Strong p-Akt Expression Research Articles

Patterns of AKT, mTOR, ERK, and STAT3 pathway activation in MGUS, smoldering multiple myeloma, and multiple myeloma.

8589 Background: Constitutive activation of key signaling pathways supports proliferation in multiple myeloma (MM). However, pathway activation in MGUS and smoldering multiple myeloma (SMM) compared to MM has not been characterized. We examined the levels of phosphorylated Akt, mTOR, ERK, and STAT3 expression by immunohistochemistry (IHC) in MGUS, SMM, and MM and assessed their impact on clinical outcomes. Methods: FFPE bone marrow biopsy samples were obtained during routine clinical care. Primary antibodies used were p-Akt (S473), p-mTOR(Ser2448), p-ERK1/2(Thr202/Tyr204), and p-STAT3 (Tyr705). Staining intensity was scored as: 0 (no staining), 1 ( < 50% of cells), 2 ( > 50%) and 3 (dark staining of all cells). Nominal variables were compared with Fisher's exact test and time to progression (TTP) was calculated with Kaplan Meier analysis. Results: Twenty-one MGUS, 20 SMM, and 18 MM patients were reviewed. Strong expression of p-Akt (2-3+ IHC score) was present in 15% of MGUS cases versus 62% of SMM and 61% of MM patients (p = 0.037). Strong p-mTOR expression was observed in SMM and MM patients compared to MGUS patients (100% and 94% versus 45%; p = 0.001). 61% of MM cases exhibited strong p-ERK expression versus 34% in SMM and 15% in MGUS (p = 0.042). There was non-significant trend toward higher p-STAT3 expression in MM versus SMM/MGUS (39% in MM versus 12% and 10% in SMM and MGUS; p = 0.15). Increased p-mTOR activity correlated with high expression of p-Akt(S473) in all patients (p = 0.023). TTP in SMM patients with high versus low p-ERK activity was significantly shorter (76 months vs 16 months; P = 0.046). No other signaling pathways demonstrated statistical differences in TTP or overall survival. Conclusions: Comprehensive IHC analysis across the spectrum from MGUS to SMM and MM demonstrated increased activity of multiple key signaling pathways as disease progression occurs. Expression of p-Akt(S473) and p-mTOR in SMM and MM is significantly higher than MGUS patients, suggesting an upregulation of these pathways is an early event in pathogenesis. Activation of p-ERK and p-STAT3 was more frequent in MM versus MGUS/SMM, suggesting they may be more active in advanced disease.

Abstract LB-517: EIF5A2, A downstream target of PI3K/Akt pathway, Is an adverse prognostic marker of melanoma patient survival by increasing cell invasion

Abstract Human cutaneous melanoma is a life-threatening skin cancer due to its invasive nature and high metastatic potential, leading to poor prognosis for melanoma patients. However, the mechanisms for melanoma invasion and metastasis are poorly understood. Human eukaryotic translation initiation factor 5A2 (EIF5A2) has been shown to be associated with tumor progression in multiple types of cancers. We examined EIF5A2 expression in 459 melanocytic lesions at different stages using tissue microarray and immunohistochemistry and analyzed the correlations between EIF5A2 expression and clinicopathologic parameters and patient survival. We found that positive EIF5A2 staining was significantly increased in primary melanomas compared to dysplastic nevi (P=0.002), and further increased in metastatic melanomas (P=0.036). EIF5A2 expression was correlated with melanoma thickness (P=0.0004) and was inversely correlated with overall and disease-specific 5-year survival of primary (P=0.008 and 0.007, respectively), especially low-risk (≤2.0mm) melanoma patients (P=0.026 and 0.044, respectively). We also examined the correlation between EIF5A2 and p-Akt protein expression and their role in regulating melanoma cell invasion. Combining the TMA data sets for EIF5A2 and p-Akt, we found that positive EIF5A2 staining directly correlated with strong p-Akt expression (P=0.026). Additionally, overexpression of PTEN or inhibition of p-Akt or ILK significantly reduced EIF5A2 expression. EIF5A2 overexpression also increased melanoma cell invasion and MMP-2 activity. We for the first time showed that the expression of EIF5A2, a downstream target of PI3K/Akt pathway, is significantly increased during melanoma invasion and is inversely correlated with patient survival, suggesting that EIF5A2 may be used as a potential therapeutic target for melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-517. doi:1538-7445.AM2012-LB-517

The expression of CXCR4, CXCL12 and CXCR7 in malignant pleural mesothelioma

The chemokine CXCL12 and its receptors, CXCR4 and CXCR7, are involved in tumour progression, metastasis, and survival. We investigated the expression of CXCR4, CXCL12, and CXCR7 in malignant pleural mesothelioma to determine if they are possible biomarkers and potential therapeutic targets. Forty-one mesothelioma tumour tissues, ten normal human pleural tissues, and two mesothelioma cell lines were stained with anti-CXCR4, anti-CXCL12, anti-CXCR7, and anti-p-Akt antibodies. RT-PCR was performed to determine the expression of CXCR4, CXCL12, and CXCR7 in six human mesothelioma cell lines (H28, 211H, H2052, ms-1, H290, and H513) and one human normal mesothelial cell line, LP9. These seven cell lines were also stained with anti-CXCR7. We found that CXCR4 and CXCL12 were expressed in 97.6% and 78.0% mesothelioma tissue samples, concurrently with strong expression of p-Akt (R(2) = 0.739 and 0.620, respectively). In addition, CXCR7 expression was weaker than CXCR4 expression in mesothelioma tissues. Furthermore, RT-PCR showed that CXCR4 and CXCL12 were overexpressed in 5/6 mesothelioma cell lines (211H, H2052, ms-1, H290, and H513), whereas CXCR7 was overexpressed in only 2/6 (H513 and H2052). Moreover, we found that the CXCR4 antagonist AMD3100 inhibited the growth of all five mesothelioma cell lines that overexpress CXCR4 and CXCL12. Our results suggest that the Akt-mTOR pathway is involved during the interruption of the CXCL12/CXCR4 axis in these five mesothelioma cell lines. In conclusion, CXCR4 and CXCL12 are highly expressed in most mesothelioma cell lines and tumour tissues, suggesting that CXCR4 and CXCL12 may be used as biomarkers for patients with mesothelioma. The CXCL12-CXCR4 interaction may be a potential therapeutic target for mesothelioma.

Association between AKT/mTOR signalling pathway and malignancy grade of human gliomas

The mammalian target of rapamycin (mTOR) signaling pathway has emerged as a major effector of cell growth and proliferation, and is an attractive target for cancer therapy. However, the association between mTOR pathway and the malignancy grade of human gliomas has not been thoroughly investigated. Tumor tissues from 87 Chinese patients (49 males, average age of 51.7 ± 13.0 years, range 15-78) with glioma were prospectively collected. The expression of three key proteins of the mTOR pathway, pAKT, pmTOR and p-p70S6 kinase (p-p70S6K) was measured by semi-quantitative immunohistochemical techniques. Grade I-II, III and IV glioma was pathologically identified in 27 (31.0%), 24 (27.6%) and 36 (41.4%) patients, respectively. Of the 87 patients, pAKT, pmTOR and p-p70S6K were found in 63 (72.4%), 65 (74.7%), and 63 (72.4%) patients, respectively. The expression of all three pAKT, pmTOR and p-p70S6K proteins was found in 42 (48.3%) patients, while only one or two of the three proteins were found in the remaining patients (51.7%). The percentage of patients with very strong expression of pAKT, pmTOR and p-p70S6K in grade IV glioma was 13 (36.1%), 16 (44.4%) and 15 (41.7%), respectively, which was greater than in grade I or II tumors (0-3.7%, P < 0.01). In conclusion, expression of mTOR pathway proteins pAKT, pmTOR and p-p70S6K can be found in human glioma of all malignancy grades. However, higher levels of these proteins were associated with advanced malignancy grades of the tumor.

PTEN/p-AKT expression as predictive markers for cetuximab in colorectal cancer.

e14031 Background: Alterations in the Ras/Raf/ERK and PTEN/PI3K/AKT signaling pathways frequently occur in colorectal cancer (CRC). KRAS or BRAF mutations preclude responsiveness to EGFR-targeted therapies for CRC patients (pts). However, the biologic impact of the PTEN/PI3K/AKT pathway on anti-EGFR treatment is still controversial. This study aims to validate whether PTEN/p-AKT expression status is predictive of response to the anti-EGFR monoclonal antibody, cetuximab. Methods: We retrospectively investigated the role of PTEN/p-AKT expression in predicting the activity of cetuximab in CRC pts by immunohistochemistry (IHC). Results: As of today, PTEN/p-AKT IHC was performed on specimens from 40 CRC pts with wild-type KRAS/BRAF. All patients received cetuximab-based regimens including cetuximab plus irinotecan combination therapy (n = 33; 82.5%) and cetuximab monotherapy (n = 5; 12.5%). High PTEN expression were associated with decreased progression-free survival (PFS) compared with low PTEN (4.1 months vs. 9.6 months, HR 2.742, 95% CI, 1.001-7.508; p = 0.050). The response rate (RR) in patients with high PTEN was lower than in those with low expression, but not significantly (20% vs. 40%, p = 0.1675). Furthermore, strong p-AKT expression were significantly associated with a poor PFS compared with weak p-AKT (2.9 months vs. 6.9 months, HR = 4.14, 95% CI 1.206-14.23, p = 0.024). The RR in patients with strong and weak p-AKT expression was 0% and 34%, respectively. There were significant correlation between high p-AKT and high PTEN expression (Fisher's exact test, p = 0.0168). Conclusions: Our analysis suggests that the level of PTEN/p-AKT expression may predict the activity of cetuximab treatment. However, our data regarding PTEN contradicts previous reports. One possible explanation could be negative feedback loop between p-AKT and PTEN gene expression. Further analyses on PTEN and p-AKT immunostaining are ongoing. No significant financial relationships to disclose.

Transplantation of sendai viral angiopoietin-1-modified mesenchymal stem cells for ischemic limb disease

Sendai viral vector (SeV) is emerging as a promising vector for gene therapy. However, little information is available regarding the combination of SeV-mediated gene and mesenchymal stem cell (MSC) therapy in dealing with ischemic diseases. In this study, we infected SeV to the MSCs in vitro; and injected MSCs modified with SeV harboring human angiopoietin-1 gene (SeVhAng-1) into the ischemic limb of rats in vivo. We found SeV had high transductive efficiency to the MSCs. Both MSCs and SeVhAng-1-modified MSCs improved the blood flow recovery and increased the capillary density of the ischemic limb, compared with the control. However, in contrast to MSCs, SeVhAng-1-modified MSCs had a better improvement of blood flow recovery in the ischemic limb. We further found the ischemic limb injected with SeVhAng-1-modified MSCs had strong expression of p-Akt, which improved survival of MSCs injected into the ischemic limb. This indicated SeVhAng-1 modification enhanced angiogenetic effect of MSCs by both angiogenesis and cell protection. We conclude that SeVhAng-1-modified MSCs may serve as a more effective tool in dealing with ischemic limb disease.

Signaling pathways in adenoid cystic cancers: Implications for treatment

Adenoid cystic cancers (ACC) in the head and neck are rare yet present a clinical dilemma. Although 5 year survivals are excellent, their have a propensity for late recurrences. Most of these cancers are initially treated with surgery followed by radiation. When recurrences happen, treatment options are limited both by the morbidity and low efficacy of re-irradiation and repeated surgical resection. Reported response rates to chemotherapy are low and targeted therapies may be one option. We, therefore, investigated signaling pathways that may be active in adenoid cystic cancers. Tissues from the last 9 ACC patients resected at the University of Iowa were immunohistochemically stained with antibodies for EGFR, phosphorylated (P) Akt, and P-MAPK in order to molecularly characterize these tumors. An ACC cell line (ACC3) was also characterized by Western blot. We found that seven of the 9 tumor samples had strong expression of P-Akt and 5/9 had P-MAPK. None of them had EGFR expression. In the ACC3 cell line, similar data was found in that there was P-Akt and P-MAPK but no EGFR expression. We tested the HIV protease inhibitor nelfinavir (NFV) which has been shown to inhibit Akt signaling to see its effect on ACC3 cells. Both P-Akt and P-MAPK were inhibited with NFV in ACC3 cells and this resulted in growth inhibition and clonogenic death. In patients where re-irradiation or further surgery is not an option, a trial of NFV may be warranted.

Role of p53 Up-regulated Modulator of Apoptosis and Phosphorylated Akt in Melanoma Cell Growth, Apoptosis, and Patient Survival

Malignant melanoma is an aggressive and chemoresistant form of skin cancer characterized by rapid metastasis and poor patient prognosis. The development of innovative therapies with improved efficacy is critical to treatment of this disease. Here, we show that aberrant expression of two proteins, p53 up-regulated modulator of apoptosis (PUMA) and phosphorylated Akt (p-Akt), is associated with poor patient survival. Using tissue microarray analysis, we found that patients exhibiting both weak PUMA expression and strong p-Akt expression in their melanoma tumor tissue had significantly worse 5-year survival than patients with either weak PUMA or strong p-Akt expression alone (P < 0.001). Strikingly, no patients exhibiting strong PUMA expression and weak p-Akt expression in primary tumor tissue died within 5 years of diagnosis. We propose a two-pronged therapeutic strategy of (a) boosting PUMA expression and (b) inhibiting Akt phosphorylation in melanoma tumor tissue. Here, we report that a recombinant adenovirus containing human PUMA cDNA (ad-PUMA) efficiently inhibits human melanoma cell survival in vitro, rapidly induces apoptosis, and dramatically suppresses human melanoma tumor growth in a severe combined immunodeficient mouse xenograft model. In melanoma cells strongly expressing p-Akt, we show that Akt/protein kinase B signaling inhibitor-2 (API-2; a small-molecule Akt inhibitor) reduces cell survival in a dose- and time-dependent manner and enhances ad-PUMA-mediated growth inhibition of melanoma cells. Finally, we show that, by combining ad-PUMA and API-2 treatments, human melanoma tumor growth can be inhibited by >80% in vivo compared with controls. Our results suggest that a strategy to correct dysregulated PUMA and p-Akt expression in malignant melanoma may be an effective therapeutic option.

Cooperation between FGF8b Overexpression and PTEN Deficiency in Prostate Tumorigenesis

Two commonly occurring genetic aberrations of human prostate cancer [i.e., overexpression of a mitogenic polypeptide (fibroblast growth factor 8, isoform b or FGF8b) and loss of function of PTEN tumor suppressor] were recapitulated into a new combinatorial mouse model. This model harboring the Fgf8b transgene and haploinsufficiency in Pten, both in a prostate epithelium-specific manner, yielded prostatic adenocarcinoma with readily detectable lymph node metastases, whereas single models with each of the defects were shown earlier to progress generally only up to prostatic intraepithelial neoplasia (PIN). In addition to late age-related development of typical adenocarcinoma, the model also displayed a low incidence of mucinous adenocarcinoma, a rare variant type of human prostatic adenocarcinoma. The cooperation between FGF8b activation and PTEN deficiency must be linked to acquisition of additional genetic alterations for the progression of the lesions to primary adenocarcinoma. Here, we identified loss of heterozygosity at the Pten gene leading to bialleic loss, as a necessary secondary event, indicating that a complete loss of PTEN function is required in the development of invasive cancer in the model. Analyses of expression of downstream mediators phospho-AKT (p-AKT) and p27(KIP1), in various types of lesions, however, revealed a complex picture. Although PIN lesions displayed relatively strong expression of p-AKT and p27(KIP1), there was a notable heterogeneity with variable decrease in their immunostaining in adenocarcinomas. Together, the results further underscore the notion that besides activation of AKT by loss of PTEN function, other PTEN-regulated pathways must be operative for progression of lesions from PIN to adenocarcinoma.

Prognostic Significance of Activated Akt Expression in Melanoma: A Clinicopathologic Study of 292 Cases

Akt is a serine/threonine kinase that leads to stimulation of cell cycle progression, cell proliferation, and inhibition of apoptosis. To investigate the role of Akt in melanoma pathogenesis, we examined the expression of phospho-Akt (p-Akt; Ser-473) in melanocytic lesions at different stages and analyzed the correlations between the p-Akt expression level and clinicopathologic factors and patient survival. We evaluated the p-Akt expression in 12 cases of normal nevi, 58 cases of dysplastic nevi, 170 cases of primary melanomas, and 52 cases of melanoma metastases using tissue microarray and immunohistochemistry. Strong p-Akt expression was observed in 17%, 43%, 49%, and 77% of the biopsies in normal nevi, dysplastic nevi, primary melanoma, and melanoma metastases, respectively. Significant differences for p-Akt staining pattern were observed between normal nevi and primary melanomas (P < .05), and between primary melanomas and melanoma metastases (P < .001). Furthermore, our Kaplan-Meier survival curves showed that strong p-Akt expression is inversely correlated with both overall and disease-specific 5-year survival of patients with primary melanoma (P < .05 for both). Strikingly, our multivariate Cox regression analysis revealed that p-Akt is an independent prognostic factor in low-risk melanomas (thickness < or = 1.5 mm; relative risk, 6.44; 95% CI, 1.28 to 32.55; P = .018). The expression of p-Akt increases dramatically with melanoma invasion and progression and is inversely correlated with patient survival. In addition, p-Akt may serve as an independent prognostic marker and help to identify those patients with low-risk melanomas who are at increased risk of death.

Simultaneous Activation of Multiple Signal Transduction Pathways Confers Poor Prognosis in Acute Myelogenous Leukemia.

The disregulation of signal transduction pathways (STPs) provides AML cells with proliferative and survival advantages in vivo. Redundancy and/or “crosstalk” within STPs could trigger the activation of multiple downstream effectors. In retrospective studies we have demonstrated that high levels of PKCα and phosphorylated ERK2 (pERK2) in AML blasts were adverse prognostic indicators. Therefore, we conducted a prospective study to determine if the activation of three STPs (i.e., PKCα, ERK2, and AKT) was associated with the clinical response of 188 newly diagnosed AML patients. The STP protein expression levels in patient samples were evaluated by Western blot analysis. Patients whose AML cells expressed high levels of PKCa had a lower incidence of complete remission (CR, 31% vs. 58%, p=0.01), tended to have higher relapse rates (60% vs. 44%), and experienced a shorter duration of CR (29 vs. 38 weeks) resulting in a lower overall median survival (12 vs. 41 weeks, P= 0.00003). Higher expression of ERK2 did not affect the CR (53% vs. 53%) or relapse rates (48% vs. 44%) of AML, but was associated with a trend for shorter CR durations (32 vs. 44 weeks) combining to result in inferior overall median survival (44 vs. 59 weeks, p= 0.02). pERK2 was undetectable in 27% of the patient samples, and was weakly or strongly expressed in 38.5% or 34% respectively. Strong expression of pERK2 was associated with trends for lower CR rates (45% vs. 57%), higher relapse rates (57% vs. 42%), somewhat shorter CR durations (30 vs. 38 weeks), and statistically significantly shorter overall median survival (32 vs. 44 weeks, p= 0.04). pAKT was undetectable, weakly, moderately, or strongly expressed in 9%, 27%, 38%, or 32% of the samples, respectively. Patients with low levels of pAKT had better CR rates (59% vs. 45%), and a statistically significant longer median survival (not reached vs. 26 weeks, p= 0.02). There were no significant differences in gender, Zubrod PS, or cytogenetics between the favorable and adverse groups for any of these proteins. We hypothesized that activation of multiple pathways would be a more adverse finding. We observed adverse expression levels of 0, 1 or 2+ STP proteins in 24%, 45% and 31% of the cases, respectively. Isolated high PKCa expression did not occur. Isolated strong pERK2 or strong pAKT expression occurred in 8% or 35% of the patient samples. Only 6% of the patients had high levels of all 3 proteins. Survival was inversely correlated with the number of proteins with adverse expression with long-term survival approximating 64%, 46%, and 13% for those with none, one or two or more proteins with adverse expression. This study demonstrates that the downstream activation of STP effectors is common in AML, and that multiple effector activation is common, occurring in approximately 33% of the cases. Agents targeting individual STPs are undergoing clinical evaluation. Our data suggest that, to improve the efficacy of therapy for AML, it may be crucial to match the agent to the STP(s) activated in a given patient, and that combined blockade may be required when multiple STPs are activated. [Display omitted]