Glioblastoma stem cells generate vascular pericytes to support vessel function and tumor growth.

Glioblastomas (GBMs) are highly vascular and lethal brain tumors that display cellular hierarchies containing self-renewing tumorigenic glioma stem cells (GSCs). Because GSCs often reside in perivascular niches and may undergo mesenchymal differentiation, we interrogated GSC potential to generate vascular pericytes. Here, we show that GSCs give rise to pericytes to support vessel function and tumor growth. In vivo cell lineage tracing with constitutive and lineage-specific fluorescent reporters demonstrated that GSCs generate the majority of vascular pericytes. Selective elimination of GSC-derived pericytes disrupts the neovasculature and potently inhibits tumor growth. Analysis of human GBM specimens showed that most pericytes are derived from neoplastic cells. GSCs are recruited toward endothelial cells via the SDF-1/CXCR4 axis and are induced to become pericytes predominantly by transforming growth factor ß. Thus, GSCs contribute to vascular pericytes that may actively remodel perivascular niches. Therapeutic targeting of GSC-derived pericytes may effectively block tumor progression and improve antiangiogenic therapy.

Periostin promotes immunosuppressive premetastatic niche formation to facilitate breast tumour metastasis.

Periostin (POSTN) is a limiting factor in the metastatic colonization of disseminated tumour cells. However, the role of POSTN in regulating the immunosuppressive function of immature myeloid cells in tumour metastasis has not been documented. Here, we demonstrate that POSTN promotes the pulmonary accumulation of myeloid-derived suppressor cells (MDSCs) during the early stage of breast tumour metastasis. Postn deletion decreases neutrophil and monocytic cell populations in the bone marrow of mice and suppresses the accumulation of MDSCs to premetastatic sites. We also found that POSTN-deficient MDSCs display reduced activation of ERK, AKT and STAT3 and that POSTN deficiency decreases the immunosuppressive functions of MDSCs during tumour progression. Moreover, the pro-metastatic role of POSTN is largely limited to ER-negative breast cancer patients. Lysyl oxidase contributes to POSTN-promoted premetastatic niche formation and tumour metastasis. Our findings indicate that POSTN is essential for immunosuppressive premetastatic niche formation in the lungs during breast tumour metastasis and is a potential target for the prevention and treatment of breast tumour metastasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

iTRAQ based quantitative proteomics approach validated the role of calcyclin binding protein (CacyBP) in promoting colorectal cancer metastasis.

Keeping continuity with our previous study that revealed direct correlations between CRC metastasis and enhanced CacyBP protein levels, here we attempt to improve our understanding of the mechanisms involved within this enigmatic process. Overexpression of CacyBP (CacyBP-OE) in primary CRC cell and its knock down (CacyBP-KD) in the metastatic CRC cells revealed (through phenotypic studies) the positive impact of the protein on metastasis. Additionally, two individual 4-plex iTRAQ based comparative proteomics experiments were carried out on the CacyBP-OE and CacyBP-KD cells, each with two biological replicates. Mining of proteomics data identified total 279 (63.80% up-regulated and 36.20% down-regulated) proteins to be significantly altered in expression level for the OE set and in the KD set, this number was 328 (48.78% up-regulated and 51.22% down-regulated). Functional implications of these significantly regulated proteins were related to metastatic phenotypes such as cell migration, invasion, adhesion and proliferation. Gene ontology analysis identified integrin signaling as the topmost network regulated within CacyBP-OE. Further detection of caveolar mediated endocytosis in the top hit list correlated this phenomenon with the dissociation of integrins from the focal adhesion complex which are known to provide the traction force for cell movement when transported back to the leading edge. This finding was further supported by the data obtained from CacyBP-KD data set showing down-regulation of proteins necessary for integrin endocytosis. Furthermore, intracellular calcium levels (known to influence integrin mediated cell migration) were found to be lowered in CacyBP-KD cells indicating decreased cell motility and vice versa for the CacyBP-OE cells. Actin nucleation by ARP-WASP complex, known to promote cell migration, was also identified as one of the top regulated pathways in CacyBP-OE cells. In short, this study presents CacyBP as a promising candidate biomarker for CRC metastasis and also sheds light on the underlying molecular mechanism by which CacyBP promotes CRC metastasis.

Twist2 promotes self-renewal of liver cancer stem-like cells by regulating CD24.

Twist2 is a highly conserved basic helix-loop-helix transcription factor that plays a critical role in embryogenesis. Recent evidence has revealed that aberrant Twist2 expression contributes to tumor progression; however, the role of Twist2 in human hepatocellular carcinoma (HCC) and its underlying mechanisms remain undefined. In this report, we demonstrate that Twist2 is overexpressed in human HCC tumors. We show that ectopic expression of Twist2 induces epithelial-mesenchymal transition phenotypes, augments cell migration and invasion and colony-forming abilities in human HCC cells in vitro, and promotes tumor growth in vivo. Moreover, we found a higher percentage of CD24(+) liver cancer stem-like cells in Twist2-transduced HCC cells. Twist2-expressing cells exhibited an increased expression of stem cell markers Bmi-1, Sox2, CD24 and Nanog and an increased capacity for self-renewal. Knockdown of CD24 in HepG2/Twist2 cells decreased the levels of Sox2, pSTAT3 and Nanog, and reversed the cancer stem-like cell phenotypes induced by ectopic expression of Twist2. Furthermore, Twist2 regulated the CD24 expression by directly binding to the E-box region in CD24 promoter. Therefore, our data demonstrated that Twist2 augments liver cancer stem-like cell self-renewal in a CD24-dependent manner. Twist2-CD24-STAT3-Nanog pathway may play a critical role in regulating liver cancer stem-like cell self-renewal. The identification of the Twist2-CD24 signaling pathway provides a potential therapeutic approach to target cancer stem cells in HCCs.

D-peptide inhibitors of the p53-MDM2 interaction for targeted molecular therapy of malignant neoplasms.

The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53, conferring tumor development and survival. Antagonists targeting the p53-binding domains of MDM2 and MDMX kill tumor cells both in vitro and in vivo by reactivating the p53 pathway, promising a class of antitumor agents for cancer therapy. Aided by native chemical ligation and mirror image phage display, we recently identified a D-peptide inhibitor of the p53-MDM2 interaction termed (D)PMI-alpha (TNWYANLEKLLR) that competes with p53 for MDM2 binding at an affinity of 219 nM. Increased selection stringency resulted in a distinct D-peptide inhibitor termed (D)PMI-gamma (DWWPLAFEALLR) that binds MDM2 at an affinity of 53 nM. Structural studies coupled with mutational analysis verified the mode of action of these D-peptides as MDM2-dependent p53 activators. Despite being resistant to proteolysis, both (D)PMI-alpha and (D)PMI-gamma failed to actively traverse the cell membrane and, when conjugated to a cationic cell-penetrating peptide, were indiscriminately cytotoxic independently of p53 status. When encapsulated in liposomes decorated with an integrin-targeting cyclic-RGD peptide, however, (D)PMI-alpha exerted potent p53-dependent growth inhibitory activity against human glioblastoma in cell cultures and nude mouse xenograft models. Our findings validate D-peptide antagonists of MDM2 as a class of p53 activators for targeted molecular therapy of malignant neoplasms harboring WT p53 and elevated levels of MDM2.

Tear dynamics testing and quantitative proteomics analysis in patients with chronic renal failure.

Ocular surface changes may develop in patients with chronic renal failure (CRF) undergoing hemodialysis. In recent years, an association of CRF with dry eye syndrome has been emphasized. However, tear proteomics of CRF patients has not been analyzed. Here, we performed systematic profiling of the tear film proteins in CRF patients through use of isobaric tags for relative and absolute quantitative (iTRAQ) MS/MS, aiming to identify associations between dry eye symptoms and expression of tear proteomic changes in patients with CRF undergoing hemodialysis. Twenty CRF patients and ten healthy subjects underwent a series of ophthalmic examinations. Tear samples from the participants were analyzed by iTRAQ approach. A total of 1139 tear proteins were screened, and 212 differentially expressed proteins were identified. The pattern changes included 77 whose expression levels were upregulated (fold increase >1.2) whereas 135 others that were downregulated (fold decrease <1/1.2). Bioinformatics analysis showed that these proteins were significantly enriched in lipid metabolism, inflammatory, and immune response pathways. Furthermore, APOA1, APOA4, APOB, APOE, S100A8, S100A9, S100A4, HSP90B and other molecules were significantly changed. Our study elucidated the characteristics of tear dynamics and protein markers in CRF patients undergoing hemodialysis. Significance: Despite the association of chronic renal failure (CRF) with dry eye disease, there are no reports describing potentially important differentially expressed tear proteins in CRF patients undergoing hemodialysis. It is still a challenge to obtain a comprehensive description of the pathogenesis of dry eye in CRF patients which hinders establishing a patient specific therapeutic scheme. Our study is the first iTRAQ proteomics analysis of the tears of patients with CRF, which reveals the changes in the protein expression profile in CRF patients afflicted with dry eye disease. The identity was verified of some relevant differentially expressed proteins, and they may be candidate diagnostic markers of dry eye disease in patients with CRF. These tear film protein constituents found in hemodialysis patients can be of important clinical significance in treating this condition. SIGNIFICANCE: Despite the association of chronic renal failure (CRF) with dry eye disease, there are no reports describing potentially important differentially expressed tear proteins in CRF patients undergoing hemodialysis. It is still a challenge to obtain a comprehensive description of the pathogenesis of dry eye in CRF patients which hinders establishing a patient specific therapeutic scheme. Our study is the first iTRAQ proteomics analysis of the tears of patients with CRF, which reveals the changes in the protein expression profile in CRF patients afflicted with dry eye disease. The identity was verified of some relevant differentially expressed proteins, and they may be candidate diagnostic markers of dry eye disease in patients with CRF. These tear film protein constituents found in hemodialysis patients can be of important clinical significance in treating this condition.

Osteopontin promotes gastric cancer metastasis by augmenting cell survival and invasion through Akt-mediated HIF-1alpha up-regulation and MMP9 activation.

Osteopontin (OPN) is a secreted, integrin-binding matrix phosphorylated glycoprotein. OPN has been shown to facilitate the progression and metastasis of malignancies and has prognostic value in several types of cancer, including gastric cancer. However, the functional mechanism of OPN mediated metastatic growth in gastric cancer remains unclear. Here, using multiple in vitro and in vivo models, we report that OPN strongly promoted the progression and metastasis of gastric cancer. Immunohistochemical staining revealed that OPN, matrix metalloproteinase (MMP)9 and hypoxia-inducible factor (HIF)-1alpha have statistically significant different expression patterns between well- and poorly differentiated tissue samples (P < 0.05). Correlations existed between OPN and MMP9, and between OPN and HIF-1 (r(1) = 0.872, p(1) < 0.01 and r(2) = 0.878, p(2) < 0.01). Furthermore, OPN dramatically increased colony formation and invasion of gastric cancer cells in vitro and promoted tumour growth and metastasis in vivo. In addition, OPN potently protected gastric cancer cells from serum depletion-induced apoptosis. Further study shows that OPN activated phosphoinositide 3-kinase/Akt survival pathway and up-regulated HIF-1alpha via binding to v3 integrins in gastric cancer cells. Moreover, we found that OPN could activate MMP9 and upregulate MMP2. Taken together, our results suggest that the survival-promoting function is crucial for OPN to promote the development of gastric cancer, and HIF-1 and MMP9 may play key roles during this process. Thus, targeting OPN and its related signalling network may develop an effective therapeutic approach for the management of gastric cancer.

Genistein induces G2/M cell cycle arrest and apoptosis of human ovarian cancer cells via activation of DNA damage checkpoint pathways.

Genistein is a major isoflavonoid in dietary soybean, commonly consumed in Asia. Genistein exerts inhibitory effects on the proliferation of various cancer cells and plays an important role in cancer prevention. However, the molecular and cellular mechanisms of genistein on human ovarian cancer cells are still little known. We show that exposure of human ovarian cancer HO-8910 cells to genistein induces DNA damage, and triggers G2/M phase arrest and apoptosis. Furthermore, we also found that checkpoint proteins ATM and ATR are phosphorylated and activated in the cells treated with genistein. It is also shown that genistein increases the phosphorylation and activation of Chk1 and Chk2, which results in the phosphorylation and inactivation of phosphatases Cdc25C and Cdc25A, and thereby the phosphorylation and inactivation of Cdc2 which arrests cells in G2/M phase. Moreover, genistein enhances the phosphorylation and activation of p53, while decreases the ratio of Bcl-2/Bax and Bcl-xL/Bax and the level of phosphorylated Akt, which result in cells undergoing apoptosis. These results demonstrate that genistein-activated ATM-Chk2-Cdc25 and ATR-Chk1-Cdc25 DNA damage checkpoint pathways can arrest ovarian cancer cells in G2/M phase, and induce apoptosis while the cellular DNA damage is too serious to be repaired. Thus, the antiproliferative, DNA damage-inducing and pro-apoptotic activities of genistein are probably responsible for its genotoxic effects on human ovarian cancer HO-8910 cells.

Ubiquitin-specific peptidase 39 regulates the process of proliferation and migration of human ovarian cancer via p53/p21 pathway and EMT.

Ovarian cancer is one of the most lethal gynecological cancers; owning to its late detection and chemoresistance, understanding the pathogenesis of this malignant tumor is much critical. Previous studies have reported that ubiquitin-specific peptidase 39 (USP39) is generally overexpressed in a variety of cancers, including hepatocellular carcinoma, gastric cancer and so forth. Furthermore, USP39 is proved to be associated with the proliferation of malignant tumors. However, the function and mechanism of USP39 in ovarian cancer have not been elucidated. In the present study, we observed that USP39 was frequently overexpressed in human ovarian cancer and was highly correlated with TNM stage. Suppression of USP39 markedly inhibited the growth and migration of ovarian cancer cell lines HO-8910 and SKOV3 and induced cell cycle G2/M arrest. Moreover, knockdown of USP39 inhibited ovarian tumor growth in a xenograft model. In addition, our findings indicated that cell cycle arrest induced by USP39 knockdown might be involved in p53/p21 signaling pathway. Furthermore, we found that the depletion of USP39 inhibited the migration of ovarian cancer cells via blocking epithelial-mesenchymal transition. Taken together, these results suggest that USP39 may play vital roles in the genesis and progression and may serve as a potential biomarker for diagnosis and therapeutic target of ovarian cancer.

Osteopontin promotes ovarian cancer progression and cell survival and increases HIF-1alpha expression through the PI3-K/Akt pathway.

Osteopontin (OPN) is a secreted, integrin-binding matrix phosphorylated glycoprotein that is overexpressed in many advanced cancers. However, the functional mechanisms by which OPN contributes to the development of ovarian cancer are poorly understood. Here, we reveal that acquired expression of OPN by HO-8910 ovarian cancer cells greatly promoted the progression of ovarian cancer. OPN expression dramatically increased the colony formation of ovarian cancer cells in vitro and tumor growth in vivo. Under the stress induced by serum depletion or curcumin treatment, OPN expression promoted the survival of ovarian cells through preventing stress-induced apoptosis. At the molecular level, both endogenous and exogenous OPN expression activated the PI3-K/Akt survival pathway and dramatically decreased p53 expression under serum depletion. In addition, HIF-1alpha was induced in OPN-producing cells under normoxia. Furthermore, we also found that inhibition of the PI3-K/Akt pathway attenuated OPN-mediated HIF-1alpha up-regulation in ovarian cancer cells. Taken together, these results indicate that OPN can increase the survival of ovarian cancer cells under stress conditions in vitro and promote the late progression of ovarian cancer in vivo, and the survival-promoting functions of OPN are mediated through Akt activation and the induction of HIF-1alpha expression.

Osteopontin prevents curcumin-induced apoptosis and promotes survival through Akt activation via alpha v beta 3 integrins in human gastric cancer cells.

Osteopontin (OPN) is a secreted, integrin-binding matrix phosphorylated glycoprotein that is overexpressed in many advanced cancers. However, the functional mechanisms by which OPN contributes to gastric cancer development are poorly understood. Here, we report that curcumin inhibited the growth of SGC7901 cell and induced apoptosis in a concentration- and time-dependent manner, while the acquired expression of OPN in SGC7901 cells dramatically promoted cell survival under serum depletion and prevented curcumin-induced apoptosis. Furthermore, PI3-K inhibitor LY294002 attenuated OPN-mediated Akt activation. Moreover, inhibiting the binding of OPN to alpha(v)beta(3) integrins reduced activation of Akt. Taken together, these results demonstrate that the pro-survival and anti-apoptosis activities of OPN in gastric cancer cells are mediated in part through PI3-K/Akt pathway via alpha(v)beta(3) integrins.

MicroRNA-543 suppresses colorectal cancer growth and metastasis by targeting KRAS, MTA1 and HMGA2.

miR-543 has been implicated as having a critical role in the development of breast cancer, endometrial cancer and hepatocellular carcinoma. However, the exact clinical significance and biological functions of miR-543 in colorectal cancer (CRC) remain unclear. Here, we found that miR-543 expression significantly downregulated in tumors from patients with CRC, APCMin mice and a mouse model of colitis-associated colon cancer. miR-543 level was inversely correlated with the metastatic status of patients with CRC and the metastatic potential of CRC cell lines. Moreover, ectopic expression of miR-543 inhibited the proliferation and metastasis of CRC cells in vitro and in vivo by targeting KRAS, MTA1 and HMGA2. Conversely, miR-543 knockdown promoted the proliferation, migration and invasion of CRC cells in vitro and augmented tumor growth and metastasis in vivo. Furthermore, we found that miR-543 expression was negatively correlated with the levels of KRAS, MTA1 and HMGA2 in clinical samples. Collectively, these data show that miR-543 inhibits the proliferation and metastasis of CRC cells by targeting KRAS, MTA1 and HMGA2. Our study highlights a pivotal role for miR-543 as a suppressor in the regulation of CRC growth and metastasis and suggests that miR-543 may serve as a novel diagnostic and prognostic biomarker for CRC metastasis.

Involvement of Wnt/ß-catenin signaling in the mesenchymal stem cells promote metastatic growth and chemoresistance of cholangiocarcinoma.

Mesenchymal stem cells (MSCs) are multi-potent progenitor cells with ability to differentiate into multiple lineages, including bone, cartilage, fat, and muscles. Recent research indicates that MSCs can be efficiently recruited to tumor sites, modulating tumor growth and metastasis. However, the underlying molecular mechanisms are not fully understood. Here, we first demonstrated that human umbilical cord-derived mesenchymal stem cells (hUC-MSCs), when mixed with human cholangiocarcinoma cell lines QBC939 in a xenograft tumor model, significantly increased the cancer cells proliferation and metastatic potency. MSCs and their conditioned media (MSC-CM) could improve the drug resistance of tumor when the compound K (CK) as an anti-cancer drug, a major intestinal bacterial metabolite of panaxoside, was administered to xenograft tumor mice. Furthermore, MSCs greatly increased the colony formation and invasion of cholangiocarcinoma cells QBC939 and Mz-ChA-1. Immunochemistry studies of cholangiocarcinoma tissue chips and transplantation tumor from nude mice showed that the expression of ß-catenin was important for cholangiocarcinoma development. We further demonstrated that MSCs and MSCs-CM could promote proliferation and migration of cholangiocarcinoma cells through targeting the Wnt/ß-catenin signaling pathway. hUC-MSCs or MSCs-CM stimulated Wnt activity by promoting the nuclear translocation of ß-catenin, and up-regulated Wnt target genes MMPs family, cyclin D1 and c-Myc. Together, our studies highlight a critical role for MSCs on cancer metastasis and indicate MSCs promote metastatic growth and chemoresistance of cholangiocarcinoma cells via activation of Wnt/ß-catenin signaling.

Twist2 promotes ovarian cancer cell survival through activation of Akt.

Hypoxia-inducible factor-1 α (HIF-1α) is an important prognostic factor in ovarian carcinoma. Hypoxia contributes to tumor progression and is involved in the epithelial-mesenchymal transition (EMT). Twist2 is an EMT regulator, however, it remains poorly understood in ovarian carcinoma. The present study evaluated the expression of HIF-1α and Twist2 and further investigated whether Twist2 is involved in hypoxia-induced apoptosis in ovarian cancer. A series of matched paraffin-embedded tissue sections from human primary ovarian cancer and normal ovarian tissues were examined through immunohistochemical analysis, a Twist2-overexpressing stable ovarian cancer cell line was established and deferoxamine (DFO) was introduced to simulate hypoxic conditions. DFO-induced apoptosis was examined by fluorescence microscopy, MTT assays and flow cytometry. In addition, a western blot analysis was performed to examine the molecular mechanism(s) of action. Twist2 increased in epithelial ovarian cancers associated with HIF-1α expression. The acquired expression of Twist2 was able to promote the survival of ovarian cancer cells through Akt phosphorylation under DFO-induced hypoxic stress. The results suggest that Twist2 activates the PI-3K-Akt pathway to protect cells from apoptosis in a hypoxic environment. Moreover, Twist2 may be involved in the HIF-1α signaling pathway in ovarian cancer.

The role of nuclear ß-catenin accumulation in the Twist2-induced ovarian cancer EMT.

BACKGROUND: Twist2 has been shown to promote human tumor invasion as in breast cancer and cervical cancer. However, whether Twist2 promotes human ovarian cancer progression remains to be elucidated. Here, we investigate the role of Twist2 in ovarian cancer invasion and metastasis as well as the underlying molecular mechanisms. METHODS: Twist2 expression was detected by Immunohistochemistry (IHC) on tissue microarray of human ovarian cancers with scoring procedure according to the staining intensity and pattern. Twist2 gene was stably introduced into SKOV-3 ovarian cancer cells to examine the changes of cellular morphology, motility, invasiveness, and EMT molecular markers. RESULTS: Twist2 expression is significantly increased in ovarian cancers along with the FIGO disease stage, indicating that Twist2 may be associated with ovarian cancer metastasis. Overexpression of Twist2 induced the EMT phenotype including downregulation of E-cadherin, and upregulation of N-cadherin and ß-catenin in human ovarian cancer cells, suggesting that Twist2 might promote ß-catenin release from the E-cadherin/ß-catenin complex through inhibition of E-cadherin. Thus, ß-catenin degradation was inhibited due to inhibition of APC, and the Wnt/ß-catenin pathway was then activated by nuclear ß-catenin accumulation, which may activate transcription of downstream target genes to promote tumor invasion and metastasis. Collectively, these data indicated that ß-catenin is involved in Twist2-induced EMT in ovarian cancer. CONCLUSION: Our data indicates that upregulation of Twist2 is correlated with the FIGO stage in human ovarian cancers. In this report, we demonstrated that nuclear ß-catenin is accumulated in Twist2-induced EMT cells to facilitates ovarian cancer invasion and metastasis.

Significance of heterogeneous Twist2 expression in human breast cancers.

BACKGROUND: Twist2 (Dermo1) has been shown to mediate the epithelial-mesenchymal transition (EMT) to promote tumor invasion and even metastasis. However, the involvement of EMT in breast cancer progression is highly debated, partially due to clinical observations showing that the majority of human breast carcinoma metastases express E-cadherin and maintain their epithelial morphology. The molecular mechanism by which Twist2 participates in EMT of breast cancer in vivo remains poorly understood. METHODS: We examined Twist2 expression pattern in human breast carcinomas by western blot and tissue microarray, and analyzed Twist2 cellular localization by confocal microscopy, cell fractionation and other approaches. RESULTS: Twist2 expression was significantly increased in breast cancer. Cytoplasmic Twist2 positive cancer cells expressing E-cadherin on the cellular membrane were mainly located at tumor center of primary carcinomas and lymph metastases, while cancer cells with nuclear Twist2 clearly showed loss of E-cadherin and were detected at the invasive front in ductal breast carcinomas. In addition, ectopically stable-expressed Twist2 was found to localize in the cytoplasm of cancer cells. Collectively, these data indicate that upregulation of cytoplasmic Twist2 is correlated with tumor histological type and tumor metastasis in human breast cancers. CONCLUSION: The differential cellular distribution of Twist2 may be associated with tumor progression. The cytoplasmic Twist2 in cancer cells at tumor center of primary carcinomas and lymph metastases contributes to the maintenance of epithelial cancer characteristics expressing E-cadherin in a noninvasive state, while the nuclear Twist2 at the cancer invasion front activates EMT to deprive epithelial property of neoplastic cells, thus facilitating invasion and metastasis. These findings suggest that heterogeneous expression of Twist2 in tumors may have a functional link to tumor progression.

Differential methylation of CpG islands within the dermo1 gene promoter in several cancer cell lines.

Inactivation of tumor-related genes by promoter hypermethylation is a common epigenetic event in the development of variety of tumors. Dermo1 (also called twist2) is a novel cancer-related gene which belongs to the basic helix-loop-helix (bHLH) transcription factor family. Herein, we report that dermo1 expression was sporadically abrogated in human cancer cells by transcriptional silencing associated with CpG island promoter hypermethylation. Direct sequencing of bisulfite-modified DNA from a panel of seven human cancer cell lines (HL60, Molm14, MV4-11, RS4:11, MDM-BA231, H358, and H1299) revealed that CpG dinucleotides in the dermo1 promoter were methylated. RT-PCR results demonstrated that dermo1 CpG island hypermethylation was accompanied by a low basal dermo1 expression level. Our data implicate dermo1 as a tumor suppressor gene and a valuable molecular marker for human cancer.

Truncated ErbB2 expressed in tumor cell nuclei contributes to acquired therapeutic resistance to ErbB2 kinase inhibitors.

ErbB2 tyrosine kinase inhibitors (TKI) block tyrosine autophosphorylation and activation of the full-length transmembrane ErbB2 receptor (p185(ErbB2)). In addition to p185(ErbB2), truncated forms of ErbB2 exist in breast cancer cell lines and clinical tumors. The contribution of these truncated forms, specifically those expressed in tumor cell nuclei, to the development of therapeutic resistance to ErbB2 TKIs has not been previously shown. Here, we show that expression of a 95-kDa tyrosine phosphorylated form of ErbB2, herein referred to as p95L (lapatinib-induced p95) was increased in ErbB2(+) breast cancer cells treated with potent ErbB2 TKIs (lapatinib, GW2974). Expressed in tumor cell nuclei, tyrosine phosphorylation of p95L was resistant to inhibition by ErbB2 TKIs. Furthermore, the expression of p95L was increased in ErbB2(+) breast cancer models of acquired therapeutic resistance to lapatinib that mimic the clinical setting. Pretreatment with proteasome inhibitors blocked p95L induction in response to ErbB2 TKIs, implicating the role of the proteasome in the regulation of p95L expression. In addition, tyrosine phosphorylated C-terminal fragments of ErbB2, generated by alternate initiation of translation and similar in molecular weight to p95L, were expressed in tumor cell nuclei, where they too were resistant to inhibition by ErbB2 TKIs. When expressed in the nuclei of lapatinib-sensitive ErbB2(+) breast cancer cells, truncated ErbB2 rendered cells resistant to lapatinib-induced apoptosis. Elucidating the function of nuclear, truncated forms of ErbB2, and developing therapeutic strategies to block their expression and/or activation may enhance the clinical efficacy of ErbB2 TKIs.

Upregulated expression of periostin by hypoxia in non-small-cell lung cancer cells promotes cell survival via the Akt/PKB pathway.

Periostin is a secreted protein and has been shown to be frequently overexpressed in various types of human cancers. We have previously reported that periostin potently promotes metastatic growth of colon cancer by augmenting cell survival. However, little is known about the functions of periostin in non-small-cell lung cancer. Here, we revealed that increased expression of periostin in non-small-cell lung cancer A549 cells was one kind of cellular responses to the stress of chemical-mimic hypoxia, and this effect could be regulated by hypoxia inducible growth factors, such as TGF-alpha and bFGF. We further demonstrated that RTK/PI3-K pathway activated by TGF-alpha and bFGF was evoked in upregulating the expression of periostin, and then periostin promoted the survival of A549 cells under hypoxic microenvironment via activation of Akt/PKB pathway. Therefore, periostin and the pathway that it involved might provide a target for lung cancer treatment.

ATPase domain of Hsp70 exhibits intrinsic ATP-ADP exchange activity.

The chaperone activity of Hsp70 in protein folding and its conformational switching are regulated through the hydrolysis of ATP and the ATP-ADP exchange cycle. It was reported that, in the presence of physiological concentrations of ATP (approximately 5 mM) and ADP (approximately 0.5 mM), Hsp70 catalyzes ATP-ADP exchange through transfer of gamma-phosphate between ATP and ADP, via an autophosphorylated intermediate, whereas it only catalyzes the hydrolysis of ATP in the absence of ADP. To clarify the functional domain of the ATP-ADP exchange activity of Hsp70, we isolated the 44-kD ATPase domain of Hsp70 after limited proteolysis with alpha-chymotrypsin (EC 3.4.21.1). The possibility of ATP-ADP exchange activity of a contaminating nucleoside diphosphate kinase (EC 2.7.4.6) was monitored throughout the experiments. The purified 44-kD ATPase domain exhibited intrinsic ATP-ADP exchange by catalyzing the transfer of gamma-phosphate between ATP and ADP with acid-stable autophosphorylation at Thr204.