5728

PTEN

phosphatase and tensin homolog;PTEN;phosphatase and tensin homolog

MMAC1 phosphatase and tensin homolog deleted on chromosome 10|mitochondrial PTENalpha|mitochondrial phosphatase and tensin protein alpha|mutated in multiple advanced cancers 1|phosphatase and tensin-like protein|phosphatidylinositol 3,4,5-trisphosphate 3-

10q23.3

protein-coding

PTEN is a novel tumor-suppressor gene located on chromosomal band 10q23. Loss of PTEN function has been implicated in the progression of several types of cancer, but the correlation between loss of PTEN expression and advanced carcinomas is not well established. The capacity for angiogenesis of a tumor is known to play a very important role in growth and metastasis, and there have been reports that PTEN relates to angiogenesis. In the present study, formalin-fixed and paraffin embedded tissues from 101 patients with breast carcinomas, including 88 cases of invasive ductal carcinomas and 13 cases of ductal carcinoma in situ (DCIS), were evaluated by immunohistochemical methods for the expression of PTEN and vascular endothelial growth factor (VEGF), as well as microvessel density (MVD). The results were compared with the clinicopathologic parameters. There was no loss of PTEN expression in any of the cases of DCIS, but 28 (32%) of the 88 invasive cases did not express PTEN. Loss of PTEN expression was associated with lymph node metastasis (P = 0.03), but did not correlate with tumor size, tumor grade, MVD or recurrence. VEGF expression significantly correlated with lymph node metastasis in invasive ductal carcinoma (P = 0.01). There was no correlation between the expression of PTEN and that of VEGF (P = 0.63). The present study suggests that loss of PTEN expression is common and correlates with tumor progression and lymph node metastasis in breast carcinoma. The relationship between loss of PTEN and progression of breast cancer may not be explained by modulation of angiogenesis.

PTEN (phosphatase and tensin homologue deleted on chromosome 10) has been shown to be inactivated in a wide variety of cancers, and the role of this gene as a tumor suppressor has been well established. On the other hand, results of recent animal studies as well as clinical evidence indicate that PTEN is also involved in tumor metastasis suppression. Although PTEN is known to play a key role in controlling cell growth and apoptosis, how PTEN exerts the metastasis suppressor function remains largely unknown. Recently, a microarray analysis identified the Drg-1 gene (differentiation related gene 1) as one of the potential targets of PTEN. The Drg-1 gene has been shown to suppress tumor metastasis in animal models of prostate and colon cancer, and the expression of this gene is significantly reduced with advancement of prostate and breast cancers in clinical setting. In this study, we explored the possibility that PTEN controls tumor metastasis by regulating the expression of the Drg-1 gene. Our results indicate that overexpression of PTEN significantly augments the endogenous expression of Drg-1 protein, whereas inhibition of PTEN by small interfering RNA decreases Drg-1 in a dose- and time-dependent manner. We also found that the control of the Drg-1 gene by PTEN seems to be at the transcriptional level, and that a phospho-Akt inhibitor restores the Drg-1 expression, indicating that PTEN controls Drg-1 by an Akt-dependent pathway. Consistent with these results, our immunohistochemical analysis revealed that PTEN expression correlates significantly with Drg-1 in both prostate and breast cancer cases. Furthermore, combination of the two markers, PTEN and Drg-1, emerged as a significantly better predictor of prostate and breast cancer patient survival than either marker alone.

Adenoid cystic carcinoma (ACC) of the breast rarely metastasizes and has been associated with excellent prognosis. We describe a patient with renal metastasis of primary breast ACC 5 years after the mastectomy. A detailed molecular genetic analysis of the primary and metastatic tumors demonstrated somatic mutations in 2 well-known cancer genes associated with regulation of PI3K/AKT signaling pathway: (1) PIK3CA, which encodes the catalytic alpha subunit of the phosphoinositide-3-kinase, and (2) PTEN, which encodes phosphatase and tensin homolog. The mutation identified in PIK3CA (Ex1+169 A>C) predicts an amino acid change from isoleucine to methionine at codon 31 (I31M) and resides in the p85-binding domain of exon 1. The mutation identified in PTEN (IVS4-3 C>T) resides in intron 4 near the splice acceptor site of exon 5 and was associated with an aberrant PTEN transcript lacking exon 5, which is necessary for protein tyrosine phosphatase function and tumor suppressor properties of PTEN. Increased promoter methylation of PTEN was present in renal metastasis, coinciding with the decrease in the level of normal PTEN transcript. These coexistent mutations/epigenetic inactivations in PI3K/AKT pathway may be responsible for the unusually aggressive course of ACC.

BACKGROUND & OBJECTIVE: tumor suppressor gene PTEN could not only inhibit the proliferation of cancer cells, but also inhibit their metastasis. However, the mechanism is still unclear. This study was to investigate the effects of PTEN gene on the proliferation and metastasis of human breast cancer ZR-75-1 cells, and explore the mechanisms. METHODS: Wild-type PTEN (wt-PTEN) plasmid and phosphatase-defective PTEN (G129R-PTEN) plasmid were transfected into ZR-75-1 cells by liposome, respectively. Cell proliferation was detected by MTT assay. Transfected cells were selected by puromycin. The expression of PTEN protein was detected by Western blot. Cell adhesion and invasion were tested by adhesion test and invasion test. RESULTS: The proliferation inhibition rate was significantly higher in wt-PTEN-transfected ZR-75-1 cells than in untransfected cells and G129R-PTEN-transfected cells (42.7% vs. 0% and 2.7%, P<0.01); there was no significant difference between untransfected cells and G129R-PTEN-transfected cells(P>0.05). The proliferation inhibition of ZR-75-1 cells was enhanced along with the increase of culture time and concentration of wt-PTEN. wt-PTEN also induced cell apoptosis. PTEN protein was expressed efficiently in the cells transfected with either wt-PTEN or G129R-PTEN. The inhibition rates of adhesion and invasion were significantly higher in wt-PTEN-transfected cells than in G129R-PTEN-transfected cells (65.7% vs. 8.8%, 70.4% vs. 6.9%, P<0.01). CONCLUSION: Wild-type PTEN gene with dual-specific phosphatase activity can inhibit the proliferation and metastasis of ZR-75-1 cells.

Phosphatase and tensin homolog (PTEN) is a key modulator of trastuzumab sensitivity in HER2-overexpressing breast cancer. Because PTEN opposes the downstream signaling of phosphoinositide 3-kinase (PI3K), we investigated the role of PTEN and other components of the PI3K pathway in trastuzumab resistance. We analyzed the status of PTEN, p-AKT-Ser473, and p-p70S6K-Thr389 using immunohistochemistry. PIK3CA mutation status was analyzed by direct sequencing. Primary tumor tissue was available from 137 patients with HER2-overexpressing metastatic breast cancer who had received trastuzumab-based chemotherapy. We observed that each of the four biomarkers alone did not significantly correlate with trastuzumab response, whereas PTEN loss alone significantly correlated with shorter survival times (P = 0.023). PI3K pathway activation, defined as PTEN loss and/or PIK3CA mutation, was associated with a poor response to trastuzumab (P = 0.047) and a shorter survival time (P = 0.015). PTEN loss was significantly associated with a poor response to trastuzumab (P = 0.028) and shorter survival time (P = 0.008) in patients who had received first-line trastuzumab and in patients with estrogen receptor- (P = 0.029) and progesterone receptor-negative tumors (P = 0.033). p-AKT-Ser473 and p-p70S6K-Thr389 each had a limited correlation with trastuzumab response. When these markers were combined with PTEN loss, an increased correlation with patient outcome was observed. In conclusion, PI3K pathway activation plays a pivotal role in trastuzumab resistance. Our findings may facilitate the evaluation of tumor response to trastuzumab-based and targeted therapies.

OBJECTIVE: Phosphatase and tensin homolog (PTEN) loss or activating mutations of phosphoinositol-3 (PI3) kinase (PIK3CA) may be related to trastuzumab resistance in in vitro studies; however, this issue in clinical studies is controversial. Therefore, we conducted a meta-analysis to assess the association between PTEN loss, PIK3CA mutation and the efficacy of trastuzumab-based treatment in HER2-positive breast cancer patients. METHODS: A computerized search was performed through the PubMed database, the online proceedings of the American Society of Clinical Oncology Annual Meetings, the San Antonio Breast cancer Symposium and the International St. Gallen Breast cancer Conference. Ten eligible studies including 1889 cases were identified. RESULTS: In HER2-positive locally advanced breast cancer patients, neither PTEN loss, PIK3CA mutation nor PI3K activation was associated with the response rate of trastuzumab-based neoadjuvant treatment (PTEN loss: RR = 0.687, 95% CI: 0.439-1.074, P = 0.099; PIK3CA mutation: RR = 1.114, 95% CI: 0.453-2.735, P = 0.814; PI3K activation: RR = 0.787, 95% CI: 0.417-1.484, P = 0.459; RR = 0.772, 95% CI: 0.387-1.539, P = 0.462). In HER2-positive early stage breast cancer patients, PTEN loss was not associated with the disease-free survival (DFS) rate of trastuzumab-based adjuvant treatment (HR = 1.096, 95% CI: 0.706-1.700, P = 0.684). In HER2-positive recurrent or metastatic breast cancer patients, PTEN loss was significantly correlated with poorer efficacy of trastuzumab-based salvage treatment (RR = 0.682, 95% CI: 0.550-0.846, P = 0.000). CONCLUSIONS: In HER2-positive recurrent or metastatic breast cancer patients PTEN loss might indicate resistance to trastuzumab-based salvage treatment. Due to the small sample size and the considerable heterogeneity in the chemotherapy treatment regimens, further research is needed to clarify the association between PTEN loss, PIK3CA mutation and the efficacy of trastuzumab-based treatment in neoadjuvant and adjuvant settings.

HER2-overexpressing breast cancers are characterized by frequent distant metastasis and often develop resistance after short-term effective treatment with the monoclonal antibody drug, trastuzumab. Here, we found that the oncogenic miRNA, miR-221, inhibited apoptosis, induced trastuzumab resistance and promoted metastasis of HER2-positive breast cancers. The tumor suppressor PTEN was identified as a miR-221 target; overexpression of PTEN abrogated the aforementioned miR-221-induced malignant phenotypes of the cells. These findings indicate that miR-221 may promote trastuzumab resistance and metastasis of HER2-positive breast cancers by targeting PTEN, suggesting its role as a potential biomarker for progression and poor prognosis, and as a novel target for trastuzumab-combined treatment of breast cancers.

The need for effective targeted therapies for renal cell carcinomas (RCCs) has fueled the interest for understanding molecular pathways involved in the oncogenesis of kidney tumors. Aiming to analyze the expression status and prognostic significance of mTOR and hypoxia-induced pathway members in patients with clear cell RCC (ccRCC), tissue microarrays were constructed from 135 primary and 41 metastatic ccRCCs. Immunoexpression levels were compared and correlated with clinicopathologic parameters and outcome. PTEN levels were significantly lower in primary and metastatic ccRCCs compared with benign tissues (P<0.001). Levels of phos-AKT, phos-S6, and 4E-binding protein-1 (4EBP1) were higher in metastatic ccRCC (Pmetastatic ccRCC (P<0.0001), and incremental p27 levels were observed in benign, primary ccRCC, and metastatic ccRCC (P<0.0001). HIF-1alpha levels were significantly higher in primary and metastatic ccRCCs compared with benign tissues (P<0.0001). In primary ccRCC, levels of all mTOR and hypoxia-induced pathway members were significantly associated with pT stage (Ptumor size (Ptumor size, HIF-1alpha, and phos-S6 levels were associated with disease-specific survival (DSS) (Ptumor progression (Ppathways were activated in primary and metastatic ccRCC. PTEN loss seems to be an early event during tumorigenesis. tumor size, HIF-1alpha, and phos-S6 expression were found to be independent predictors of both DSS and tumor progression in primary ccRCC.

BACKGROUND: The tumour suppressor phosphatase and tensin homolog (PTEN) is an important negative regulator of cell-survival signaling. To evaluate the correlation between PTEN expression and clinicopathological characteristics of colorectal cancer patients with and without liver metastases, we investigated PTEN expression in primary colorectal cancer and colorectal cancer liver metastases. METHODS: Sixty-nine pairs of primary colorectal cancer and corresponding liver metastasis specimens were analyzed immunohistochemically, and the correlation between immunohistochemical findings and clinicopathological factors was investigated. Seventy primary colorectal cancer specimens from patients without liver metastases were used as controls. RESULTS: PTEN was strongly expressed in 44 (62.9%) colorectal cancer specimens from patients without liver metastases. In contrast, PTEN was weakly expressed in 52 (75.4%) primary colorectal cancer specimens from patients with liver metastases, and was absent in liver metastases. Weak PTEN expression in colorectal cancer tissues was significantly associated with advanced TNM stage (p < 0.01) and lymph node metastasis (p < 0.05). PTEN expression was significantly stronger in primary colorectal cancer specimens from patients without liver metastases. Furthermore, among colorectal cancer patients with liver metastases, the 5-year survival rate was significantly higher in patients with positive PTEN expression compared to those with negative PTEN expression (p = 0.012). CONCLUSION: Our results suggest that loss of PTEN expression is involved with colorectal cancer aggressive capacity and that diagnostic evaluation of PTEN expression may provide valuable prognostic information to aid treatment strategies for colorectal cancer patients.

Colorectal cancer is the second leading cause of cancer-related deaths in the United States. Understanding the distinct genetic and epigenetic changes contributing to the establishment and growth of metastatic lesions is crucial for the development of novel therapeutic strategies. In a search for key regulators of colorectal cancer metastasis establishment, we have found that the serine/threonine kinase Akt2, a known proto-oncogene, is highly expressed in late-stage colorectal cancer and metastatic tumors. Suppression of Akt2 expression in highly metastatic colorectal carcinoma cells inhibits their ability to metastasize in an experimental liver metastasis model. Overexpression of wild-type Akt1 did not restore metastatic potential in cells with downregulated Akt2, thus suggesting non-redundant roles for the individual Akt isoforms. In contrast, Akt2 overexpression in wild-type PTEN expressing SW480 colorectal cancer cells led to the formation of micrometastases; however, loss of PTEN is required for sustained formation of overt metastasis. Finally, we found that the consequence of PTEN loss and Akt2 overexpression function synergistically to promote metastasis. These results support a role for Akt2 overexpression in metastatic colorectal cancer and establish a mechanistic link between Akt2 overexpression and PTEN mutation in metastatic tumor establishment and growth. Taken together, these data suggest that Akt family members have distinct functional roles in tumor progression and that selective targeting of the PI3K/Akt2 pathway may provide a novel treatment strategy for colorectal cancer metastasis.

PURPOSE: PTEN, AKT, and KRAS are epidermal growth factor receptor (EGFR) downstream regulators. KRAS mutations confer resistance to cetuximab. This retrospective study investigated the role of PTEN loss, AKT phosphorylation, and KRAS mutations on the activity of cetuximab plus irinotecan in patients with metastatic colorectal cancer (mCRC). PATIENTS AND METHODS: A cohort of patients with irinotecan-refractory mCRC who were treated with cetuximab plus irinotecan was tested for PTEN immunoreactivity (ie, immunohistochemistry; IHC), pAKT IHC, and KRAS mutations. Analyses were performed both on primary tumors and on related metastases, and the association among IHC, mutational results, and treatment outcomes was investigated. RESULTS: One-hundred two patients were eligible. Ninety-six primary tumors, 59 metastases, and 53 paired samples were available. Forty-nine primary tumors (58% of assessable samples) had a preserved PTEN expression (PTEN-positive), whereas 35 (40% of assessable samples) were pAKT-positive. Levels of concordance between primary tumors and metastases were 60%, 68%, and 95% for PTEN, pAKT, and KRAS, respectively. PTEN status on primary tumors and pAKT status both on primary tumors and on metastases did not predict response or progression-free survival (PFS). On metastases, 12 (36%) of 33 patients with PTEN-positive tumors were responders compared with one (5%) of 22 who had PTEN-negative tumors (P = .007). The median PFS of patients with PTEN-positive metastases was 4.7 months compared with 3.3 months for those with PTEN-negative metastases (hazard ratio [HR], 0.49; P = .005). Patients with PTEN-positive metastases and KRAS wild type had longer PFS compared with other patients (5.5 months v 3.8 months; HR, 0.42; P = .001). CONCLUSION: PTEN loss in metastases may be predictive of resistance to cetuximab plus irinotecan. The combination of PTEN IHC and KRAS mutational analyses could help to identify a subgroup of patients with mCRC who have higher chances of benefiting from EGFR inhibition.

BACKGROUND: Loss of phosphatase and tensin homologue deleted in chromosome 10 (PTEN) function in advanced colorectal cancer (CRC) may represent one of the resistance mechanisms to cetuximab by interfering with the epidermal growth factor receptor signal transduction pathway. METHODS: PTEN expression tested by indirect immunofluorescence was evaluated both on primary (n=43) and on metastatic (n=24) sites in CRC patients treated with cetuximab. RESULTS: The loss of PTEN expression tested on metastatic sites was negatively associated with response (100% progressive disease (PD) in PTEN-negative cases vs 30% PD in PTEN-positive cases; P<0.05), PFS (0.8 vs 8.2 months; P<0.001) and OS (2.9 vs 14.2 months; P<0.001). CONCLUSION: A potential role of PTEN in the anti-tumour activity of cetuximab could be hypothesised.

AIM: To investigate the prognostic value of KRAS mutation, and phosphatase and tensin (PTEN) expression in Chinese metastatic colorectal cancer metastatic colorectal cancer (mCRC) patients treated with cetuximab. METHODS: Ninety Chinese mCRC patients treated with cetuximab were evaluated for KRAS mutation and PTEN protein expression by DNA sequencing of codons 12 and 13 and immunohistochemistry, respectively. We then selected 61 patients treated with cetuximab, either in combination with chemotherapy, or alone as a second-line or third-line regimen to assess whether KRAS mutation or PTEN protein expression is associated with the response and the survival time of mCRC patients treated with cetuximab. RESULTS: KRAS mutation was found in 30 (33.3%) tumor samples from the 90 patients, and positive PTEN expression was detected in 58 (64.4%) of the 90 patients. Among the 61 patients who were treated with cetuximab as a second-line or third-line regimen, the resistance to cetuximab was found in 22 patients with KRAS mutation and in 39 patients without KRAS mutation, with a response rate of 4.5% and 46.1% respectively (P = 0.001), a shorter median progression-free survival (PFS) time of 14 +/- 1.3 wk and 32 +/- 2.5 wk respectively (P < 0.001), a median overall survival (OS) time of 11 +/- 1.2 mo and 19 +/- 1.8 mo respectively (P < 0.001), as well as in 24 patients with negative PTEN expression and in 37 patients with positive PTEN expression respectively (P < 0.001), with a responsive rate of 4.2% and 48.6% respectively, a shorter median PFS survival time of 17 +/- 2.0 wk and 28 +/- 1.9 wk respectively (P = 0.07), and a median OS time of 11 +/- 1.3 mo and 18 +/- 1.9 mo respectively (P = 0.004). Combined KRAS mutation and PTEN expression analysis showed that the PFS and OS time of patients with two favorable prognostic factors were longer than those of patients with one favorable prognostic factor or no favorable prognostic factor (P < 0.001). CONCLUSION: KRAS mutation and PTEN protein expression are significantly correlated with the response rate and survival time of Chinese mCRC patients treated with cetuximab.

BACKGROUND: The PTEN phosphatase acts on phosphatidylinositol 3,4,5-triphosphates resulting from phosphatidylinositol 3-kinase (PI3K) activation. PTEN expression has been shown to be decreased in colorectal cancer. Little is known however as to the specific cellular role of PTEN in human intestinal epithelial cells. The aim of this study was to investigate the role of PTEN in human colorectal cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: Caco-2/15, HCT116 and CT26 cells were infected with recombinant lentiviruses expressing a shRNA specifically designed to knock-down PTEN. The impact of PTEN downregulation was analyzed on cell polarization and differentiation, intercellular junction integrity (expression of cell-cell adhesion proteins, barrier function), migration (wound assay), invasion (matrigel-coated transwells) and on tumor and metastasis formation in mice. Electron microscopy analysis showed that lentiviral infection of PTEN shRNA significantly inhibited Caco-2/15 cell polarization, functional differentiation and brush border development. A strong reduction in claudin 1, 3, 4 and 8 was also observed as well as a decrease in transepithelial resistance. Loss of PTEN expression increased the spreading, migration and invasion capacities of colorectal cancer cells in vitro. PTEN downregulation also increased tumor size following subcutaneous injection of colorectal cancer cells in nude mice. Finally, loss of PTEN expression in HCT116 and CT26, but not in Caco-2/15, led to an increase in their metastatic potential following tail-vein injections in mice. CONCLUSIONS/SIGNIFICANCE: Altogether, these results indicate that PTEN controls cellular polarity, establishment of cell-cell junctions, paracellular permeability, migration and tumorigenic/metastatic potential of human colorectal cancer cells.

PURPOSE: To identify novel genetic markers predictive of clinical benefit from epidermal growth factor receptor-directed antibody therapy in patients with metastatic colorectal cancer. PATIENTS AND METHODS: Seventy-six consecutive patients who received cetuximab or panitumumab, either alone or in combination with chemotherapy and with available tumor tissue were included. tumor tissue was tested by pyrosequencing for mutations at known hot spots in the KRAS, BRAF, PIK3CA, PIK3R1, AKT1, and PTEN genes. PTEN promoter methylation status was analyzed by methylation-specific polymerase chain reaction, and expression was determined by immunohistochemistry (IHC). Forty-four patients had 4 weeks of therapy and were considered for clinical correlates. RESULTS: Consistent with previous studies, KRAS gene mutations were associated with a shorter progression-free survival (PFS) and overall survival (OS). Among the patients with wild-type KRAS, preservation of PTEN expression and PIK3CA wild-type status was associated with improved OS (median OS, 80.4 vs. 32.5 weeks; hazard ratio, 0.33; P = .0008) and a trend toward improved PFS (median PFS, 24.8 vs. 15.2 weeks; hazard ratio, 0.51; P = .06), compared with PTEN-negative or PIK3CA-mutant tumors. PTEN methylation was more common in the metastatic samples than in the primary samples (P = .02). The simultaneous presence of methylation and mutation in the PTEN gene was associated with IHC negativity (P = .026). CONCLUSION: In addition to KRAS mutation, loss of PTEN expression (by IHC) and PIK3CA mutation is likely to be predictive of a lack of benefit to anti-EGFR therapy in metastatic colorectal cancer. PTEN promoter methylation and mutation status was predictive of PTEN expression and may be used as an alternative means of predicting response to EGFR-targeted therapy.

BACKGROUND: mutational loss of tumor suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN) is associated with malignant progression in many cancers, including colorectal cancer (CRC). PTEN is involved in negatively regulating the phosphatidylinositol 3-kinase/AKT oncogenic signaling pathway and has been implicated in the metastatic colonization process. Few in vivo models are available to study CRC metastasis. The purpose of this study was to determine the effect of restoring PTEN activity on metastases in an orthotopic murine model. METHODS: Green fluorescent protein labeled TENN, a highly metastatic human colon cancer cell line with mutational loss of PTEN gene and TENN clones (with restoration of PTEN gene) tumors were orthotopically implanted onto the colons of BALB/c nude mice and allowed to develop primary and metastatic tumors. Seven weeks post-implantation, mice were euthanized and organs extracted for examination. RESULTS: Both TENN and TENN clone cell lines demonstrated 100% primary invasion. However, compared with the parental TENN cells, which demonstrated 62% metastases to both lungs and liver, TENN clone cells showed an approximately 50% reduction in metastasis, with only 31.6% liver metastasis and no metastasis to the lungs (P = 0.02). CONCLUSIONS: Our study shows that reactivation of PTEN tumor suppressor pathway leads to a 50% reduction in CRC metastasis without affecting primary tumor formation. Importantly, PTEN restoration also changed the organotropic homing from liver and lung metastasis to liver metastasis only. This in vivo study demonstrates that PTEN might act specifically as a metastasis suppressor and, thus, efforts to target the phosphatidylinositol 3-kinase/PTEN pathway are legitimate.

The PTEN/MMAC1/TEP1 gene (phosphatase and tensin homolog deleted on chromosome 10/mutated in multiple advanced cancers/TGF-beta regulated and epithelial cell enriched phosphatase 1), which regulates the signaling pathways of Akt, is a novel tumor suppressor gene implicated in multiple cancers. Because a number of tumor suppressor genes are known to be silenced by aberrant promoter methylation, we examined the methylation status of the 5' CpG islands of PTEN using methylation-specific PCR. The altered expression of PTEN in 310 gastric carcinomas was analyzed by immunohistochemical staining using tissue-array and clinicopathologic profiles related to PTEN expression were characterized. Of 310 consecutive gastric carcinomas, 62 cases (20%) showed expression loss of PTEN. Altered PTEN expression was significantly associated with tumor depth and size, lymphatic invasion, advanced stage, pTNM stage, and patient survival (p < 0.001). The promoter methylation frequency of PTEN was found to be present in 26 (39%) of 66 cases examined, and 19 (73%) of 26 gastric cancer tissues showing promoter methylation exhibited the loss of PTEN expression. Abnormalities in the expression of PTEN significantly correlated with promoter methylation (p < 0.001). In conclusion, silencing of the PTEN gene occurs frequently in gastric carcinoma and aberrant promoter methylation is a major mechanism of silencing of the PTEN gene. The abnormalities of the PTEN gene are associated with tumor progression, metastasis, and survival.

AIM: To investigate the loss of heterozygosity (LOH) and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions. METHODS: Thirty cases of normal gastric mucosa, advanced and early stage gastric cancer, intestinal metaplasia, atrophic gastritis, and atypical hyperplasia were analyzed for PTEN LOH and mutations within the entire coding region of PTEN gene by PCR-SSCP denaturing PAGE gel electrophoresis, and PTEN mutation was detected by PCR-SSCP sequencing followed by silver staining. RESULTS: LOH rate found in respectively atrophic gastritis was 10% (3/30), intestinal metaplasia 10% (3/30), atypical hyperplasia 13.3% (4/30), early stage gastric cancer 20% (6/30), and advanced stage gastric cancer 33.3% (9/30), None of the precancerous lesions and early stage gastric cancer showed PTEN mutations, but 10% (3/30) of the advanced stage gastric cancers, which were all positive for LOH, showed PTEN mutation. CONCLUSION: LOH of PTEN gene appears in precancerous lesions, and PTEN mutations are restricted to advanced gastric cancer, LOH and mutation of PTEN gene are closely related to the infiltration and metastasis of gastric cancer.

BACKGROUND AND AIM: Gastric carcinoma is the second most frequent cause of cancer-related death worldwide. As PTEN is a potential modifier of tumour response to trastuzumab, a recently approved therapy in metastatic HER2 positive gastric cancer, the existence of PTEN deletions in primary gastric cancer was investigated. METHODS: 230 primary gastric cancers were analysed in a tissue microarray format by dual labelling fluorescence in situ hybridisation for PTEN deletion. HER2 analysis was also performed. To study PTEN deletion heterogeneity, all available large tissue sections from primary cancer and corresponding metastases were analysed in seven patients. RESULTS: Eight of 180 interpretable primary gastric cancer spots showed PTEN deletions (4.4%), including seven hemizygous and one homozygous deletion. PTEN deletion was correlated with nodal (8 of 122 cases (6.6%); p=0.041) and distant metastases (4 of 19 (21.1%); p<0.001). Large section validation showed a homogeneous distribution of PTEN deletion. HER2 positivity was seen in one PTEN deleted case. CONCLUSION: Genomic PTEN deletion is a rare event in gastric adenocarcinoma but correlates with metastatic disease. The homogeneous distribution pattern indicates that this alteration occurs early in tumour development.

Little is reported about the role of PTEN gene in the progression and prognosis of GISTs. This study examined the clinical implications of the tumor suppressor gene PTEN as a prognostic factor in the GISTs. Immunohistological staining and immunoblotting were employed to examine the PTEN protein expression, and its association with clinical measures. Clinicopathological features were reviewed by a retrospective examination of medical records. Reduced PTEN expression was significantly associated with tumor diameter, mitotic figure count, metastasis and pathological stage of tumor (P<0.05), and was not correlated with age, gender and tumor location (P>0.05). The 3-year survival rate of the patients with reduced PTEN expression was significantly lower than those with high PTEN expression (P<0.01). These results suggest that the expression of PTEN gene was significantly linked with the progression and metastasis of GISTs and it is an independent prognostic factor.

OBJECTIVE: To explore the relationship between hypermethylation of the PTEN gene promoter and laryngeal squamous cell carcinoma (LSCC). METHOD: Promoter hypermethylation and mRNA expression were detected by methylation specific PCR and RT-PCR in LSCC and normal laryngeal tissue. RESULT: Among the 40 patients with LSCC, hypermethylation of PTEN gene promoter was detected in 16 cases (40.0%). Among the nine normal laryngeal tissues, hypermethylation of PTEN gene promoter was detected in no case. The high and moderate expression rates of PTEN gene mRNA in nine normal laryngeal tissues were 77.8% (7/9) and 22.2% (2/9), respectively; The high, moderate, low and negative expression rates of PTEN mRNA in 15 laryngeal cancers with lymph node metastasis were 26.7% (4/15), 33.3% (5/15), 26.7% (4/15) and 13.3% (2/15), respectively. The PTEN mRNA expression in normal laryngeal tissues was higher than that in the laryngeal cancers with lymph node metastasis (P < 0.05). The high, moderate and low expression rates of PTEN mRNA in 10 highly differentiated laryngeal cancers were 60.0% (6/10), 30.0% (3/10) and 10.0% (1/10), respectively; The high, moderate, low and negative expression rates of PTEN mRNA in 12 low differentiated laryngeal cancers were 16.7% (2/12), 25.0% (3/12), 41.7% (5/12) and 16.7% (2/12), respectively. The differences of PTEN mRNA expression between high and low differentiation laryngeal cancers were statistically significant (P < 0.05). The hypermethylation rate of PTEN gene promoter in the low mRNA expression laryngeal cancers was 70.0% (7/10), the hypermethylation rate of PTEN gene promoter in the high mRNA expression laryngeal cancers was 23.5% (4/17). The hypermethylation rate of PTEN gene promoter in the low mRNA expression laryngeal cancers was higher than that in the high mRNA expression laryngeal cancers (P < 0.05). CONCLUSION: Hypermethylation of PTEN gene promoter is associated with loss of its transcription in laryngeal squamous cell carcinoma, the decreasing of PTEN mRNA expression may be related to lymph node metastasis and low differentiation of laryngeal squamous cell carcinoma.

OBJECTIVE: To investigate the expression of FLIP and PTEN in laryngeal Squamous cell carcinoma (LSCC), and the relationship between FLIP and PTEN. METHOD: The protein expression of FLIP and PTEN were examined by using immunohistochemical method in 45 cases of LSCC and 15 cases of para-carcinoma tissues. RESULT: FLIP protein positive expressive rate in the laryngeal squamous cell carcinoma cases (77.8%) was higher than that in the para-carcinoma tissues cases (33.3%, P < 0.05). The protein expression of FLIP was correlated with cervical lymph node metastasis, clinical stage and prognosis. On the other hand, PTEN positive expressive rate in the laryngeal squamous cell carcinoma (65.0%) was higher than that in the para-carcinoma tissues (0%, P < 0.01). The protein expression of PTEN was associated with tumor differentiation grade, clinical stage, cervical lymph node metastasis and prognosis. There was a negative relationship between the expression of FLIP and PTEN in LSCC. CONCLUSION: The protein expression of FLIP may be an important prognostic marker for LSCC. The protein expression of PTEN was correlated with clinical stage, tumor differentiation grade, and cervical lymph node metastasis . Consequently, it could be used as a valuable marker for the prognosis of LSCC.

OBJECTIVE: To investigate the effect of the wild type phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor-suppressor gene on the proliferation and apoptosis of human chronic myeloid leukemia (CML) cells line (K562) in vitro and explore the influence of PTEN-FAK signaling pathway on invasion and metastasis of leukemia cells. METHODS: The recombinant Ad-PTEN gene containing green fluorescent protein gene (Ad-PTEN-GFP) or the empty vector (Ad-GFP) was transfected into K562 cells and fresh leukemia cells from CML patients in blast crisis. The growth of K562 cells was assayed by MTT assay; the apoptosis rate was assessed by flow cytometry (FCM). PTEN and FAK mRNA levels were detected by real-time fluorescent relative- quantification reverse transcriptional PCR (FQ-PCR) and its protein levels by Western blot. The metastasis and invasive ability was examined by transwell chamber assay. RESULTS: The growth of K562 cells was suppressed markedly when Ad-PTEN-GFP was transfected into K562 cells at the 200 multiplicity of infection (MOI). The maximum growth inhibition rate was 35.2%. Transwell results showed the number of cells entered the lower chamber in Ad-GFP group was 9.1 fold more than that in Ad-PTEN-GFP group;The ability of metastasis and invasion of fresh leukemia cells was also suppressed after transfection with Ad-PTEN-GFP. FAK and p-FAK proteins were down-regulated by 0.72 and 0.16 fold lower after transfected with Ad-PTEN-GFP compared with Ad-GFP group. CONCLUSIONS: PTEN gene might inhibit the proliferation, metastasis and invasive ability of leukemia cells via down-regulating FAK expression.

Lung cancer is the leading cause of cancer-related mortality in the United States. Only 15% of patients with this disease survive 5 years or longer. Early metastatic spread is the single most important reason for this poor outcome. The survival of patients with pathological stage I disease, that is, no evidence for metastatic spread, and molecular aberrations on chromosome 11p15.5 is equal to that of patients with stage II disease, that is, metastatic spread to hilar lymph nodes. RRM1 is a gene in this region, and it is haploinsufficient in at least 34% stage I patients. Here, we show that overexpression of RRM1 in human and mouse lung cancer cell lines induced PTEN expression, reduced phosphorylation of focal adhesion kinase (FAK), suppressed migration, invasion, and metastasis formation, and increased survival in an animal model. Increased PTEN expression was required for the RRM1-induced suppression of cell motility and FAK phosphorylation. We conclude that RRM1 functions as a metastasis suppressor gene through induction of PTEN expression.

Mitogen-activated protein kinase (MAPK) and AKT pathways are frequently co-activated in melanoma through overexpression of receptor tyrosine kinases, mutations in their signaling surrogates, such as RAS and BRAF, or loss of negative regulators such as PTEN. As RAS can be a positive upstream regulator of PI3-K, it has been proposed that the loss of PTEN and the activation of RAS are redundant events in melanoma pathogenesis. Here, in genetically engineered mouse models of cutaneous melanomas, we sought to better understand the genetic interactions between HRAS activation and PTEN inactivation in melanoma genesis and progression in vivo. We showed that HRAS activation cooperates with Pten+/- and Ink4a/Arf-/- to increase melanoma penetrance and promote metastasis. Correspondingly, gain- and loss-of-function studies established that Pten loss increases invasion and migration of melanoma cells and non-transformed melanocytes, and such biological activity correlates with a shift to phosphorylation of AKT2 isoform and E-cadherin down-regulation. Thus, Pten inactivation can drive the genesis and promote the metastatic progression of RAS activated Ink4a/Arf deficient melanomas.

The PTEN gene is one of the most frequently inactivated tumor suppressor genes in sporadic cancers. Inactivating mutations and deletions of the PTEN gene are found in many types of cancers, including melanoma. However, the exact frequency of PTEN alteration in melanoma is unknown. In this study, we comprehensively reviewed 16 studies on PTEN genetic changes in melanoma cell lines and tumor biopsies. To date, 76 PTEN alterations have been reported in melanoma cell lines and 38 PTEN alterations in melanoma biopsies. The rate of PTEN alterations in melanoma cell lines, primary melanoma, and metastatic melanoma is 27.6, 7.3, and 15.2%, respectively. Three mutations were found in both melanoma cell lines and biopsies. These mutations are scattered throughout the gene, with the exception of exon 9. A mutational hot spot is found in exon 5, which encodes the phosphatase activity domain. Evidence is also presented to suggest that numerous homozygous deletions and missense variants exist in the PTEN transcript. Studying PTEN functions and implications of its mutations and other genes could provide insights into the precise nature of PTEN function in melanoma and additional targets for new therapeutic approaches.

pathway-specific therapy is the future of cancer management. The oncogenic phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in solid tumors; however, currently, no reliable test for PI3K pathway activation exists for human tumors. Taking advantage of the observation that loss of PTEN, the negative regulator of PI3K, results in robust activation of this pathway, we developed and validated a microarray gene expression signature for immunohistochemistry (IHC)-detectable PTEN loss in breast cancer (BC). The most significant signature gene was PTEN itself, indicating that PTEN mRNA levels are the primary determinant of PTEN protein levels in BC. Some PTEN IHC-positive BCs exhibited the signature of PTEN loss, which was associated to moderately reduced PTEN mRNA levels cooperating with specific types of PIK3CA mutations and/or amplification of HER2. This demonstrates that the signature is more sensitive than PTEN IHC for identifying tumors with pathway activation. In independent data sets of breast, prostate, and bladder carcinoma, prediction of pathway activity by the signature correlated significantly to poor patient outcome. Stathmin, encoded by the signature gene STMN1, was an accurate IHC marker of the signature and had prognostic significance in BC. Stathmin was also pathway-pharmacodynamic in vitro and in vivo. Thus, the signature or its components such as stathmin may be clinically useful tests for stratification of patients for anti-PI3K pathway therapy and monitoring therapeutic efficacy. This study indicates that aberrant PI3K pathway signaling is strongly associated with metastasis and poor survival across carcinoma types, highlighting the enormous potential impact on patient survival that pathway inhibition could achieve.

BACKGROUND: mutations in the p53 gene are amongst the most frequent aberrations seen in human cancer. Our objective was to characterize the clinical characteristics associated with p53 mutation in patients with advanced cancer. METHODS: We retrospectively reviewed and analyzed the clinical features and response to standard systemic therapy of 145 patients with documented tumor p53 mutational status (mutant-type [mtp53] vs. wild-type [wtp53]) referred to the Clinical Center for Targeted Therapy. RESULTS: Sixty-six (45.5%) patients had mtp53. mutations in p53 occurred more frequently in older patients (p= 0.015) and in Caucasians (p=0.024). The incidence of liver metastases was 69.2% vs. 43%, p=0.002 in mtp53 and wtp53, respectively. PTEN loss by immunohistochemistry was found more frequently in mtp53-bearing tumors compared to wtp53 (33.3% vs. 10%, p=0.007). The best progression-free survival (PFS) on standard systemic therapy was significantly longer with bevacizumab-containing regimens as compared to non-bevacizumab containing regimen in patients with mtp53 (median 11.0 [95%

The multistep process of carcinogenesis implies the accumulation of multiple molecular defects. Alteration of tumor suppressor and metastasis suppressor genes are the important steps. Increasing experimental evidence indicates that decreased expression of tumor-metastasis/suppressor genes and gene products are involved in the progression of a variety of human malignancies. In the present study, we have extended this analysis to non-small cell lung carcinomas (NSCLC). The expression and prognostic significance of the tumor suppressor gene PTEN and metastasis suppressor genes nm23-H1 and KAI-1 was evaluated in NSCLCs. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissues from 53 bronchogenic adenocarcinomas and 51 squamous cell carcinomas using monoclonal antibodies against PTEN, nm23H-1, and KAI-1 proteins. Immunohistochemical results were correlated with tumor stage, grade, lymph nodes positivity, metastasis, and patient survival. Significant co-expression of PTEN, nm23-H1 and KAI-1 was observed in NSCLC (P<.001 to .002). The immunohistochemical expression of these proteins was significantly higher in stages 1 and 2 compared with stages 3 and 4 (P=.04 for PTEN and KAI-1, P=.039 for nm23-H1). When all stages were considered together, loss of immunoreactivity for PTEN, nm23-H1 and KAI-1 was found in advanced NCSCLs (P=.015 for PTEN, P=.001 for KAI-1, P=.004 for nm23-H1), which is suggestive of co-downregulation of these proteins in the process of tumor progression. On multivariate analysis, negative staining for PTEN (P=.014), KAI-1 (P=.034), and nm23-H1 (a trend toward association for nm23-H1 reached near significance P=.08) correlated with disease-related death. Positive lymph node status was associated with negative immunostaining for PTEN (P=.007) but no correlation was observed for nm23-H1 and KAI-1. Loss of expression was linked to distant metastasis (P=.006 for PTEN, P=.002 for nm23H1, P=.001 for KAI-1). On multivariate analysis, co-downregulation of PTEN (P=.009), KAI-1 (P=.02), and nm23-H1 (P=.011) independently predicted shortened survival in NSCLC. Although NSCLC exhibits strong co-expression of PTEN, nm23-H1 and KAI-1, there is a loss of these proteins in high-stage tumors. Co-downregulation of PTEN, KAI-1, and nm23-H1 significantly correlates with distant metastasis and predicts shortened survival. Our study supports a role of these tumor suppressor and metastasis suppressor genes in the evolution and progression of NSCLC.

BACKGROUND AND OBJECTIVE: It has been proved that mTOR was an important signal transduction molecular and protein kinase regulating cell growth and proliferation, and mTOR could activate the downstream protein effector. PTEN could negatively regulate mTOR signal pathway and inhibit its activity. The aim of this study is to detect the mRNA expression levels of mTOR and PTEN gene, which are the key genes of mTOR signaling pathway in human non-small cell lung cancer (NSCLC) tissue. The relationship between mTOR signaling pathway and NSCLC is also explored. METHODS: Lung cancer tissue specimens were obtained from 65 patients. Adjacent-tumor non-small cell lung cancer tissues from the 30 patients were served as control. The RT-PCR technique was used to detect the mTOR and PTEN gene expression levels. RESULTS: The average mRNA expression levels of mTOR gene were significantly higher (0.23 +/- 0.16) in lung cancer than in adjacent-tumor tissue (0.12 +/- 0.09)(P < 0.01). The average mRNA expression levels of PTEN gene were (0.19 +/- 0.28) in lung cancer, while the mRNA expression levels of PTEN gene were (0.53 +/- 0.28) in adjacent-tumor tissue (P < 0.01). The levels of PTEN gene expression in non-small cell lung cancer were significantly lower than that in adjacent-tumor lung tissue. There are not significant relationship between mTOR and PTEN gene expression levels and patients' age, gender, pathological type, differentiation, lymph node metastasis, except tumor size. CONCLUSIONS: The expression of mTOR is activated in NSCLC. The expression of PTEN is absent or decreased. The mTOR activated in NSCLC may be correlate with the absent or decreased of PTEN. The absent or decreased expression of PTEN and the actived mTOR may play important roles in carcinogenesis and metastasis of NSCLC.

A high frequency of mutations at the PTEN locus has been noticed in carcinoma of lung. However, the role of PTEN alternations and its association with outcome variables in the genesis of lung carcinoma are not understood fully. The purpose of our study was to examine the impact of EGFR, TGF-alpha, P-AKT and PTEN in the genesis of non-small cell lung cancer (NSCLC). Total numbers of 66 histopathologically confirmed cases of NSCLC and 10 cases of benign control samples embedded with wax were studied. We assessed EGFR, TGF-alpha and P-AKT by the use of specific antibody through immunohistochemistry as directed by the manufacturer, and detected PTEN expression by in situ hybridization. There were progressive loss of PTEN expression and significant increasing in EGFR, TGF-alpha, P-AKT expression from benign samples to NSCLC (p<0.05). The overexpression of EGFR, TGF-alpha, P-AKT and loss of PTEN expression were correlated to differentiation extent of cancer tissue, metastasis of lymph nodes and histological classification. Thus, alteration of EGFR, TGF-alpha, P-AKT and PTEN are likely important molecular events in pathogenesis and carcinogenesis of NSCLC.

BACKGROUND: Type-1 insulin-like growth factor (IGF-1) up-regulates cell proliferation and invasiveness through activation of PI3K/Akt signaling pathway. IGF-1 also down-regulates the tumor suppressor chromosome 10 (PTEN). We investigated the mechanism by which IGF-1 affects cell proliferation and invasion by suppression of PTEN phosphorylation and interaction with PI3K/PTEN/Akt/NF-small ka, CyrillicB signaling pathway in pancreatic cancer. MATERIALS AND METHODS: The expression of IGF-1 receptor (IGF-1R) and PTEN in five pancreatic cancer cell lines was determined by RT-PCR and Western blot. Proliferation and invasion were investigated by WST-1 assay and Matrigel-double chamber assay. Pancreatic cancer cells were transfected with PTEN siRNA to investigate which signaling pathway correlates in regulation of cancer cell proliferation and invasion. RESULTS: Five pancreatic cancer cell lines expressed PTEN and IGF-1R in mRNA and protein levels. Suppression of PTEN phosphorylation strongly enhanced cell proliferation and invasion stimulated with IGF-1 via activation of PI3K/Akt/NF-small ka, CyrillicB signaling pathway. In addition, knockdown of PTEN by siRNA transfection also enhanced activation of PI3K/Akt/NF-small ka, CyrillicB pathway, subsequently up-regulating cell invasiveness and proliferation. CONCLUSIONS: The IGF-1/PI3K/PTEN/Akt/NF-small ka, CyrillicB cascade may be a key pathway stimulating metastasis of pancreatic cancer cells. We suggest that interfering with the functions of IGF-1/PI3K/Akt/NF-small ka, CyrillicB might be a novel therapeutic approach to inhibit aggressive spread of pancreatic cancer.

BACKGROUND AND AIMS: We investigated PTEN expression in primary pancreatic cancer and pancreatic cancer liver metastasis in order to evaluate the interrelationship between PTEN expression and clinicopathological characteristics of pancreatic cancer patients with and without liver metastasis. METHODS: Eighty five primary pancreatic cancer specimens without liver metastasis were analyzed as controls. Eighty seven pancreatic cancer specimens and homologous liver metastasis specimens were investigated immunohistochemically, and the correlation between immunohistochemical findings and clinicopathological factors was investigated. RESULTS: A strong PTEN expression was observed in 52 (61.2%) specimens from patients without liver metastasis. In contrast, only 26 (29.9%) specimens were observed in patients with liver metastasis. A strong PTEN expression was apparently associated with low-grade lymph node metastasis (p <0.05) and TNM stage (p <0.05). PTEN expression in patients without liver metastasis was apparently stronger than that with liver metastasis. In addition, among patients with liver metastasis, the 5-year survival rate was markedly higher in patients with strong PTEN expression compared to those with weak PTEN expression. CONCLUSIONS: Our results suggest that a high level of PTEN expression is associated with low-grade liver metastasis and satisfactory patient survival in pancreatic cancer. The diagnostic evaluation of PTEN expression may provide valuable prognostic information to aid treatment strategies for pancreatic cancer patients.

PTEN (phosphatase and tensin homologue deleted on chromosome 10) has been shown to be inactivated in a wide variety of cancers, and the role of this gene as a tumor suppressor has been well established. On the other hand, results of recent animal studies as well as clinical evidence indicate that PTEN is also involved in tumor metastasis suppression. Although PTEN is known to play a key role in controlling cell growth and apoptosis, how PTEN exerts the metastasis suppressor function remains largely unknown. Recently, a microarray analysis identified the Drg-1 gene (differentiation related gene 1) as one of the potential targets of PTEN. The Drg-1 gene has been shown to suppress tumor metastasis in animal models of prostate and colon cancer, and the expression of this gene is significantly reduced with advancement of prostate and breast cancers in clinical setting. In this study, we explored the possibility that PTEN controls tumor metastasis by regulating the expression of the Drg-1 gene. Our results indicate that overexpression of PTEN significantly augments the endogenous expression of Drg-1 protein, whereas inhibition of PTEN by small interfering RNA decreases Drg-1 in a dose- and time-dependent manner. We also found that the control of the Drg-1 gene by PTEN seems to be at the transcriptional level, and that a phospho-Akt inhibitor restores the Drg-1 expression, indicating that PTEN controls Drg-1 by an Akt-dependent pathway. Consistent with these results, our immunohistochemical analysis revealed that PTEN expression correlates significantly with Drg-1 in both prostate and breast cancer cases. Furthermore, combination of the two markers, PTEN and Drg-1, emerged as a significantly better predictor of prostate and breast cancer patient survival than either marker alone.

Little is known about the role of the tumor suppressor gene phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in prostate cancer bone metastasis. To explore this, we used a pTetOn PTEN cell line in which PTEN expression was reconstituted in a PTEN-null bone metastatic human prostate cancer cell line, LnCaP-C4-2. We found that C4-2 cells selectively migrated toward conditioned medium from primary mouse calvaria cells compared with that derived from lung fibroblasts. Further evaluation with conditioned medium from an established mouse calvaria osteoblast cell line and control non-osteoblast cell line indicates that osteoblastic characteristics convey this specific migration to C4-2 cells. We evaluated promiscuously metastatic PC-3 prostate as well as T24T and UMUC-3 bladder cells and found they did not have a specific migratory response to calvaria-conditioned medium as did C4-2. Induction of PTEN expression inhibited the motility of C4-2 cells toward calvaria-conditioned medium but had no effect on migration toward lung-conditioned medium and this inhibitory effect was dependent on the PTEN lipid phosphatase activity. Calvaria- but not lung-conditioned medium induced activation of the small GTPase Rac1. Constitutively active Rac1 but not focal adhesion kinase or Cdc42 could rescue cells from the inhibitory effect of PTEN on cell migration and PTEN induction was observed to inhibit Rac1 activation in response to calvaria-conditioned medium. Our results support the notion that loss of PTEN function in human prostate cancer may specifically facilitate bone rather than other organ metastasis and suggest that Rac1, as a PTEN effector, may contribute to this metastatic tropism.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is frequently inactivated in metastatic prostate cancer, yet the molecular consequences of this and their association with the metastatic phenotype are incompletely understood. We performed transcriptomic analysis and identified genes altered by conditional PTEN reexpression in C4-2, a human metastatic prostate cancer cell line with inactive PTEN. PTEN-regulated genes were disproportionately represented among genes altered in human prostate cancer progression and metastasis but not among those associated with tumorigenesis. From the former set, we identified two novel putative PTEN targets, cdc6 and cyclin E2, which were overexpressed in metastatic human prostate cancer and up-regulated as a function of PTEN depletion in poorly metastatic DU145 human prostate cancer cells harboring a wild type PTEN. Inhibition of cdc6 and cyclin E2 levels as a consequence of PTEN expression was associated with cell cycle G(1) arrest, whereas use of PTEN activity mutants revealed that regulation of these genes was dependent on PTEN lipid phosphatase activity. Computational and promoter-reporter evaluations implicated the E2F transcription factor in PTEN regulation of cdc6 and cyclin E2 expression. Our results suggest a hypothetical model whereby PTEN loss upregulates cell cycle genes such as cdc6 and cyclin E2 that in turn promote metastatic colonization at distant sites.

PTEN loss or PI3K/AKT signaling pathway activation correlates with human prostate cancer progression and metastasis. However, in preclinical murine models, deletion of Pten alone fails to mimic the significant metastatic burden that frequently accompanies the end stage of human disease. To identify additional pathway alterations that cooperate with PTEN loss in prostate cancer progression, we surveyed human prostate cancer tissue microarrays and found that the RAS/MAPK pathway is significantly elevated in both primary and metastatic lesions. In an attempt to model this event, we crossed conditional activatable K-ras(G12D/WT) mice with the prostate conditional Pten deletion model. Although RAS activation alone cannot initiate prostate cancer development, it significantly accelerated progression caused by PTEN loss, accompanied by epithelial-to-mesenchymal transition (EMT) and macrometastasis with 100% penetrance. A novel stem/progenitor subpopulation with mesenchymal characteristics was isolated from the compound mutant prostates, which was highly metastatic upon orthotopic transplantation. Importantly, inhibition of RAS/MAPK signaling by PD325901, a mitogen-activated protein (MAP)-extracellular signal-regulated (ER) kinase (MEK) inhibitor, significantly reduced the metastatic progression initiated from transplanted stem/progenitor cells. Collectively, our findings indicate that activation of RAS/MAPK signaling serves as a potentiating second hit to alteration of the PTEN/PI3K/AKT axis, and cotargeting both the pathways is highly effective in preventing the development of metastatic prostate cancers.

Multilocular cystic renal cell carcinoma is a rare renal cell carcinoma with an excellent prognosis. To clarify the relationship with typical clear cell renal cell carcinoma, we evaluated 15 cases of multilocular cystic renal cell carcinomas diagnosed according to the 2004 WHO classification. Von Hippel Lindau (VHL) gene mutations were determined by whole genome amplification and direct sequencing. Carbonic anhydrase 9 (CAIX), a hypoxia-inducible factor (HIF) target, paired box gene 2 (PAX2), cyclin-dependent kinase inhibitor p27 and glycogen synthase kinase 3-beta (GSK3beta) were immunohistochemically evaluated as members of the VHL protein (pVHL)- and phosphatase and tensin homolog (PTEN)-controlled pathways. VHL mutations were identified in 3 of 12 (25%) tumors. Inactivated GSK3beta, decreased PTEN expression and PAX2 positivity were observed in the vast majority of the multilocular cystic renal cell carcinomas. Strong nuclear staining of p27 was seen in 14 of 15 cases. Compared with multilocular cystic renal cell carcinomas, expression frequencies of PAX2, p-GSK3beta, PTEN and CAIX were similar in a set of low-grade, early-stage clear cell renal cell carcinomas, whereas only 30% had strong p27 positivity. These results are consistent with the hypothesis that multilocular cystic renal cell carcinomas are related at the molecular level with clear cell renal cell carcinomas. Maintenance of a strong subcellular p27 expression in all multilocular cystic renal cell carcinomas analyzed may in part explain the excellent prognosis of these tumor patients.

CONCLUSION: Down-regulation of metastasis inhibitor genes PTEN and thrombospondin 2 may play a role in the lymphatic metastasis of supraglottic carcinoma. OBJECTIVES: To investigate differentially expressed genes in supraglottic squamous cell carcinoma with and without lymphatic metastasis using cDNA microarray. MATERIALS AND METHODS: cDNA microarray was used to detect the expression profiles of supraglottic carcinoma and the results were validated with real-time PCR. RESULTS: Compared with normal laryngeal tissues, the expression of 15.6% (15/96) genes including cathepsin B, cathepsin D, cathepsin L, Ets1, c-Ets2, and PEA3 were up-regulated in both cases of supraglottic carcinoma. Compared with the supraglottic carcinoma with no lymph node metastasis, the expression of metastasis inhibitor genes PTEN and thrombospondin 2 was down-regulated in the supraglottic carcinoma tissue with lymph node metastasis.