observed when FoxO3a was knocked down in UM cells. The results of the present study indicated that FoxO3a may exhibit a negative role in UM development and formation, which is consistent with its role as a tumor suppressor.

Expression of hsa-let-7b-5p, hsa-let-7f-5p, and hsa-miR-222-3p and their putative targets HMGA2 and CDKN1B in typical and atypical carcinoid tumors of the lung.

Typical and atypical carcinoid tumors belong to the neuroendocrine lung tumors. They have low recurrence and proliferation rate, lymph node, and distant metastases. Nevertheless, these tumors have shown a more aggressive behavior. In the last years, microRNAs were screened as new tumor markers for their potential diagnostic and therapeutic relevance. The expression of hsa-let-7b-5p, hsa-let-7f-5p, hsa-miR-222-3p, and their targets HMGA2 (high-mobility group A2) and CDKN1B (cyclin-dependent kynase inhibitor 1B, p27kip1) was evaluated in this rare small group of patients. We analyzed the clinical data of all typical and atypical carcinoid tumors of patients who underwent surgical operation at Marburg University Hospital (n = 18) from 2000. Quantitative reverse transcription polymerase chain reaction was performed in formalin-fixed paraffin-embedded tumor tissue versus four tumor-free lung tissue samples. HMGA2 was stable or downregulated; only one patient showed a significant overexpression. CDKN1B showed a significant overexpression or a stable level; it was downregulated in two samples only. Hsa-miR-222-3p resulted almost stable or overexpressed except for two samples (significantly downregulated). Hsa-let-7f-5p was stable or overexpressed in the majority of analyzed samples, whereas hsa-let-7b-5p was significantly downregulated. HMGA2 and CDKN1B are differently expressed between atypical and typical carcinoid tumors, thus representing valid biomarkers for the classification of the two tumor groups. Hsa-let-7f-5p and HMGA2 are inversely correlated. Hsa-miR-222-3p does not correlate with its predicted target CDKN1B.

Inhibition of Curcumin on ZAKα Activity Resultant in Apoptosis and Anchorage-Independent Growth in Cancer Cells.

Curcumin, a popular yellow pigment of the dietary spice turmeric, has been reported to inhibit cell growth and to induce apoptosis in a wide variety of cancer cells. Although numerous studies have investigated anticancer effects of curcumin, the precise molecular mechanism of action remains unidentified. Whereas curcumin mediates cell survival and apoptosis through mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling cascades, its impact on the upstream regulation of MAPK is unclear. The leucine-zipper and sterile-α motif kinase alpha (ZAKα), a mitogen-activated protein kinase kinase kinase (MAP3K), activates the c-Jun N-terminal kinase (JNK) and NF-κB pathway. This paper investigated the prospective involvement of ZAKα in curcumin-induced effects on cancer cells. Our results suggest that the antitumor activity of curcumin is mediated via a mechanism involving inhibition of ZAKα activity.

Protein expression patterns of cell cycle regulators in operable breast cancer.

BACKGROUND-AIM: To evaluate the prognostic role of elaborate molecular clusters encompassing cyclin D1, cyclin E1, p21, p27 and p53 in the context of various breast cancer subtypes. METHODS: Cyclin E1, cyclin D1, p53, p21 and p27 were evaluated with immunohistochemistry in 1077 formalin-fixed paraffin-embedded tissues from breast cancer patients who had been treated within clinical trials. Jaccard distances were computed for the markers and the resulted matrix was used for conducting unsupervised hierarchical clustering, in order to identify distinct groups correlating with prognosis. RESULTS: Luminal B and triple-negative (TNBC) tumors presented with the highest and lowest levels of cyclin D1 expression, respectively. By contrast, TNBC frequently expressed Cyclin E1, whereas ER-positive tumors did not. Absence of Cyclin D1 predicted for worse OS, while absence of Cyclin E1 for poorer DFS. The expression patterns of all examined proteins yielded 3 distinct clusters; (1) Cyclin D1 and/or E1 positive with moderate p21 expression; (2) Cyclin D1 and/or E1, and p27 positive, p53 protein negative; and, (3) Cyclin D1 or E1 positive, p53 positive, p21 and p27 negative or moderately positive. The 5-year DFS rates for clusters 1, 2 and 3 were 70.0%, 79.1%, 67.4% and OS 88.4%, 90.4%, 78.9%, respectively. CONCLUSIONS: It seems that the expression of cell cycle regulators in the absence of p53 protein is associated with favorable prognosis in operable breast cancer.

Regulation of p27 by ubiquitin ligases and its pathological significance in human lung carcinomas.

Down-regulation of cyclin-dependent kinase inhibitor protein p27, due to enhanced degradation, is frequently observed in various cancers. The ubiquitin ligases that mediate this degradation have been identified as S-phase kinase-associated protein-2 (Skp2), Kip1 ubiquitylation-promoting complex (KPC), and p53-inducible protein with RING-H2 domain (Pirh2) as well. We investigated the correlation among expression of these 3 ligases and p27 status in surgical specimens of human lung carcinomas by immunohistochemical analysis. Among 93 cases, expressions of p27, Skp2, KPC, and Pirh2 were found in 89.2%, 59.1%, 59.1%, and 67.7%, respectively. Down-regulation of p27 in cancer cells was frequently observed in adenocarcinoma (AC) and squamous cell carcinoma (SCC), but not in small cell carcinoma (SmCC). Overexpression of ubiquitin ligases was variously observed among histological types: Skp2 was more frequently observed in SCC and SmCC, KPC in SCC and Pirh2 in AC, followed by SCC. Several novel findings were obtained: (i) cytoplasmic p27 was observed in 8.6%, most frequently in SCC (13.3%), and correlated with nodal metastasis (P=.0044), (ii) significant inverse correlation between nuclear p27 and Pirh2 expression was observed by statistical analysis and at the cellular level, and (iii) cytoplasmic Pirh2 and total (cytoplasmic and/or nuclear) Pirh2 were significantly correlated with the nodal status (P=.0225, 0.0314), the pathological stage (P=.0213, 0.0475) and recurrence-free survival (P=.0194, 0.0482, respectively) in AC. Altogether, our data suggests that p27 and its cognate ubiquitin ligases are specifically involved in the clinical profiles, and thus, molecular targeting of these ubiquitin ligases, in particular, Pirh2, may have therapeutic value for human lung carcinomas.

PTEN loss and p27 loss differ among morphologic patterns of prostate cancer, including cribriform.

The presence and extent of cribriform pattern of prostate cancer portend recurrence and cancer death. The relative expressions within this morphology of the prognostically adverse loss of PTEN, and the downstream inactivation of cell cycle inhibitor p27/Kip1 had been uncertain. In this study, we examined 52 cases of cribriform cancer by immunohistochemistry for PTEN, p27, and CD44 variant (v)7/8, and a subset of 17 cases by chromogenic in situ hybridization (ISH) using probes for PTEN or CDKN1B (gene for p27). The fractions of epithelial pixels positive by immunohistochemistry and ISH were digitally assessed for benign acini, high-grade prostatic intraepithelial neoplasia, and 8 morphologic patterns of cancer. Immunostaining results demonstrated that (1) PTEN loss was significant for fused small acini, cribriform-central cells, small cribriform acini, and Gleason grade 5 cells in comparison with other acini; (2) p27 loss was significant only for cribriform-peripheral cells and borderline significant for fused small acini in comparison with benign acini; and (3) CD44v7/8 showed expression loss in cribriform-peripheral cells; other comparisons were not significant. ISH showed that cribriform cancer had significant PTEN loss normalized to benign acini (P<.02), whereas Gleason 3 cancer or fused small acini did not. With CDKN1B, the degree of signal loss among various cancer morphologies was insignificant. In conclusion, molecular disparities emerged between the fused small acini and cribriform patterns of Gleason 4 cancer. PTEN or p27 loss as prognostic factors demands distinct assessment in the varieties of Gleason 4 cancer, and in the biphenotypic peripheral versus central populations in cribriform structures.

Angiogenic and lymphangiogenic profiles in histological variants of papillary thyroid carcinoma.

INTRODUCTION: Papillary thyroid carcinoma (PTC) is a well­differentiated tumor that occurs in several histological variants whose biological behaviors remain unclear. Angiogenesis and lymphangiogenesis are critical processes that enable tumor progression. OBJECTIVES: The aim of this study was to evaluate the angiogenic and lymphangiogenic phenotypes of PTC, considering the differences between histological variants. PATIENTS AND METHODS: Angiogenic and lymphangiogenic profiles were analyzed by determining microvascular density (MVD) and lymphatic vessel density (LVD) in 73 cases of PTC, using immunohistochemistry. To assess the biological markers involved in blood and lymph vessel formation, the expression of vascular endothelial growth factor (VEGF), cyclooxygenase 2 (COX­2), and p27kip1 (p27) was determined. RESULTS: MVD was significantly higher in patients with high­risk PTC and in those with local extrathyroidal and vascular invasion. Positive VEGF expression was strongly associated with high MVD and age­related tumor enlargement. The presence of lymph vessel invasion was associated with the expression of either VEGF or COX­2. The analysis of angiogenesis and lymphangiogenesis in different histological variants of PTC revealed elevated LVD rather than MVD in the follicular variant of PTC (FV­PTC).Lower MVD was observed in FV­PTC relative to the classic variant of PTC (CV­PTC). The frequency of VEGF­positive tumors was higher in CV­PTC than in FV­PTC. A significant association between COX­2 and p27 expression was observed in FV­PTC but not in CV­PTC. CONCLUSIONS: These results suggest that VEGF, COX­2, and p27 may be important biological markers that determine the angiogenic and lymphangiogenic potentials of PTC, particularly between the follicular and classic variants.

Cellular interactions of the phosphorylated form of AKT in prostate cancer.

Phospho-Akt (P-Akt1) promotes proliferation and increased survival in vitro and plays an important role in prostate cancer (PCa) progression as well as the prediction of the probability of recurrence. In this study, the goal was to demonstrate the involvement and impact of P-Akt1 on cellular interactions, biomechanisms, and pathways in PCa. Tissue microarrays from 640 PCa patients were immunostained with various antibodies. Ki-67 was used to measure proliferation index, and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling was used for apoptotic index. Increased expression of P-Akt1 was associated with an increased proliferation but inversely correlated with apoptotic index. Higher levels of P-Akt1 are associated with both higher levels of cytoplasmic p27 and higher levels of nuclear p27, suggesting an involvement in both cytoplasmic entrapment and phosphorylation of p27. P-Akt1 expression significantly correlated with nuclear and cytoplasmic staining of FHKR and GSK. The strongest correlations were found with the P- forms of both, suggesting enzyme kinetics in the latter. Here, phosphorylation is the principal method of FHKR and GSK inactivation. P-Akt1 correlated with nuclear transcription factor kappa B, suggesting a role in the inhibition through phosphorylation of nuclear transcription factor kappa B. The results of the current study are unique because of the scope of the markers and the size of the population used. In vitro- and in vivo-derived information of P-Akt1 and its downstream effectors demonstrates significant involvement in PCa. Our data suggest that PCa uses multiple mechanisms to regulate this pathway and substantiate the concept of redundancy in cancer pathway regulation. Consequently, new hypothesis-driven studies can be derived from this information.

Serum vitamin D (25OHD3) levels and the risk of different subtypes of breast cancer: A nested case-control study.

BACKGROUND: Previous studies regarding the association between serum 25-hydroxyvitamin D (25OHD3) and breast cancer risk have not been conclusive. The aim of this study was to investigate the potential association between pre-diagnostic serum 25OHD3 levels and the risk of different subtypes of breast cancer. MATERIALS AND METHODS: The study was based on The Malmö Diet and Cancer Study recruiting 17,035 women from 1991 to 1996. A total of 764 incident breast cancers with matched controls were analysed for 25OHD3 in samples collected at baseline, before diagnosis. A logistic regression analysis was used to calculate odds ratios with 95% confidence intervals for tertiles of 25OHD3 in relation to different subtypes of breast cancer, i.e. defined according to tumour type, tumour size, lymph node involvement, histological grade, oestrogen receptor (ER) status, progesterone receptor (PgR) status, Ki67, cyclin D1 and p27. RESULTS: As compared to the 1st tertile of 25OHD3, the second tertile had a statistically significantly lower risk of ER negative tumours, PgR negative tumours and tumours with a high expression of Ki67, A similar pattern was seen in relation to large tumours (≥21 mm), grade III tumours, and tumours with low p27 expression, but these associations did not reach statistical significance. The third tertile had a similar risk as the first tertile. CONCLUSIONS: We found that women with low levels of 25OHD3, as compare to women in the middle tertile, had a high risk of breast tumours with an unfavourable prognosis.

Encorafenib (LGX818), a potent BRAF inhibitor, induces senescence accompanied by autophagy in BRAFV600E melanoma cells.

Encorafenib (LGX818) is a new-generation BRAF inhibitor that is under evaluation in clinical trials. However, the underlying mechanism remains to be elucidated. Here we show that LGX818 potently decreased ERK phosphorylation and inhibited proliferation in BRAFV600E melanoma cell lines. Moreover, LGX818 downregulated CyclinD1 in a glycogen synthase kinase 3ß-independent manner and induced cell cycle arrest in the G1 phase, Surprisingly, LGX818 triggered cellular senescence in BRAFV600E melanoma cells, as evidenced by increased ß-galactosidase staining, while no appreciable induction of apoptosis was detected, as determined by Annexin V and propidium iodide staining and immunoblot analysis of caspase-3 processing and poly (ADP-ribose) polymerase cleavage. Increased p27KIP1 expression and retinoblastoma protein activation were detected during LGX818-induced senescence. Additionally, inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 1B by AZ191 reversed LGX818-induced CyclinD1 turnover and senescence. Interestingly, autophagy is triggered through inhibition of the mTOR/70S6K pathway during LGX818-induced senescence. Moreover, autophagy inhibition by pharmacological and genetic regulation attenuates LGX818-induced senescence. Notably, combining LGX818 with autophagy modulators has anti-proliferative effect in LGX818-resistant BRAF mutant melanoma cells. Altogether, we uncovered a mechanism by which LGX818 exerts its anti-tumor activity in BRAFV600E melanoma cells.

DNA methyltransferase immunohistochemical expression in odontogenic tumours.

BACKGROUND: Odontogenic tumours are a heterogeneous group of lesions formed from tissues that give rise to the tooth. DNA methylation, a covalent addition of a methyl group to the 5-carbon position of a cytosine nucleotide, is considered an important regulator of gene expression. The addition of the methyl radical is catalysed by DNA methyltransferases (DNMTs). Although some epigenetic studies have been conducted in odontogenic tumours, a study with the three types of DNMTs in several different members of this group is missing. This study analyses the expression of DNMTs in odontogenic tumours. METHODS: Formalin-fixed and paraffin-embedded tissue samples of 20 ameloblastomas, 10 calcifying cystic odontogenic tumours, 10 calcifying epithelial tumours, 10 adenomatoid odontogenic tumours, 10 keratocystic odontogenic tumours, five ameloblastic fibromas, two ameloblastic fibro-odontomas, four central odontogenic fibromas, seven peripheral odontogenic fibromas and 10 odontogenic myxomas were included. Immunohistochemical expression of DNMT1, 3A and 3B was assessed using a semi-quantitative analysis, and also a correlation with p21, p27 and E-cadherin immunoexpression was made. RESULTS: DNMT1, 3A and 3B were expressed in the nucleus and/or cytoplasm of all odontogenic tumours. DNMT1 expression was directly correlated with p27 expression in ameloblastomas. CONCLUSION: The high expression of DNMTs in odontogenic tumour cells suggests methylation as an important mechanism for this group of tumours.

Delayed luminescence to monitor programmed cell death induced by berberine on thyroid cancer cells.

Correlation between apoptosis and UVA-induced ultraweak photon emission delayed luminescence (DL) from tumor thyroid cell lines was investigated. In particular, the effects of berberine, an alkaloid that has been reported to have anticancer activities, on two cancer cell lines were studied. The FTC-133 and 8305C cell lines, as representative of follicular and anaplastic thyroid human cancer, respectively, were chosen. The results show that berberine is able to arrest cell cycle and activate apoptotic pathway as shown in both cell lines by deoxyribonucleic acid fragmentation, caspase-3 cleavage, p53 and p27 protein overexpression. In parallel, changes in DL spectral components after berberine treatment support the hypothesis that DL from human cells originates mainly from mitochondria, since berberine acts especially at the mitochondrial level. The decrease of DL blue component for both cell lines could be related to the decrease of intra-mitochondrial nicotinamide adenine dinucleotide and may be a hallmark of induced apoptosis. In contrast, the response in the red spectral range is different for the two cell lines and may be ascribed to a different iron homeostasis.

Smad2 overexpression reduces the proliferation of the junctional epithelium.

The overexpression of the intracellular signaling molecule of the transforming growth factor-beta family (TGF-ß) Smad2 was found to induce apoptosis and inhibit the proliferation rate of oral epithelial cells. Therefore, the aim of this study was to investigate in vivo the effect of Smad2 overexpression on the proliferation rate of the junctional epithelium (JE). Smad2 overexpression was driven by the cytokeratin 14 promoter (K14-Smad2) in transgenic mice. The K14-Smad2 mice were compared with wild-type (WT) mice selected as the control group. Samples were stained with hematoxylin and eosin stains and analyzed by image analysis. Immunohistochemistry was conducted for proliferating cell nuclear antigen (PCNA) and c-Myc as markers of cell proliferation. The expression of cyclin-dependent kinase inhibitors (P15, P21, and P27) was determined by real-time polymerase chain-reaction (RT-PCR). The quantity of phosphorylated retinoblastoma (pRB) was determined with Western blots. The overexpression of Smad2 altered the area of the junctional epithelial cells in one-year-old K14-Smad2 mice. The area was 32,768 (± 3,473) µm(2) for the WT and 24,937.25 (± 1,965) µm(2) for the K14-Smad2 mice. There was a significant difference in the proliferation rates of the JE (PCNA-positive cells) between the WT and K14-Smad2 mice, 20.7% (± 1.1) and 2.1% (± 0.5), respectively. A significant difference in c-Myc expression occurred between experimental and control samples. The K14-Smad2 mice had a mean of 2.3% (± 0.6), and the WT mice had a mean of 20.1% (± 3.6). Smad2 overexpression up-regulated the mRNA expression of P15 by 2.3-fold and that of P27 by 5.5-fold in the K14-Smad2 mice. Finally, the pRB protein showed a 2.3 (± 0.5)-fold increase in K14-Smad2 mice when compared with WT mice. Smad2 overexpression inhibits the proliferation of JE cells by down-regulating c-Myc and up-regulating P15 and P27, which resulted in an increase in pRB, leading to cell-cycle arrest.

Tiam1 siRNA enhanced the sensitivity of sorafenib on esophageal squamous cell carcinoma in vivo.

Our previous study demonstrated that Tiam1 was highly expressed in esophageal squamous cell carcinoma (ESCC) tissues. In the present study, we investigated the therapeutic role of Tiam1 siRNA in combination with sorafenib in xenografted human ESCC. Our results demonstrated that expression of Tiam1 protein in EC9706 cells was significantly higher than those in ESCC cells (Eca109 and EC1) and normal esophageal epithelial cells Het-1A (P < 0.05). Tiam1 siRNA markedly suppressed Tiam1 protein expression in tumor tissues of nude mice, but sorafenib did not alter Tiam1 level. In addition, Tiam1 siRNA or sorafenib alone evidently inhibited tumor growth, reduced Ki-67 proliferation index, and induced cell apoptosis in xenografted nude mice, and their combinations had the strongest effect. Notably, Tiam1 siRNA or sorafenib alone obviously increased p27 level, but reduced Mcl-1 and bcl-2 levels in xenografted nude mice, and their combinations reached the best effect. These findings suggest that combination of Tiam1 siRNA with sorafenib may be the novel molecular therapy target for the patients with ESCC.

Revisiting the role of MCL1 in tumorigenesis of solid cancer: gene expression correlates with antiproliferative phenotype in breast cancer cells and its functional regulatory variants are associated with reduced cancer susceptibility.

Compared to the well-defined anti-apoptotic role of myeloid cell leukemia sequence 1 (MCL1), its antiproliferative function in tumorigenesis is less studied. We had recently reported that regulatory variants of MCL1 contribute to enhanced promoter activity but reduced risk of lung cancer. We hypothesized that MCL1 expression may manifest antiproliferative phenotype and its functional variations may have etiological relevance for breast cancer. We manipulated MCL1 expression in MCF-7 cells and MDA231 with overexpression and knockdown, analyzed the effects on cell viability and cell cycling phase, and characterized the correlation with expression profiles of key regulators of cell cycle. We further genotyped the -190 insertion polymorphism and the neighboring single nucleotide polymorphisms (SNPs) in 745 breast cancer patients and 537 controls and analyzed their association with cancer risk. We confirmed that heightened expression of MCL1 resulted in decreased proliferation ability of breast cancer cells. We further observed that MCL1 overexpression in breast cancer cells resulted in cell cycle progression arresting in S phase and concomitant enhanced expression of p27, which could be rescued by p27 knockdown with co-transfection of small interfering RNA (siRNA). Furthermore, we found a significant reduction in breast cancer risk [odds ratio (OR) = 0.74; 95 % confidence interval (CI) = 0.59-0.93] associated with -190 insertion genotype; the expression-enhancing regulatory haplotype (OR 0.79; 95 % CI 0.66-0.95) and diplotype (OR 0.71; 95 % CI 0.57-0.89) were consistently associated with decreased cancer susceptibility. The study demonstrates that the expression-enhancing regulatory variants of MCL1 are protective modifiers of breast cancer risk, and reduced cell proliferation and arrested cell cycle progression partly mediated by p27 might be the underlying mechanism.

Differential expression of Pim-3, c-Myc, and p-p27 proteins in adenocarcinomas of the gastric cardia and distal stomach.

Gastric cardia adenocarcinoma (GCA) is distinct from adenocarcinoma of the distal stomach because of its different etiological factors, tumor characteristics, and biological behavior. However, its pathogenesis is not fully understood. The purpose of this study is to characterize the role of Pim-3, c-Myc, and p-p27 in the tumorigenesis and progression of different sites of gastric adenocarcinoma by determining its pathogenetic significance. The expression of Pim-3, c-Myc, and p-p27 proteins was evaluated by immunohistochemistry in 140 resection specimens of gastric adenocarcinomas (78 GCAs , 62 DGAs and 20 normal gastric tissues). The level of expression of Pim-3, c-Myc, and p-p27 and the co-expression of all three markers (Pim-3+/c-Myc+/p-p27+) in GCA were significantly lower than that in DGA tumors (P < 0.05). Detailed analysis of the immunoreactivity patterns showed that in DGA, Pim-3 immunoreactivity was associated significantly with poor tumor differentiation, advanced tumor stage, and presence of lymph node metastasis. In addition, c-Myc overexpression correlated with tumor stage and lymph node metastasis, and positive p-p27 expression correlated with poor differentiation and tumor stage. The phenotype of Pim-3(+)/c-Myc(+)/p-p27(+) co-expression was closely correlated with tumor stage and lymph node metastasis (P < 0.05). In contrast, GCA only demonstrated a close correlation of Pim-3 overexpression with poor tumor differentiation and tumor stage (P < 0.05). Our results demonstrate the presence of different expression patterns of Pim-3, c-Myc, p-p27, and Pim-3(+)/c-Myc(+)/p-p27(+) and their clinicopathologic significance in GCA and DGA tumors. Our results add support to the notion that distinct molecular mechanisms may be involved in the development and progression of adenocarcinomas from the gastric cardia and distal portion of stomach.