Activation of the phosphatidylinositol 3'-kinase/AKT pathway in neuroblastoma and its regulation by thioredoxin 1

Neuroblastoma is a malignant pediatric tumor with poor survival. The phosphatidylinositol 3'-kinase/AKT pathway is a crucial regulator of cellular processes including apoptosis. Thioredoxin 1, an inhibitor of tumor-suppressor phosphatase and tensin homolog, is overexpressed in many tumors. The objective of this study was to explore phosphatidylinositol 3'-kinase/AKT pathway activation and regulation by thioredoxin 1 to identify potential therapeutic targets. Immunohistochemical analysis was done on tissue microarrays from tumor samples of 101 patients, using antibodies against phosphatidylinositol 3'-kinase, AKT, activated AKT, phosphatase and tensin homolog, phosphorylated phosphatase and tensin homolog, thioredoxin 1, epidermal growth factor receptor, vascular endothelial growth factor and receptors (vascular endothelial growth factor 1 and vascular endothelial growth receptor 2), platelet-derived growth factor receptors, insulin-like growth factor 1 receptor, neurotrophic tyrosine kinase receptor type 2, phosphorylated 70-kd S6 protein kinase, 4E-binding protein 1, and phosphorylated mammalian target of rapamycin. Using 3 neuroblastoma cell lines, we investigated cell viability with AKT-specific inhibitors (LY294002, RAD001) and thioredoxin 1 alone or in combination. We found activated AKT and AKT expressed in 97% and 98%, respectively, of neuroblastomas, despite a high expression of phosphatase and tensin homolog correlated with thioredoxin 1. AKT expression was greater in metastatic than primary tumors. Insulin-like growth factor 1 receptor, tyrosine kinase receptor type 2, vascular endothelial growth receptor 1, and downstream phosphorylated 70-kd S6 protein kinase were correlated with activated AKT. LY294002 and RAD001 significantly reduced AKT activity and cell viability and induced a G(1) cell cycle arrest. Thioredoxin 1 decreased cytotoxicity of AKT inhibitors and doxorubicin, up-regulated AKT activation, and induced cell growth. Thus, vascular endothelial growth receptor 1, tyrosine kinase receptor type 2, insulin-like growth factor 1 receptor, and thioredoxin 1 emerged as preferentially committed to phosphatidylinositol 3'-kinase/AKT pathway activation as observed in neuroblastoma. Thioredoxin 1 is a potential target for therapeutic intervention.     

Tumor-associated macrophage (TAM)-derived CCL22 induces FAK addiction in esophageal squamous cell carcinoma (ESCC)

Tumor cell dependence on activated oncogenes is considered a therapeutic target, but protumorigenic microenvironment-mediated cellular addiction to specific oncogenic signaling molecules remains to be further defined. Here, we showed that tumor-associated macrophages (TAMs) produced an abundance of C-C motif chemokine 22 (CCL22), whose expression in the tumor stroma was positively associated with the level of intratumoral phospho-focal adhesion kinase (pFAK Tyr³⁹⁷), tumor metastasis and reduced patient survival. Functionally, CCL22-stimulated hyperactivation of FAK was correlated with increased malignant progression of cancer cells. CCL22-induced addiction to FAK was demonstrated by the persistent suppression of tumor progression upon FAK-specific inhibition. Mechanistically, we identified that diacylglycerol kinase α (DGKα) acted as a signaling adaptor to link the CCL22 receptor C-C motif chemokine receptor 4 (CCR4) and FAK and promoted CCL22-induced activation of the FAK/AKT pathway. CCL22/CCR4 signaling activated the intracellular Ca²⁺/phospholipase C-γ1 (PLC-γ1) axis to stimulate the phosphorylation of DGKα at a tyrosine residue (Tyr³³⁵) and promoted the translocation of DGKα to the plasma membrane to assemble the DGKα/FAK signalosome, which critically contributed to regulating sensitivity to FAK inhibitors in cancer cells. The identification of TAM-driven intratumoral FAK addiction provides opportunities for utilizing the tumor-promoting microenvironment to achieve striking anticancer effects.     

MicroRNA‐137‐mediated Src oncogenic signaling promotes cancer progression

The tyrosine kinase c‐Src is frequently overexpressed and activated in a wide variety of human cancers. However, the molecular mechanisms responsible for the upregulation of c‐Src remain elusive. To examine whether microRNA‐mediated c‐Src upregulation promotes cancer progression, we screened miRNAs with complementarity to the 3′‐UTR of c‐Src mRNA. Among these miRNAs, down‐regulation of miR‐137 was tightly associated with c‐Src‐mediated tumor progression of human colon cancer cells/tissues. Re‐expression of miR‐137 in human colon cancer cells suppressed tumor growth and caused the disruption of focal contacts, suppression of cell adhesion, and invasion, although restoration of c‐Src in miR‐137‐treated cells could not fully rescue the tumor‐suppressive effect of miR‐137. We found that miR‐137 targets AKT2 and paxillin also and miR‐137‐mediated regulation of c‐Src /AKT2 is crucial for controlling tumor growth, whereas that of c‐Src/paxillin contributes to malignancy. miR‐137 suppressed Src‐related oncogenic signaling and changed the expression of miRNAs that are regulated by Src activation. miR‐137 controls the expression of c‐Src/AKT2/paxillin and synergistically suppresses Src oncogenic signaling evoked from focal adhesions. In various human cancers that harbor c‐Src upregulation, the dysfunction of this novel mechanism would serve as a critical trigger for tumor progression.     

Solid Stress Facilitates Fibroblasts Activation to Promote Pancreatic Cancer Cell Migration

Pancreatic fibroblasts are continuously gaining ground as an important component of tumor microenvironment that dynamically interact with cancer cells to promote tumor progression. In addition, these tumor-infiltrated fibroblasts can acquire an activated phenotype and produce excessive amounts of extracellular matrix creating a highly dense stroma, a situation known as desmoplasia. Desmoplasia, along with the uncontrolled proliferation of cancer cells, leads to the development of compressive forces within the tumor, generating the so-called solid stress. Solid stress is previously shown to affect cancer cell proliferation and migration, however there is no pertinent study taking into account the effects of solid stress on fibroblasts and whether these effects contribute to tumor progression. In this work, we applied a defined compressive stress on pancreatic fibroblasts, similar in magnitude to that experienced by cells in native pancreatic tumors. Our results suggest that solid stress stimulates fibroblasts activation and strongly upregulates Growth Differentiation Factor-15 (GDF15) expression. Moreover, co-culture of compression-induced activated fibroblasts with pancreatic cancer cells significantly promotes cancer cell migration, which is inhibited by shRNA-mediated silencing of GDF15 in fibroblasts. Conclusively, our findings highlight the involvement of biophysical factors, such as solid stress, in tumor progression and malignancy revealing a novel role for GDF15.     

PI3K/AKT inhibition induces caspase-dependent apoptosis in HTLV-1-transformed cells

The phosphatidylinositol-3-kinase (PI3K) and AKT (protein kinase B) signaling pathways play an important role in regulating cell cycle progression and cell survival. In previous studies, we demonstrated that AKT is activated in HTLV-1-transformed cells and that Tax activation of AKT is linked to p53 inhibition and cell survival. In the present study, we extend these observations to identify regulatory pathways affected by AKT in HTLV-1-transformed cells. We demonstrate that inhibition of AKT reduces the level of phosphorylated Bad, an important member of the pro-apoptotic family of proteins. Consistent with the decrease of phosphorylated Bad, cytochrome c is released from the mitochondria and caspase-9 is activated. Pretreatment of the cells with caspase-9 specific inhibitor z-LEHD-FMK or pan caspase inhibitor Ac-DEVD-CHO prevented LY294002-induced apoptosis. Of interest, p53 siRNA prevents LY294002-induced apoptosis in HTLV-1-transformed cells, suggesting that p53 reactivation is linked to apoptosis. In conclusion, the AKT pathway is involved in targeting multiple proteins which regulate caspase- and p53-dependent apoptosis in HTLV-1-transformed cells. Since AKT inhibitors simultaneously inhibit NF-kappaB and activate p53, these drugs should be promising candidates for HTLV-1-associated cancer therapy.     

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

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

Macrophage infiltration promotes invasiveness of breast cancer cells via activating long non-coding RNA UCA1

There is now considerable evidence supporting the view that macrophage infiltration is playing a critical role in the proliferation and progression of breast cancer but the underlying molecular mechanisms remain largely unknown. To this end, using long non-coding RNA (lncRNA) expression profiling, we examined changes in lncRNA expression in breast cancer cells treated with conditioned medium (CM) from cultured human THP-1 macrophages. We found that treatment with macrophage CM induced the expression of numerous lncRNAs, including urothelial cancer associated 1 (UCA1). Knockdown of UCA1 using shRNA inhibited AKT phosphorylation and abolished invasiveness of tumor cells induced by macrophage CM. Consistent with these results; we further showed that UCA1 level was significantly enhanced in human primary breast tumors and correlated with advanced clinical stage, supporting its role in promoting carcinogenesis and progression of breast cancer. Together, these results suggest that macrophage could promote invasiveness of breast cancer cells by enhancing expression of lncRNA UCA1.     

Stat3在多种恶性肿瘤中的作用

Stat3是转录信号转导子与激活子家族（Stats）的重要成员,广泛表达于不同类型的细胞和组织中,受大量细胞因子、生长因子和癌基因调节,其生物学功能广泛,在调节细胞增殖、存活和分化方面起着关键作用。持续激活的Stat3（p-Stat3）在许多恶性肿瘤中已经发现,包括人类白血病、头颈部鳞状细胞癌、乳腺癌、前列腺癌、肺癌、胰腺癌和黑色素瘤等,p-Stat3与恶性肿瘤的发生、发展及转移密切相关。因此,Stat3已成为近年来肿瘤治疗研究探讨的新靶点,以Stat3为靶点的分子治疗有可能为肿瘤治疗翻开新的一页。     

Two autopsy cases of desmoplastic small round cell tumor

Desmoplastic small round cell tumor (DSRCT) is a rare aggressive malignant tumor. It is a refractory tumor and the median overall survival is very short. We report two autopsy cases of DSRCT, both of which were already advanced and metastasized at the first medical examination. Both cases showed typical DSRCT findings in terms of localization of the lesions, histopathology and genetics, but the rate of disease progression was quite different. Survival after initial symptoms in Case 1 was only 12 months. On the other hand, survival after primary hospitalization in Case 2 was 42 months. The Case 2 patient initially received chemotherapy for advanced pancreatic carcinoma, because a nodule of the pancreatic tail was found on computed tomography (CT) scan. After chemotherapy, tumor regression was observed on CT scan. It is thus implied that adoption of the regimen for pancreatic carcinoma might have been one of reasons of the long survival in Case 2.     

Tissue microarray analysis reveals strong clinical evidence for a close association between loss of annexin A1 expression and nodal metastasis in gastric cancer

Aims Annexin A1 (ANXA1) is a calcium- and phospholipid-binding protein that has been implicated in the regulation of inflammation, cell proliferation, and apoptosis. Its role in tumor development and progression is controversial, whereas its role in gastric cancer is unknown. We investigated ANXA1 expression and determined its clinical significance in gastric cancer. Methods and results Tissue microarray blocks containing primary gastric cancer, lymph node metastasis, and adjacent normal mucosa specimens obtained from 1,072 Chinese patients were constructed. Expression of ANXA1 in these specimens was analyzed using immunohistochemistry. Complete loss of ANXA1 expression was observed in 691 (64%) of the 1,072 primary tumors and 146 (86%) of 169 nodal metastases. Loss of ANXA1 expression was significantly associated with advanced T stage, lymph node metastasis, advanced disease stage, and poor histological differentiation. Loss of ANXA1 expression correlated significantly with poor survival rates in both univariate and multivariate analyses. Conclusions ANXA1 expression decreased significantly as gastric cancer progressed and metastasized, suggesting the importance of ANXA1 as a negative biomarker for gastric cancer development and progression.     

The ADAMTS1 protease gene is required for mammary tumor growth and metastasis

A disintegrin and metalloprotease with thrombospondin motifs protein 1 (ADAMTS1) is a protease commonly up-regulated in metastatic carcinoma. Its overexpression in cancer cells promotes experimental metastasis, but whether ADAMTS1 is essential for metastatic progression is unknown. To address this question, we investigated mammary cancer progression and spontaneous metastasis in the MMTV-PyMT mouse mammary tumor model in Adamts1 knockout mice. Adamts1(-/-)/PyMT mice displayed significantly reduced mammary tumor and lung metastatic tumor burden and increased survival, compared with their wild-type and heterozygous littermates. Histological examination revealed an increased proportion of tumors with ductal carcinoma in situ and a lower proportion of high-grade invasive tumors in Adamts1(-/-)/PyMT mice, compared with Adamts1(+/+)/PyMT mice. Increased apoptosis with unaltered proliferation and vascular density in the Adamts1(-/-)/PyMT tumors suggested that reduced cell survival accounts for the lower tumor burden in ADAMTS1-deficient mice. Furthermore, Adamts1(-/-) tumor stroma had significantly lesser amounts of proteolytically cleaved versican and increased numbers of CD45(+) leukocytes. Characterization of immune cell gene expression indicated that cytotoxic cell activation was increased in Adamts1(-/-) tumors, compared with Adamts1(+/+) tumors. This finding is supported by significantly elevated IL-12(+) cell numbers in Adamts1(-/-) tumors. Thus, in vivo ADAMTS1 may promote mammary tumor growth and progression to metastasis in the PyMT model and is a potential therapeutic target to prevent metastatic breast cancer.     

PRMT4 promotes hepatocellular carcinoma progression by activating AKT/mTOR signaling and indicates poor prognosis

Background: Protein arginine methyltransferase 4 (PRMT4) has been reported to play a role in several common cancers; however, the function and mechanism of PRMT4 in hepatocellular carcinoma (HCC) are not fully understood. This study aimed to investigate the role and mechanism of PRMT4 in the progression of HCC.Methods: PRMT4 expression and clinicopathological characteristics were investigated using an HCC tissue microarray (TMA) consisting of 140 patient samples analyzed by immunohistochemistry. CCK-8, crystal violet and Transwell assays were used to determine cell proliferation, colony formation, migration, and invasion of HCC cell lines in which PRMT4 was overexpressed or downregulated. The underlying mechanism of PRMT4 function was explored by Western blot assays.Results: PRMT4 was highly expressed in HCC tumor tissues compared to adjacent nontumor tissues. PRMT4 expression was significantly associated with alpha-fetoprotein levels, tumor size, satellite nodules, and microvascular invasion. Patients with higher PRMT4 expression had a shorter survival time and higher recurrence rate. Functional studies demonstrated that PRMT4 overexpression promoted HCC cell proliferation, migration, and invasion in vitro, while knocking down PRMT4 inhibited these malignant behaviors. Additional results revealed that PRMT4 promoted the progression of HCC cells via activation of the AKT/mTOR signaling pathway. Furthermore, inhibition of the AKT/mTOR signaling by MK2206 or rapamycin significantly attenuated PRMT4-mediated malignant phenotypes.Conclusions: This study suggests that PRMT4 may promote the progression of HCC cells by activating the AKT/mTOR signaling pathway, which may be a valuable biomarker and potential target for HCC.     

The Akt pathway in human breast cancer: a tissue-array-based analysis.

The Akt pathway, an important regulator of cell proliferation and survival, is deregulated in many cancers. The pathway has achieved considerable importance due to the development of kinase inhibitors that are able to successfully reduce tumor growth. This study was conducted to determine the status of the Akt pathway in human breast cancers and to study the relationship between the different component proteins. Expression levels of PTEN, phosphorylated forms of the constituent proteins (Akt, FKHR, mTOR, and S6) and cyclin D1 were evaluated by immunohistochemistry, on consecutive sections from a tissue microarray containing 145 invasive breast cancers and 140 pure ductal carcinomas in-situ. Aberrant expression was correlated statistically with tumor characteristics and disease outcome. The Akt pathway was found to be activated early in breast cancer, in the in-situ stage. In all, 33, 15, 32, and 60% of ductal carcinoma in-situ showed overexpression of Akt, FKHR, mTOR, and cyclin D1. PTEN loss did not correlate statistically with expression of AKT or any of the other proteins with the exception of S6, indicating that Akt activation was not a result of PTEN loss. Expression levels of PTEN and S6 were significantly different in in-situ and invasive cancers, indicating association with disease progression. Loss of PTEN was noted in 11% of in-situ as compared to 26% of invasive cancers, while S6 overexpression was seen in 47% in-situ and in 72% invasive cancers. High-grade carcinomas were associated with PTEN loss, while low-grade carcinomas with good prognostic features showed cyclin D1 overexpression and were associated with longer disease free survival. Additionally, cancers with mTOR overexpression showed a three times greater risk for disease recurrence. Overall, a large proportion of in-situ and invasive breast cancers overexpressed cyclinD1 and S6. Our results may have significant implications in the development and application of targeted therapy.     

Role of tumor associated macrophages in regulating pancreatic cancer progression

Pancreatic cancer has an overall 5-year survival rate of less than 5%. Unfortunately, patient survival has not substantially improved in the last couple of decades despite advances in treatment modalities that have been successful in other cancer types. The poor response of pancreatic cancer to therapy is a major obstacle faced by clinicians. Increasing attention is being paid to how tumor cells and non-tumor cells influence each other in the pancreatic tumor microenvironment. Tumor-associated macrophages (TAMs) are a highlight in this field because of their vast presence in the tumor microenvironment. TAMs promote angiogenesis, metastasis, and suppress the anti-tumor immune response. Here we review the current understanding of the role of TAMs in regulating the progression of pancreatic cancer.     

Luteolin Reduces Aqueous Extract PM2.5-induced Metastatic Activity in H460 Lung Cancer Cells

Fine particulate matter (PM2.5) is the critical cause of lung cancer and can further promote tumor cell migration and invasion. This study investigated the effects of luteolin, an antiangiogenic flavonoid agent, on blocking aqueous extract PM2.5-prompted cancer progression. We observed that luteolin reduced cell migration and the expression of pro-metastatic factors pro-matrix metalloproteinase (MMP)-2 and intercellular adhesion molecule (ICAM)-1 in PM2.5-exposed H460 lung cancer cells. Luteolin treatment also reduced the transduction of PM2.5-induced epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) cascade signaling. Furthermore, the reduction of MMP-2 expression and ICAM-1 production by luteolin in PM2.5-stimulated H460 cells is EGFR-PI3K-AKT pathway dependent. These results suggest that luteolin exhibits antitumor progression by inhibiting EGFR-PI3K-AKT pathway.     

Deciphering the role of hedgehog signaling in pancreatic cancer

Pancreatic cancer, mostly pancreatic ductal adenocarcinoma (PDAC), is a leading cause of cancer-related death in the US, with a dismal median survival of 6 months. Thus, there is an urgent unmet need to identify ways to diagnose and to treat this deadly cancer. Although a number of genetic changes have been identified in pancreatic cancer, their mechanisms of action in tumor development, progression and metastasis are not completely understood. Hedgehog signaling, which plays a major role in embryonic development and stem cell regulation, is known to be activated in pancreatic cancer; however, specific inhibitors targeting the smoothened molecule failed to improve the condition of pancreatic cancer patients in clinical trials. Furthermore, results regarding the role of Hh signaling in pancreatic cancer are controversial with some reporting tumor promoting activities whereas others tumor suppressive actions. In this review, we will summarize what we know about hedgehog signaling in pancreatic cancer, and try to explain the contradicting roles of hedgehog signaling as well as the reason(s) behind the failed clinical trials. In addition to the canonical hedgehog signaling, we will also discuss several non-canonical hedgehog signaling mechanisms.     

eEF1A2 promotes cell migration, invasion and metastasis in pancreatic cancer by upregulating MMP-9 expression through Akt activation

eEF1A2 is a protein translation factor involved in protein synthesis that is overexpressed in various cancers, with important functions in tumor genesis and progression. We have previously showed that the ectopic expression of eEF1A2 is correlated with lymph node metastasis and perineural invasion in pancreatic cancer. In this study, we investigated the functional role of eEF1A2 in the regulation of cell migration, invasion, and metastasis in pancreatic cancer. Furthermore, we investigated the potential molecular mechanisms involved. By evaluating the invasive ability of a panel of pancreatic cancer cell lines with different metastatic potentials, eEF1A2 expression in cells was positively associated with their invasive ability. The knockdown of eEF1A2 by siRNA decreased the migration and invasion of PANC-1 cells. By contrast, the ectopic expression of exogenous eEF1A2 significantly promoted the migration and invasion of SW1990 cells. Stable eEF1A2 overexpression in a nude mouse model of peritoneal metastasis likewise dramatically enhanced the intraperitoneal metastatic ability of SW1990 cells. In addition, eEF1A2 overexpression could upregulate MMP-9 expression and activity. A significant positive correlation between the overexpression of both eEF1A2 and MMP-9 was observed in pancreatic cancer tissues. The inhibition of MMP-9 activity reduced the promoting effect of eEF1A2 on cell migration and invasion. Furthermore, eEF1A2-mediated cell migration and invasion, as well as MMP-9 expression and upregulation, were largely dependent on the eEF1A2-induced Akt activation. The findings suggested the potentially important role of eEF1A2 in pancreatic cancer migration, invasion, and metastasis. Thus, the results provide evidence of eEF1A2 as a potential therapeutic target in the treatment of aggressive pancreatic cancer.     

NSCLC and the alternative pathway of NF-κB: uncovering an unknown relation

Lung cancer is the leading cause of cancer-related deaths worldwide. Although our knowledge on the pathobiology of the disease has increased in the last decades, the prognosis of lung cancer patients has hardly changed. Many signaling pathways are implicated in lung carcinogenesis, but the role of the alternative pathway of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in lung cancer pathogenesis and progression has not been investigated. The aim of our study was to investigate the role of this pathway in non-small cell lung cancer (NSCLC) patients. NF-κB2 and RelB protein expression was retrospectively assessed by immunohistochemistry in tissue samples from 109 NSCLC patients. RelB and NF-κB2 protein levels differed between tumors and adjacent nonneoplastic lung parenchyma. Cytoplasmic immunoreactivity of NF-κB2 and RelB was correlated with tumor stage (p = 0.03 and p = 0.016, respectively). In addition, cytoplasmic NF-κB2 levels were related to tumor grade (p = 0.046). Expression of RelB in the cytoplasm was tumor histologic type-specific, with squamous cell carcinomas having the highest protein levels. Nuclear expression of RelB and NF-κB2 differed between tumor and nonneoplastic tissues, possibly indicating activation of the alternative pathway of NF-κB in cancer cells. Moreover, lymph node metastasis was related to nuclear NF-κB2 expression in tumor cells. The deregulation of the alternative NF-κB pathway in NSCLC could play a role in the development and progression of the disease.