Rho-associated coiled-coil kinase (ROCK) signaling and disease

Abstract

The small Rho GTPase family of proteins, encompassing the three major G-protein classes Rho, Rac and cell division control protein 42, are key mitogenic signaling molecules that regulate multiple cancer-associated cellular phenotypes including cell proliferation and motility. These proteins are known for their role in the regulation of actin cytoskeletal dynamics, which is achieved through modulating the activity of their downstream effector molecules. The Rho-associated coiled-coil kinase 1 and 2 (ROCK1 and ROCK2) proteins were the first discovered Rho effectors that were primarily established as players in RhoA-mediated stress fiber formation and focal adhesion assembly. It has since been discovered that the ROCK kinases actively phosphorylate a large cohort of actin-binding proteins and intermediate filament proteins to modulate their functions. It is well established that global cellular morphology, as modulated by the three cytoskeletal networks: actin filaments, intermediate filaments and microtubules, is regulated by a variety of accessory proteins whose activities are dependent on their phosphorylation by the Rho-kinases. As a consequence, they regulate many key cellular functions associated with malignancy, including cell proliferation, motility and viability. In this current review, we focus on the role of the ROCK-signaling pathways in disease including cancer.     

The effects of adipose-derived stem cells on CD133-expressing bladder cancer cells

Mesenchymal stem cells (MSCs) hold great promise as therapeutic agents in regenerative medicine. They are also considered as a preferred cell source for urinary tract reconstruction. However, as MSCs exhibit affinity to tumor microenvironment, possible activation of tumor-initiating cells remains a major concern in the application of stem cell-based therapies for patients with a bladder cancer history. To analyze the influence of adipose-derived stem cells (ASCs) on bladder cancer cells with stem cell-like properties, we isolated CD133-positive bladder cancer cells and cultured them in conditioned medium from ASCs (ASC-CM). Our results showed that parental 5637 and HB-CLS-1 cells showed induced clonogenic potential when cultured in ASC-CM. Soluble mediators secreted by ASCs increased proliferation and viability of unsorted cells as well as CD133+ and CD133− subpopulations. Furthermore, incubation with ASC-CM modulated activation of intracellular signaling pathways. Soluble mediators secreted by ASCs increased phosphorylation of AKT1/2/3 (1.4-fold, P < 0.05), ERK1/2 (1.6-fold, P < 0.02), and p70 S6K (1.4-fold) in CD133+ cells isolated from 5637 cell line. In turn, decreased phosphorylation of those three proteins involved in PI3K/Akt and MAPK signaling was observed in CD133+ cells isolated from HB-CLS-1 cell line. Our results revealed that bladder cancer stem-like cells are responsive to signals from ASCs. Paracrine factors secreted by locally-delivered ASCs may, therefore, contribute to the modulation of signaling pathways involved in cancer progression, metastasis, and drug resistance.     

Focal adhesion kinase (FAK) activates and stabilizes IGF-1 receptor

Recent studies have shown a direct association between IGF-1R and FAK, two important mediators of cell growth, survival and migration. However, the mechanism by which FAK affects IGF-1R function remains unknown. This study investigates the potential role of FAK in mediating activation and stability of IGF-1R. Autophosphorylation and phosphorylation capacities of wild type and mutant IGF-1R were studied. Surprisingly, we found that the mutant IGF-1R lacking the three core tyrosine residues in the activation-loop can be phosphorylated although it is unable to undergo autophosphorylation, suggesting that another kinase possesses the ability to phosphorylate IGF-1R. By using wild type MEFs and FAK−/− MEFs we could demonstrate that FAK mediates activation-loop independent phosphorylation, as well as Akt and ERK activation. Furthermore, the stability of IGF-1R was decreased upon FAK siRNA or inactivation. Taken together, our data suggest a role for FAK in phosphorylation, signaling and stability of the IGF-1R.     

Identification of cancer stem cells from human glioblastomas: growth and differentiation capabilities and CD133/prominin-1 expression

CD133 can be a marker of tumorigenic CSCs (cancer stem cells) in human GBM (glioblastoma multiforme), although tumorigenic CD133-negative CSCs have been also isolated. Additional evidence indicates that CSCs from GBM exhibit different phenotypes, with increasing interest in the potential significance of the different CSCs with respect to diagnosis, prognosis and the development of novel targets for treatment. We have analysed the expression of CD133 in freshly isolated cells from 15 human GBM specimens. Only 4 of them contained cells positive for AC133 by FACS analysis, and all of them yielded distinct CSC lines, whereas only 6 CSC lines were obtained from the other 11 GBMs. Of these 10 CSCs lines, we further characterized 6 CSC lines. Three CSCs grew as fast-growing neurospheres with higher clonogenic ability, whereas the remaining 3 grew as slow-growing semi-adherent spheres of lower clonogenicity. In addition, the former CSC lines displayed better differentiation capabilities than the latter ones. PCR and Western blot analysis showed that all 6 GBM CSC lines expressed CD133/prominin-1, suggesting that cells negative by FACS analysis may actually represent cells expressing low levels of CD133 undetected by FACS. Nevertheless, all the 6 CSC lines were tumorigenic in nude mice. In conclusion, CSCs from human primary GBMs show different phenotypes and variable levels of CD133 expression, but these parameters did not directly correlate with the tumorigenic potential.     

Proteomic analysis of cancer stem cells in human prostate cancer cells

Results from recent studies support the hypothesis that cancer stem cells (CSCs) are responsible for tumor initiation and formation. Here, we applied a proteome profiling approach to investigate the mechanisms of CSCs and to identify potential biomarkers in the prostate cancer cell line DU145. Using MACS, the DU145 prostate cancer cell line was isolated into CD44+ or CD44− cells. In sphere culture, CD44+ cells possessed stem cell characteristics and highly expressed genes known to be important in stem cell maintenance. In addition, they showed strong tumorigenic potential in the clonogenic assay and soft agar colony formation assay. We then analyzed and identified proteins that were differentially expressed between CD44+ and CD44− using two-dimensional gel electrophoresis and LC-MS/MS. Cofilin and Annexin A5, which are associated with proliferation or metastasis in cancer, were found to be positively correlated with CD44 expression. These results provide information that will be important to the development of new cancer diagnostic tools and understanding the mechanisms of CSCs although a more detailed study is necessary to investigate the roles of Cofilin and Annexin A5 in CSCs.     

Jagged1 protein enhances the differentiation of mesenchymal stem cells into cardiomyocytes

BACKGROUND: Mesenchymal stem cells (MSCs) can differentiate into cardiomyocytes if an appropriate cellular environment is provided. Notch signals exchanged between neighboring cells through the Notch receptor can eventually dictate cell differentiation. In our study, we show that MSC differentiation into cardiomyocytes is dependent on the Notch signal. METHODS: We created a myocardial infarction model in rat by coronary ligation, administered direct intramyocardial injection of DAPI-labeled MSC immediately, and observed the differentiation of MSCs after 14 days by immunofluorescence staining against troponin T. We cultured MSCs and cardiomyocytes in four ways, respectively, in vitro. (1) MSCs cocultured with cardiomyocytes obtained from neonatal rat ventricles in a ratio of 1:10. (2) The two types of cells were cultured in two chambers separated by a semipermeable membrane as indirect coculture group. (3) Notch receptor-soluble jagged1 protein was added to indirect coculture group. (4) Both jagged1 protein and gamma-secretase inhibitor-DAPT were added to indirect coculture group. Two weeks later, we observed the differentiation percentage, respectively, by immunofluorescence staining. RESULTS: We found the differentiation of MSCs which were close to cardiomyocytes in vivo. The differentiation percentage of the four cell culture group was 30.13+/-2.16%, 12.52+/-1.18%, 26.33+/-2.20%, and 13.08+/-1.15%. CONCLUSIONS: MSCs can differentiate into cardiomyocytes in vitro and in vivo if a cardiomyocyte microenvironment is provided. 2. Cell-to-cell interaction is very important for the differentiation of MSCs into cardiomyocytes. 3. Jagged1 protein can activate Notch signal and enhance the differentiation of MSC into cardiomyocyte, while the effect can be inhibited by DAPT.     

Distribution of CD133 reveals glioma stem cells self-renew through symmetric and asymmetric cell divisions

Malignant gliomas contain a population of self-renewing tumorigenic stem-like cells; however, it remains unclear how these glioma stem cells (GSCs) self-renew or generate cellular diversity at the single-cell level. Asymmetric cell division is a proposed mechanism to maintain cancer stem cells, yet the modes of cell division that GSCs utilize remain undetermined. Here, we used single-cell analyses to evaluate the cell division behavior of GSCs. Lineage-tracing analysis revealed that the majority of GSCs were generated through expansive symmetric cell division and not through asymmetric cell division. The majority of differentiated progeny was generated through symmetric pro-commitment divisions under expansion conditions and in the absence of growth factors, occurred mainly through asymmetric cell divisions. Mitotic pair analysis detected asymmetric CD133 segregation and not any other GSC marker in a fraction of mitoses, some of which were associated with Numb asymmetry. Under growth factor withdrawal conditions, the proportion of asymmetric CD133 divisions increased, congruent with the increase in asymmetric cell divisions observed in the lineage-tracing studies. Using single-cell-based observation, we provide definitive evidence that GSCs are capable of different modes of cell division and that the generation of cellular diversity occurs mainly through symmetric cell division, not through asymmetric cell division.     

Migration and invasion of NSCLC suppressed by the downregulation of Src/focal adhesion kinase using single,double and tetra domain anti- CEACAM6 antibodies

Carcinoembryonic antigen-related cell adhesion molecules 6 (CEACAM6) is a cell adhesion receptor. Expression of CEACAM6 in non-small cell lung cancer (NSCLC) associated with tumor progression and metastatic condition via Src/FAK signaling pathway. We established three anti-CEACAM6 antibodies with valences, which were designed to be monomeric sdAb, bivalent sdAb (2Ab), and tetravalent sdAb (4Ab). The anti-CEACAM6 antibodies can be used to target CEACAM6 overexpressing NSCLC. Anti-CEACAM6 antibodies, sdAb, 2Ab and 4Ab, were modified with different valency via protein engineering. sdAb and multivalent sdAbs (2Ab & 4Ab) were expressed and purified from E.coli and CHO cells, respectively. We compared the effect of anti-CEACAM6 antibodies with doxorubicin in NSCLC cell line both in vitro and in vivo. The 4Ab showed significant effect on cell viability. In addition, A549 cells treated with 2Ab and 4Ab inhibited the invasion and migration. In western blot, the 2Ab and 4Ab showed significant inhibition of phospho FAK domain Ty397 that is essential for activation of Src kinase family. Meanwhile, overall protein analysis revealed that 2Ab and 4Ab potently inhibited the phosphorylation of pSRC, pERK, pFAK, pAKT, MMP-2, MMP-9 and N-cadherin. Anti-tumor effect was observed in an A549 NSCLC xenograft model treated with 2Ab or 4Ab compared with doxorubicin. Confocal analysis showed higher targeting ability of 4Ab than that of 2Ab at 4 h incubation. Our data suggests that 2Ab and 4Ab inhibits EMT-mediated migration and invasion via suppression of Src/FAK signaling, which exhibits therapeutic efficiency for NSCLC treatment.     

p56(lck) Controls phosphorylation of filamin (ABP-280) and regulates focal adhesion kinase (pp125(FAK))

Transformation of cells by src -like kinases leads to altered cell morphology associated with the disassembly of focal contacts and concomitant increase in tyrosine phosphorylation of pp125(FAK) x p56(lck) is a lymphocyte-specific member of the src family of protein tyrosine kinases that associates with cell surface glycoproteins such as CD4 and CD8. It phosphorylates and activates pp125(FAK) and increases its autokinase activity, thus pretreatment of pp125(FAK) with protein kinase C (PKC) markedly attenuates its phosphorylation and activation, suggesting a potential regulatory pathway of pp125(FAK) activation in focal contacts. p56(lck) further phosphorylates and activates actin binding protein (ABP-280; filamin) and controls its association with cell surface receptors such as beta-2 integrins, actin filament cross-linking, and possibly lipid membrane insertion.     

Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer

Aldehyde dehydrogenase 1 (ALDH1) has been considered to be a marker for cancer stem cells. However, the role of ALDH1 in head and neck squamous cell carcinoma (HNSCC) has yet to be determined. In this study, we isolated ALDH1-positive cells from HNSCC patients and showed that these HNSCC-ALDH1⁺ cells displayed radioresistance and represented a reservoir for generating tumors. Based on microarray findings, the results of Western blotting and immunofluorescent assays further confirmed that ALDH1⁺-lineage cells showed evidence of having epithelial-mesenchymal transition (EMT) shifting and endogenously co-expressed Snail. Furthermore, the knockdown of Snail expression significantly decreased the expression of ALDH1, inhibited cancer stem-like properties, and blocked the tumorigenic abilities of CD44⁺CD24⁻ALDH1⁺ cells. Finally, in a xenotransplanted tumorigenicity study, we confirmed that the treatment effect of chemoradiotherapy for ALDH1⁺ could be improved by Snail siRNA. In summary, it is likely that ALDH1 is a specific marker for the cancer stem-like cells of HNSCC.     

干扰FSCN1基因表达对前列腺癌细胞凋亡、活性氧水平影响的研究

目的 探讨干扰FSCN1基因表达对前列腺癌细胞凋亡、活性氧(ROS)水平影响及机制.方法 以正常前列腺上皮细胞RWPE-1为对照细胞,通过RT-PCR及Western blot检测前列腺癌LNCaP、DU145和PC-3细胞中FSCN1 mRNA及蛋白表达;以LipofectamineTM 2000为载体,DU145细...     

High expression of sphingosine kinase 1 and S1P receptors in chemotherapy-resistant prostate cancer PC3 cells and their camptothecin-induced up-regulation

Although most of pharmacological therapies for cancer utilize the apoptotic machinery of the cells, the available anti-cancer drugs are limited due to the ability of prostate cancer cells to escape from the anti-cancer drug-induced apoptosis. A human prostate cancer cell line PC3 is resistant to camptothecin (CPT). To elucidate the mechanism of this resistance, we have examined the involvement of sphingosine kinase (SPHK) and sphingosine 1-phosphate (S1P) receptor in CPT-resistant PC3 and -sensitive LNCaP cells. PC3 cells exhibited higher activity accompanied with higher expression levels of protein and mRNA of SPHK1, and also elevated expression of S1P receptors, S1P(1) and S1P(3), as compared with those of LNCaP cells. The knockdown of SPHK1 by small interfering RNA and inhibition of S1P receptor signaling by pertussis toxin in PC3 cells induced significant inhibition of cell growth, suggesting implication of SPHK1 and S1P receptors in cell proliferation in PC3 cells. Furthermore, the treatment of PC3 cells with CPT was found to induce up-regulation of the SPHK1/S1P signaling by induction of both SPHK1 enzyme and S1P(1)/S1P(3) receptors. These findings strongly suggest that high expression and up-regulation of SPHK1 and S1P receptors protect PC3 cells from the apoptosis induced by CPT.     

New emerging roles of CD133 in cancer stem cell: Signaling pathway and miRNA regulation

Cancer stem cells (CSC) are rare immortal cells within a tumor that are able to initiate tumor progression, development, and resistance. Advances studies show that, like normal stem cells, CSCs can be both self-renewed and given rise to many cell types, therefore form tumors. A number of cell surface markers, such as CD44, CD24, and CD133 are frequently used to identify CSCs. CD133, a transmembrane glycoprotein, either alone or in collaboration with other markers, has been mainly considered to identify CSCs from different solid tumors. However, the exactness of CD133 as a cancer stem cell biomarker has not been approved yet. The clinical importance of CD133 is as a CSC marker in many cancers. Also, it contributes to shorter survival, tumor progression, and tumor recurrence. The expression of CD133 is controlled by many extracellular or intracellular factors, such as tumor microenvironment, epigenetic factors, signaling pathways, and miRNAs. In this study, it was attempted to determine: 1) CD133 function; 2) the role of CD133 in cancer; 3) CD133 regulation; 4) the therapeutic role of CD133 in cancers.     

Triptolide inhibits CD133+/CD44+ colon cancer stem cell growth and migration through triggering apoptosis and represses epithelial-mesenchymal transition via downregulating expressions of snail,slug, and twist

High recurrence and metastatic behavior patterns are the most important reasons for the failure of treatment strategies in patients with colon cancer. Cancer stem cells (CSCs), which are considered root of cancer, are thought to be associated with therapy resistance, relapse, and metastasis, and, therefore, targeting CSCs rather than the bulk population may be an effective approach. In cancer studies, there is an increasing interest in close friendship between epithelial-mesenchymal transition (EMT) and CSCs. Triptolide (TPL) isolated from Chinese herb Tripterygium wilfordii has important effects on the prevention of migration and metastasis as well as cytotoxic effect against cancer cells. The potential lethal efficacy of TPL on CSCs that is highly resistant to the drug is an unsolved mystery. Fundamentally, the present study basically aims to find answers to two questions: (a) is it possible to target colon CSCs with TPL? and (b) what are the mechanisms underlying TPL's potential to eliminate CSCs? Cytotoxic effects of TPL on CSCs were evaluated by WST-1 and Muse count and viability assays. Apoptosis assay and cell-cycle analysis were performed to investigate the inhibitory effect of TPL. Moreover, the effects of TPL on spheroid formation capacity, migration, and EMT processes, which are associated with CSC phenotype, were also investigated. The results revealed that TPL triggered cell death and apoptosis and altered cell cycle distribution. Moreover, TPL significantly reduced the snail slug and twist expressions associated with EMT. TPL has been shown to be effective in colon CSCs by in vitro experiments, and it might be a highly effective agent against colon cancer has been implicated in need of supporting in vivo and clinical studies.     

GHRH antagonist inhibits focal adhesion kinase (FAK) and decreases expression of vascular endothelial growth factor (VEGF) in human lung cancer cells in vitro

Lung cancers which show increased vascularization and high microvessel density are considered highly metastatic and with poor prognosis. Growth hormone releasing hormone (GHRH) antagonists are anticancer agents without adverse events in lung cancer tumor models. In the present study we investigated the in vitro effect of GHRH antagonist, MZ-5-156, on focal adhesion kinase (FAK) activity, on the expression of MMP-2 and MMP-9 metalloproteinases, as well as on vascular endothelial growth factor (VEGF) levels in A549 non-small cell lung (NSCLC) cancer cells and H727 bronchial carcinoid cells. We demonstrate for the first time that GHRH antagonist, MZ-5-156, inhibits FAK signaling in lung cancer cells and decreases the expression of additional factors involved in angiogenesis and invasion. In contrast, GHRH itself counteracted these effects. Our study contributes to the further understanding of the processes which govern the mechanism of action of GHRH and its antagonists in cancers.     

Phage display screening identifies a prostate specific antigen (PSA)–/lo prostate cancer cell specific peptide to retard castration resistance of prostate cancer

Patients with prostate cancer (PCa) will eventually progress to castrate-resistant prostate cancer (CRPC) after androgen deprivation therapy (ADT) treatment. Prostate-specific antigen (PSA)^−/lo cells which harbor self-renewing long-term tumor-propagating cells that can be enriched using ALDH+CD44+α2β1+ and can initiate tumor development may represent a critical source of CRPC cells. Our purpose was to find a peptide that specifically targets PSA^−/lo PCa cells to retard the development of CRPC. PSA⁺ and PSA^−/lo cells were successfully separated from LNCaP xenograft tumors after prostate- PSAP-GFP vector infection and FACS. A variety of PSA^−/lo cells specifically targeting peptide (named as “TAP1” targeted affinity peptide 1) was identified by using phage display library screening. The highest binding rate in TAP1 binding cell subpopulations are identified to be among ALDH⁺CD44⁺CXCR4⁺CD24⁺ cells. TAP1 significantly inhibited PCa growth both in vitro and in vivo. TAP1 significantly improved the anti-proliferation effect of the anti-androgens (Charcoal dextran-stripped serum (CDSS)+Bicalutamide, Enzalutamide) and chemotherapeutic agents (Abiraterone, Docetaxel, Etoposide) in vitro. TAP1 treatment shortens the length of telomeres in ALDH⁺CD44⁺CXCR4⁺CD24⁺ cells and significantly reduces the expression of Homeobox B9 (HOXB9) and TGF-β2. In conclusion, PSA^−/lo PCa cell-specific targeting peptide (TAP1) that suppressed PCa cell growth both in vitro and in vivo and improved the drug sensitivities of anti-androgens and chemotherapeutic agents at least through shortening the length of telomere and reducing the expression of HOXB9 and TGF-β2. Therapeutic peptides that specifically target prostate cancer stem cell might be a very valuable and promising approach to overcome chemoresistance and prevent recurrence in patients with PCa.     

Androgen receptor expression reduces stemness characteristics of prostate cancer cells (PC3) by repression of CD44 and SOX2

Studies have shown that a subgroup of tumor cells possess stemness characteristics having self-renewal capacity and the ability to form new tumors. We sought to identify the plausible stemness factor that determines the “molecular signature” of prostate cancer (PCa) cells derived from different metastases (PC3, PCa2b, LNCaP, and DU145) and whether androgen receptor (AR) influences the maintenance of stemness features. Here we show sex-determining region Y (SRY)-box 2 (SOX2) as a putative stem cell marker in PC3 PCa cells and not in DU145, PCa2b, or LNCaP cells. PCa2b and PC3 cells were derived from bone metastases. PCa2b cells which are positive for the AR failed to demonstrate the expression of either cluster of differentiation 44 (CD44) or SOX2. Knockdown (KD) of AR in these cells did not affect the expression of either CD44 or SOX2. Conversely, PC3 cells, which are negative for AR, expressed both CD44 and SOX2. However, the expression of AR downregulated the expression of both CD44 and SOX2 in PC3 cells. CD44 regulates SOX2 expression as KD of CD44 and reduces SOX2 levels considerably. SOX2 KD attenuated not only the expression of SNAIL and SLUG but also the migration and tumorsphere formation in PC3 cells. Collectively, our findings underscore a novel role of CD44 signaling in the maintenance of stemness and progression of cancer through SOX2 in AR-independent PC3 cells. SOX2 has a role in the regulation of expression of SNAIL and SLUG. SOX2 could be a potential therapeutic target to thwart the progression of SOX2-positive cancer cells or recurrence of androgen-independent PCa.