Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

Background

Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK). Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK). Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC) lines.

Methods

Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays.

Results

In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity.

Conclusions

We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.

     

Evidence of p38γ and p38δ involvement in cell transformation processes

The p38 mitogen-activated protein kinase (p38MAPK) signal transduction pathway is an important regulator of cell processes, whose deregulation leads to the development and progression of cancer. Defining the role of each p38MAPK family member in these processes has been difficult. To date, most studies of the p38MAPK pathways focused on function of the p38α isoform, which is widely considered to negatively regulate malignant transformation; nonetheless, few reports address the p38γ and p38δ isoforms. Here, we used embryonic fibroblasts derived from mice lacking p38γ or p38δ and show evidence that these isoforms participate in several processes involved in malignant transformation. We observed that lack of either p38γ or p38δ increased cell migration and metalloproteinase-2 secretion, whereas only p38δ deficiency impaired cell contact inhibition. In addition, lack of p38γ in K-Ras-transformed fibroblasts led to increased cell proliferation as well as tumorigenesis both in vitro and in vivo. Our results indicate that p38γ and p38δ have a role in the suppression of tumor development.     

Statin-dependent activation of protein kinase Cδ in acute promyelocytic leukemia cells and induction of leukemic cell differentiation

Statins are HMG-CoA (3-hydroxy-3-methyl-glutaryl-coenzyme A) reductase inhibitors, which block the conversion of HMG-CoA to mevalonate and have potent cholesterol lowering properties. Beyond their importance in the generation of lipid lowering effects, the regulatory effects of statins on the mevalonate pathway have a significant impact on multiple other cellular functions. There is now extensive evidence that statins have anti-inflammatory and anti-neoplastic properties, but the precise mechanisms by which such responses are generated are not well understood. In the present study we demonstrate that statins engage a member of the protein kinase C (PKC) family of proteins, PKCδ, in acute promyelocytic leukemia (APL) cells. Our study shows that atorvastatin and fluvastatin induce proteolytic activation of PKCδ in the APL NB4 cell line, which expresses the t(15;17) translocation. Such engagement of PKCδ results in induction of its kinase domain and downstream regulation of pathways important for statin-dependent leukemia cell differentiation. Our research shows that the function of PKCδ is essential for statin-induced leukemic cell differentiation, as demonstrated by studies involving selective targeting of PKCδ using siRNAs. We also demonstrate that the potent enhancing effects of statins on all-trans retinoic acid (ATRA)-induced gene expression for CCL3 and CCL4 requires the function of PKCδ, suggesting a mechanism by which statins may promote ATRA-induced antileukemic responses. Altogether, our data establish a novel function for PKCδ as a mediator of statin-induced differentiation of APL cells and antileukemic effects.     

Targeting cytosolic phospholipase A2 α in colorectal cancer cells inhibits constitutively activated protein kinase B (AKT) and cell proliferation

A constitutive activation of protein kinase B (AKT) in a hyper-phosphorylated status at Ser⁴⁷³ is one of the hallmarks of anti-EGFR therapy-resistant colorectal cancer (CRC). The aim of this study was to examine the role of cytosolic phospholipase A₂α (cPLA₂α) on AKT phosphorylation at Ser⁴⁷³ and cell proliferation in CRC cells with mutation in phosphoinositide 3-kinase (PI3K). AKT phosphorylation at Ser⁴⁷³ was resistant to EGF stimulation in CRC cell lines of DLD-1 (PIK3CA^E545K mutation) and HT-29 (PIK3CA^P499T mutation). Over-expression of cPLA₂α by stable transfection increased basal and EGF-stimulated AKT phosphorylation and proliferation in DLD-1 cells. In contrast, silencing of cPLA₂α with siRNA or inhibition with Efipladib decreased basal and EGF-stimulated AKT phosphorylation and proliferation in HT-29. Treating animals transplanted with DLD-1 with Efipladib (10 mg/kg, i.p. daily) over 14 days reduced xenograft growth by >90% with a concomitant decrease in AKT phosphorylation. In human CRC tissue, cPLA₂α expression and phosphorylation were increased in 63% (77/120) compared with adjacent normal mucosa determined by immunohistochemistry. We conclude that cPLA₂α is required for sustaining AKT phosphorylation at Ser⁴⁷³ and cell proliferation in CRC cells with PI3K mutation, and may serve as a potential therapeutic target for treatment of CRC resistant to anti-EGFR therapy.     

Protein kinase C isoform α overexpression in C6 glioma cells and its role in cell proliferation

Summary Previous studies from this laboratory have demonstrated that protein kinase C (PKC) enzyme activity is highly correlated with the proliferation rate of glioma cells, and that glioma cells of both human and rat origin have very high PKC enzyme activity when compared to non-malignant glia including astrocytes, the antecedents of most gliomas. In the present study, by contrasting the rat C6 glioma cells with non-malignant rat astrocytes, we have sought to determine whether the high PKC enzyme activity of glioma cells was due to the overexpression of a specific isoform of PKC. By Western blot analyses, both C6 glioma cells and astrocytes were found to express PKC, , , and , but not . Enzyme activity measurements revealed that the elevated PKC activity of glioma cells compared to glia was calcium-dependent, thereby implicating abnormal activity of the or isoforms. On Western blots, when compared to astrocytes, glioma cells were determined to overexpress PKCa but not . An antisense oligonucleotide to PKC, directed at the site of initiation of translation, inhibited the proliferation rate of glioma cells when compared to cells treated with control oligonucleotides; PKC enzyme activity and PKCa protein expression were significantly reduced by the antisense treatment. These results suggest that the high PKC enzyme activity of glioma cells, and its correspondence with proliferation rate, is the result of overexpression of isozyme a. Targetting PKCa in glioma cells may provide a refinement of therapy of glioma patients, some of which are already showing clinical stabilization when treated with drugs with PKC-inhibitory effects.     

microRNA-107 functions as a candidate tumor-suppressor gene in head and neck squamous cell carcinoma by downregulation of protein kinase Cɛ

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide with about 600?000 new cases diagnosed each year. Understanding the molecular pathways that lead to HNSCC is crucial to identify new targets for anti-cancer drug development. Protein kinase C? (PKC?) is elevated in HNSCC and regulates the activation of Akt, Stat3 and Rho GTPases. To date, the molecular mechanism of PKC? dysregulation in HNSCC remains to be elucidated. In silico analysis identified three putative microRNA-107 (miR-107) binding sites in the 3'-untranslated region (UTR) of PKC?. An inverse relationship was revealed between miR-107 and PKC? in HNSCC cell lines. Delivery of miR-107 reduced PKC? levels in SCC15, SCC25 and CAL27, three HNSCC cell lines with high PKC? and low miR-107. The activity of a luciferase reporter construct containing the 3'-UTR of PKC? was downregulated by miR-107 and mutations in the three cognate miR-107 binding sites completely ablated the regulation by miR-107. Treatment with miR-107 significantly blocked cell proliferation, DNA replication, colony formation and invasion in SCC25 and CAL27 cells. Ectopic expression of miR-resistant PKC? was sufficient to partially rescue the loss-of-function phenotype in miR-107-overexpressing SCC25 cells. Tumor growth in nude mice was retarded by 93±7% in CAL27/miR-107 cells compared with CAL27/miR-control cells. Last, human primary HNSCC tumors with elevated PKC? had reduced miR-107 expression. Our results demonstrate that PKC? is directly regulated by miR-107 and, moreover, suggest that miR-107 may be a potential anti-cancer therapeutic for HNSCC.     

The protein kinase C inhibitor Gö6976 is a potent inhibitor of DNA damage-induced S and G2 cell cycle checkpoints

In response to DNA damage, cells arrest progression through the cell cycle at either G(1), S, or G(2). We have reported that UCN-01 (7-hydroxystaurosporine) abrogates DNA damage-induced S and G(2) arrest and enhances cytotoxicity selectively in p53 mutant cells, thus providing a potential, tumor-targeted therapy. Unfortunately, UCN-01 binds avidly to human plasma proteins, limiting bioavailability. Because UCN-01 also inhibits protein kinase C (PKC), we screened other PKC inhibitors, expecting them to be unable to abrogate arrest. However, G?6976 potently abrogated S and G(2) arrest and enhanced the cytotoxicity of the topoisomerase I inhibitor SN38 only in p53-defective cells. Importantly, G?6976 was nearly as potent at abrogating S and G(2) arrest in human serum, a property not possessed by UCN-01. Cell viability studies demonstrated that G?6976 was impressively nontoxic as a single agent. Analysis of proteins that regulate cell cycle arrest suggested that both drugs inhibit the checkpoint kinases Chk1 and/or Chk2. Additionally, G?6976 abrogated S and G(2) arrest at a concentration substantially lower than that required to inhibit PKC; UCN-01 did not demonstrate this selectivity for checkpoint inhibition. These properties make G?6976 a promising candidate for preclinical and clinical studies.     

Dexamethasone inhibits proliferation and stimulates ecto-5′-nucleotidase/CD73 activity in C6 rat glioma cell line

Purpose To review interpathologist diagnosis variability and survival of children treated for either anaplastic mixed glioma (AMG) or anaplastic oligodendroglioma (AO) with surgery, irradiation and chemotherapy. Patients and methods Two hundred and fifty patients with an institutional diagnosis of malignant glioma were enrolled on Children’s Cancer Group CCG-945 between 1985 and 1991, and administered vincristine during involved field radiotherapy, then six cycles of prednisone, lomustine and, vincristine; or two cycles of “eight-drugs-in-one-day” (8-in-1) chemotherapy then involved-field radiotherapy followed by six cycles of 8-in-1 chemotherapy. Central review of institutional pathology was post hoc by five experienced neuropathologists. Results Twenty-six children had institutional diagnoses of AMG and four had AO. Complete resection and cerebral tumor location was associated with better overall survival (OS) in patients with institutional diagnoses of AMG. However, central review established that only nine of 26 children had AMG: either mixed oligoastrocytoma (MOA) or anaplastic mixed oligoastrocytoma (AOA) and only one had AO. Central review revealed five more patients with AMG, but none with AO. Institutional and CCG central review diagnoses of AMG or AO had poor Jaccard reliabilities of 0.29 and 0.25 respectively. Five-year EFS and OS for five children with centrally confirmed MOA was 50 ± 20%, with four centrally confirmed AOA was 37.5 ± 17%. After central review, small samples made tests for differences in survival between regimes impossible. Conclusion Diagnosis of these tumors is challenging, with only 35% of institutional diagnoses confirmed for AMG and 25% for AO, and survival among children with these tumors is poor, despite intensive therapy. This suggests reliable diagnostic markers and new therapeutic approaches are needed.     

Enhancement of in vitro cell motility and invasiveness of human malignant pleural mesothelioma cells through the HIF-1α-MUC1 pathway

In this study, we examined the effects of hypoxia on the malignancy of human malignant pleural mesothelioma (MPM) cell lines, and found (1) hypoxia enhanced motility and invasiveness of human malignant pleural mesothelioma (MPM) cells; (2) this phenomenon resulted from increased expression of sialylated MUC1 through the activation of HIF-1 pathway; (3) two HIF-binding sites located in the promoter region of MUC1 were important for MUC1 transactivation under hypoxia. These findings are useful for better understanding molecular mechanisms of aggressive behavior of MPM cells and for targeting them in the clinical therapies for MPM patients.     

Cell surface heat shock protein 90 modulates prostate cancer cell adhesion and invasion through the integrin-β1/focal adhesion kinase/c-Src signaling pathway

Heat shock protein (Hsp) 90 is a molecular chaperone that maintains the active conformation and function of numerous client oncoproteins in cancer cells. Hsp90 has also been detected on the plasma membrane of cells, and its expression has been suggested to correlate with metastatic potential. We studied the PC3 cell line, which is a highly invasive human prostate cancer cell line, and confirmed that Hsp90 is present on the cell surface of PC3 cells. Interestingly, cell surface Hsp90 was also specifically localized at the leading edge of migrating cells. By using a specific antibody that inhibited cell surface Hsp90, adhesion and invasion of PC3 cells were significantly suppressed in?vitro. Concomitantly with these findings, we demonstrated that the inhibition of cell surface Hsp90 not only inhibited the FN-dependent association between FAK, c-Src and integrin β1, but also significantly inhibited the phosphorylation of FAK and c-Src, as well as their downstream targets paxillin and p130Cas. Additionally, the Hsp90 antibody reversed cell invasion stimulated by overexpression of FAK. These data indicate that cell surface Hsp90 is involved in prostate cancer cell invasion through the integrin β1/FAK/c-Src signaling pathway. Our study provides new insights into the mechanisms of cell surface Hsp90 in cancer invasion. These results suggest that molecular targeting of cell surface Hsp90 may therefore be a novel target for the effective treatment of metastatic prostate cancer.     

N-α-acetyltransferase 10 protein suppresses cancer cell metastasis by binding PIX proteins and inhibiting Cdc42/Rac1 activity

N-α-acetyltransferase 10 protein, Naa10p, is an N-acetyltransferase known to be involved in cell cycle control. We found that Naa10p was expressed lower in varieties of malignancies with lymph node metastasis compared with non-lymph node metastasis. Higher Naa10p expression correlates the survival of lung cancer patients. Naa10p significantly suppressed migration, tumor growth, and metastasis independent of its enzymatic activity. Instead, Naa10p binds to the GIT-binding domain of PIX, thereby preventing the formation of the GIT-PIX-Paxillin complex, resulting in reduced intrinsic Cdc42/Rac1 activity and decreased cell migration. Forced expression of PIX in Naa10-transfected tumor cells restored the migration and metastasis ability. We suggest that Naa10p functions as a tumor metastasis suppressor by disrupting the migratory complex, PIX-GIT- Paxillin, in cancer cells.     

Assessment of p38α in peripheral blood mononuclear cells (PBMC): a potential blood protein marker of head and neck squamous cell carcinoma

Purpose

Solid tumors involve an inflammatory microenvironment portrayed by immune cells playing role in cancer progression via inflammatory p38α mitogen-activated protein kinase (MAPK) molecule that produces pro-inflammatory cytokines-TNFα, IL1β and IL6. This study quantified and compared the expression of p38α in peripheral blood mononuclear cells (PBMCs) of HNSCC patients with the healthy subjects.

Methods

The PBMC were isolated from the 35 control and 83 HNSCC patients. The expression of p38α in PBMCs was assessed using surface plasmon resonance (SPR), ELISA and western blot analysis.

Results

p38α levels were found to be over-expressed in HNSCC patients 0.98 ng/μl (95 % CI 0.95–1.02) as compared to controls 0.46 ng/μl (95 % CI 0.42–0.50) (p < 0.0001).

Conclusion

p38α is over-expressed in PBMCs of HNSCC patients and may play a role in the progression of cancer. This research may translate a protein marker for HNSCC to clinical oncologist for therapeutic intervention and use as a predictive marker.     

Valproic acid induces p21 and topoisomerase-II (α/β) expression and synergistically enhances etoposide cytotoxicity in human glioblastoma cell lines

Object Etoposide, a topoisomerase-II inhibitor promotes DNA damage and apoptosis of cancer cells. In this study, we have examined the ability of the histone deacetylase inhibitor, valproic acid (VPA) to modulate gene expression and sensitize glioblastoma cell lines to the cytotoxic effects of etoposide in vitro. Methods The effect of VPA and etoposide alone or a combination of the two drugs on the growth of three different glioblastoma cell lines (U87, LN18, and U251) were measured by MTT assays. Drug treated cells were analyzed for their cell cycle profile, gene expression, differentiation status, and induction of apoptosis by flow-cytometry, western blotting, immunofluorescence assays, and caspase activity measurements. Results We observed that while VPA and etoposide independently inhibited the growth of U87, U251, and LN18 cells, exposure of tumor cells to both drugs significantly enhanced the cytotoxicity of etoposide in all cell lines. VPA promoted a G₁ accumulation of U87, while an increase in the G₂/M population of U251 and LN18 cells was observed upon exposure to the drug. Treatment with etoposide resulted in a G₂/M arrest of U87, U251, and LN18 cells, whereas, exposure to both drugs increased the fraction of cells with a G2/M and sub-G1 DNA content. Further, VPA and not etoposide, promoted acetylation of histone H4 and induced the expression of the cyclin-dependent kinase inhibitor (CDKI), p21/WAF1. VPA also up-regulated the expression of the α and β isoforms of topoisomerase-II, as well as the glial differentiation marker, glial fibrillary acidic protein. Finally, a significant increase in caspase-3 activity and apoptosis was observed in the presence of both VPA and etoposide compared to either agent alone. Conclusion Our study demonstrates that VPA sensitizes U87, U251, and LN18 cells to the cytotoxic effects of etoposide in vitro by inducing differentiation and up-regulating the expression of p21/WAF1 and both isoforms of topoisomerase-II.     

C1B domain peptide of protein kinase Cγ significantly suppresses growth of human colon cancer cells in vitro and in an in vivo mouse xenograft model through induction of cell cycle arrest and apoptosis

Two peptides derived from the C1B domain of protein kinase Cγ (PKCγ) were shown to associate with classical PKC isozymes and modulate their activities. These C1B peptides are designated C1B1 (amino acid residues 101-112) and C1B5 (residues 141-151). Since PKC enzyme activity is shown to be involved in colon cancer development, the effect of C1B peptides on the growth of various human colon cancer cell lines was examined in vitro and in vivo. Sub-micromolar to micromolar levels of both C1B peptides induced approximately 60-70% growth attenuation in multiple colon cancer cell lines in a soft agar tumor colony assay; however, C1B5 peptide was not cytotoxic to normal colon epithelial cells in two dimensional culture. The effect of C1B5 peptide on colony growth of COLO205 cells was reversed by treatment with the PKCα/β inhibitor, Ro-32-0432. C1B peptide treatment attenuated COLO205 cells via two mechanisms: 1) cell cycle arrest and 2) stimulation of apoptosis. This is evident in G 2 arrest and increases in levels of cleaved caspase 3 and p53 phosphorylated at serine 20. Intratumoral injection of C1B5 peptide (20 mg/kg/day, every three days) markedly attenuated the growth of subcutaneous xenografts of COLO205 cells in SCID mice by 76% compared with the control. Taken together, these results strongly suggest that C1B peptides have negligible effects on normal tissues but are potentially effective chemotherapeutic agents for colon cancer.