Adenoviral-Vector Based siRNA for Mutant K-ras as a Promising Tool for Lung Cancer Gene Therapy: A License to Kill.
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Adenoviridae↗
Biomedical subjects
Dihua Yu
Publications and source records attributed to Dihua Yu.
Tumor progression and metastasis from genetic to microenvironmental determinants: a workshop of the tumor progression and metastasis NIH study section in honor of Dr. Martin L. Padarathsingh, May 31, 2006, Georgetown, Washington, DC.
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Animals↗
Wild-type p53 inhibits nuclear factor-kappaB-induced matrix metalloproteinase-9 promoter activation: implications for soft tissue sarcoma growth and metastasis.
Human soft tissue sarcoma (STS) is a highly lethal malignancy in which control of metastasis determines survival. Little is known about the molecular determinants of STS dissemination. Here, we show that human STS express high levels of matrix metalloproteinase-9 (MMP-9) and that MMP-9 expression levels correlate with sequence analysis-defined p53 mutational status. Reintroduction of wild-type p53 (wtp53) into mutant p53 STS cell lines decreased MMP-9 mRNA and protein levels, decreased zymography-assessed MMP-9 proteolytic activity, and decreased tumor cell invasiveness. Reintroduction of wtp53 into STS xenografts decreased tumor growth and MMP-9 protein expression. Luciferase reporter studies showed that reintroduction of wtp53 into mutant p53 STS cells decreased MMP-9 promoter activity. Deletion constructs of the MMP-9 promoter identified a region containing a p53-responsive element that lacked a p53 consensus binding site but did contain a nuclear factor-kappaB (NF-kappaB) site. Mutating this NF-kappaB binding site eliminated the wtp53-repressive effect. Electrophoretic mobility shift assays confirmed decreased NF-kappaB binding in STS cells in the presence of wtp53. Our findings suggest a role for MMP-9 in STS progression and expand the role of p53 in molecular control of STS growth and metastasis. Therapeutic interventions in human STS targeting MMP-9 activity directly or via reintroduction of wtp53 merit further investigation.
Cell Line, Tumor↗
Vascular endothelial growth factor overexpression by soft tissue sarcoma cells: implications for tumor growth, metastasis, and chemoresistance.
To better elucidate the role of vascular endothelial growth factor (VEGF)(165) in soft tissue sarcoma (STS) growth, metastasis, and chemoresistance, we generated stably transfected human STS cell lines with VEGF(165) to study the effect of VEGF(165) on STS cells in vitro and the effect of culture medium from these cells on human umbilical vascular endothelial cells. Severe combined immunodeficient mice bearing xenografts of transfected cell lines were used to assess the effect of VEGF overexpression and the effect of VEGF receptor (VEGFR) 2 inhibition on STS growth, metastasis, and response to doxorubicin. VEGF(165)-transfected xenografts formed highly vascular tumors with shorter latency, accelerated growth, enhanced chemoresistance, and increased incidence of pulmonary metastases. Blockade of VEGFR2 signaling using DC101 anti-VEGFR2 monoclonal antibody enhanced doxorubicin chemoresponse; this combined biochemotherapy inhibited tumor growth and decreased pulmonary metastases without overt toxicity. Combined therapy reduced microvessel counts while increasing vessel maturation index. VEGF overexpression did not affect on the sarcoma cells per se; however, conditioned medium from VEGF transfectants caused increased endothelial cell proliferation, migration, and chemoresistance. Addition of DC101 induced endothelial cell sensitivity to doxorubicin and suppressed the activity of matrix metalloproteinases secreted by endothelial cells. We therefore conclude that VEGF is a critical determinant of STS growth and metastasis and that STS chemoresistance, in our model, is a process induced by the interplay between STS cells and tumor-associated endothelial cells. STS growth and metastasis can be interrupted by combined low-dose doxorubicin and anti-VEGFR2, a strategy that attacks STS-associated endothelial cells. In the future, such therapeutic approaches may be useful in treating STS before the development of clinically apparent metastases.
Animals↗
Selective inhibition of ErbB2-overexpressing breast cancer in vivo by a novel TAT-based ErbB2-targeting signal transducers and activators of transcription 3-blocking peptide.
ErbB2 is an excellent target for cancer therapies. Unfortunately, the outcome of current therapies for ErbB2-positive breast cancers remains unsatisfying due to resistance and side effects. New therapies for ErbB2-overexpressing breast cancers continue to be in great need. Peptide therapy using cell-penetrating peptides (CPP) as peptide carriers is promising because the internalization is highly efficient, and the cargoes delivered can be bioactive. However, the major obstacle in using these powerful CPPs for therapy is their lack of specificity. Here, we sought to develop a peptide carrier that could introduce therapeutics specifically to ErbB2-overexpressing breast cancer cells. By modifying the HIV TAT-derived CPP and conjugating anti-HER-2/neu peptide mimetic (AHNP), we developed the peptide carrier (P3-AHNP) that specifically targeted ErbB2-overexpressing breast cancer cells in vitro and in vivo. A signal transducers and activators of transcription 3 (STAT3)-inhibiting peptide conjugated to this peptide carrier (P3-AHNP-STAT3BP) was delivered more efficiently into ErbB2-overexpressing than ErbB2 low-expressing cancer cells in vitro and successfully decreased STAT3 binding to STAT3-interacting DNA sequence. P3-AHNP-STAT3BP inhibited cell growth in vitro, with ErbB2-overexpressing 435.eB breast cancer cells being more sensitive to the treatment than the ErbB2 low-expressing MDA-MB-435 cells. Compared with ErbB2 low-expressing MDA-MB-435 xenografts, i.p. injected P3-AHNP-STAT3BP preferentially accumulated in 435.eB xenografts, which led to more reduction of proliferation and increased apoptosis and targeted inhibition of tumor growth. This novel peptide delivery system provided a sound basis for the future development of safe and effective new-generation therapeutics to cancer-specific molecular targets.
Amino Acid Sequence↗
ErbB2 increases vascular endothelial growth factor protein synthesis via activation of mammalian target of rapamycin/p70S6K leading to increased angiogenesis and spontaneous metastasis of human breast cancer cells.
ErbB2 overexpression in breast tumors results in increased metastasis and angiogenesis and reduced survival. To study ErbB2 signaling mechanisms in metastasis and angiogenesis, we did a spontaneous metastasis assay using MDA-MB-435 human breast cancer cells stably transfected with constitutively active ErbB2 kinase (V659E), a kinase-dead mutant of ErbB2 (K753M), or vector control (neo). Mice injected with V659E had increased metastasis incidence and tumor microvessel density than mice injected with K753M or control. Increased angiogenesis in vivo from the V659E transfectants paralleled increased angiogenic potential in vitro. V659E produced increased vascular endothelial growth factor (VEGF) through increased VEGF protein synthesis. This was mediated through signaling events involving extracellular signal-regulated kinase, phosphatidylinositol 3-kinase/Akt, mammalian target of rapamycin (mTOR), and p70S6K. The V659E xenografts also had significantly increased phosphorylated Akt, phosphorylated p70S6K, and VEGF compared with controls. To validate the clinical relevance of these findings, we examined 155 human breast tumor samples. Human tumors that overexpressed ErbB2, which have been previously shown to have higher VEGF expression, showed significantly higher p70S6K phosphorylation as well. Increased VEGF expression also significantly correlated with higher levels of Akt and mTOR phosphorylation. Additionally, patients with tumors having increased p70S6K phosphorylation showed a trend for worse disease-free survival and increased metastasis. Our findings show that ErbB2 increases VEGF protein production by activating p70S6K in cell lines, xenografts, and in human cancers and suggest that these signaling molecules may serve as targets for antiangiogenic and antimetastatic therapies.
Animals↗
Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer.
Trastuzumab is a monoclonal antibody targeted against the human epidermal growth factor receptor (HER) 2 tyrosine kinase receptor, which is overexpressed in approximately 25% of invasive breast cancers. The majority of patients with metastatic breast cancer who initially respond to trastuzumab, however, demonstrate disease progression within 1 year of treatment initiation. Preclinical studies have indicated several molecular mechanisms that could contribute to the development of trastuzumab resistance. Increased signaling via the phosphatidylinositol 3-kinase/Akt pathway could contribute to trastuzumab resistance because of activation of multiple receptor pathways that include HER2-related receptors or non-HER receptors such as the insulin-like growth factor 1 receptor, which appears to be involved in a cross-talk with HER2 in resistant cells. Additionally, loss of function of the tumor suppressor PTEN gene, the negative regulator of Akt, results in heightened Akt signaling that leads to decreased sensitivity to trastuzumab. Decreased interaction between trastuzumab and its target receptor HER2, which is due to steric hindrance of HER2 by cell surface proteins such as mucin-4 (MUC4), may block the inhibitory actions of trastuzumab. Novel therapies targeted against these aberrant molecular pathways offer hope that the effectiveness and duration of response to trastuzumab can be greatly improved.
Antibodies, Monoclonal↗
Prostate tumor cells infected with a recombinant influenza virus expressing a truncated NS1 protein activate cytolytic CD8+ cells to recognize noninfected tumor cells.
Many viral oncolytic approaches against cancer are based on the ability of specific viruses to replicate in tumors expressing components of the constitutively activated Ras/mitogen-activated protein kinase (MAPK) pathways and/or inhibited or dysregulated alpha/beta interferon (IFN-alpha/beta) response pathways. A major issue when considering these approaches is their applicability to tumors that lack activated Ras. To identify the effector mechanisms activated by oncolytic viruses, we investigated inhibition of proliferation of the prostate cancer line LNCap by the recombinant TR-NS1 influenza A virus, a genetically attenuated influenza A/PR8/34 virus expressing a truncated nonstructural protein (NS1) of 126 amino acids. LNCap cells lack constitutively activated MAPK, extracellular signal-regulated kinase (ERK), and p38 and are resistant to death by IFN-alpha. Truncation of the NS1 protein of influenza viruses is known to result in viral attenuation due to a reduced ability of the NS1 to inhibit the IFN-alpha/beta response. Infection with TR-NS1 virus rapidly activated ERK-1 more than ERK-2 in LNCap cells. Importantly, TR-NS1 virus infection transiently inhibited cell proliferation and induced apoptosis in LNCap cells. Addition of peripheral blood mononuclear cells (PBMC) and interleukin 12 (IL-12) to TR-NS1 virus-infected LNCap cells (TR-NS1-LNCap) resulted in faster elimination of TR-NS1-LNCap cells compared with LNCap cells. Moreover, TR-NS1-LNCap cells induced IFN-gamma in PBMC. The levels of IFN-gamma were amplified by IL-12. TR-NS1-LNCap cells also induced tumor-lytic cytotoxic T lymphocytes (CTL). These CTL lysed noninfected LNCap cells in a CD8-dependent manner. Activation of cellular immunity to tumor cells by viruses is an intriguing effector pathway, which should be especially significant for elimination of human tumors that lack activated Ras.
CD8-Positive T-Lymphocytes↗
Synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid induces growth arrest in HER2-overexpressing breast cancer cells.
HER2 overexpression is one of the most recognizable molecular alterations in breast tumors known to be associated with a poor prognosis. In the study described here, we explored the effect of HER2 overexpression on the sensitivity of breast cancer cells to the growth-inhibitory effects of 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), a synthetic triterpenoid, both in vitro and in vivo in a xenograft model of breast cancer. Both cell growth and colony formation in the soft agar assay, a hallmark of the transformation phenotype, were preferentially suppressed in HER2-overexpressing cell lines at low concentrations of CDDO, whereas growth-inhibitory effects at high concentrations did not correlate with the expression level of HER2. CDDO dose-dependently inhibited phosphorylation of HER2 in HER2-overexpressing cells and diminished HER2 kinase activity in vitro. CDDO induced the transactivation of the nuclear receptor peroxisome proliferator-activated receptor-gamma in both vector control and HER2-transfected MCF7 cells. Dose-response studies showed that the growth inhibition seen at lower concentrations of CDDO correlated with induction of the tumor suppressor gene caveolin-1, which is known to inhibit breast cancer cell growth. CDDO also reduced cyclin D1 mRNA and protein expression. In vivo studies with liposomally encapsulated CDDO showed complete abrogation of the growth of the highly tumorigenic MCF7/HER2 cells in a xenograft model of breast cancer. These findings provide the first in vitro and in vivo evidence that CDDO effectively inhibits HER2 tyrosine kinase activity and potently suppresses the growth of HER2-overexpressing breast cancer cells and suggest that CDDO has a therapeutic potential in advanced breast cancer.
Animals↗
Cytoplasmic expression of p21CIP1/WAF1 is correlated with IKKbeta overexpression in human breast cancers.
Regulation of cytoplasmic p21CIP/WAF1 (p21) is of great clinical significance in molecular oncology due to its identification as an antiapoptotic factor, a poor survival predictor as well as a drug-resistance inducer. A retrospective study of the immunohistochemical (IHC) profiles of 128 human primary breast cancers showed that increased total and cytoplasmic p21 expression were highly associated with the expression of IkappaB kinase beta (IKKbeta), the major catalytic subunit of the IKK complex and another crucial player in tumorigenesis and drug resistance. The causal relationship study based on cultured cell lines, MDA-MB-453 and MCF-7, confirmed that IKKbeta overexpression did upregulate protein levels of total and cytoplasmic p21. Mechanistic investigation demonstrated that IKKbeta increased p21 expression through upregulation of p21 mRNA level. Moreover, by Western blotting, IKKbeta was found to be able to upregulate Akt phosphorylation on Ser 473. This novel finding indicated that IKKbeta could mediate cytoplasmic p21 accumulation via activation of its downstream target Akt, which was known to phosphorylate p21 and lead to cytoplasmic localization of p21.
Breast Neoplasms↗
ErbB2 promotes Src synthesis and stability: novel mechanisms of Src activation that confer breast cancer metastasis.
Activation of Src kinase plays important roles in the development of many neoplasias. Most of the previous Src studies focused on the deregulation of Src kinase activity. The deregulated Src protein synthesis and stability in mediating malignant phenotypes of cancer cells, however, have been neglected. While investigating the signal transduction pathways contributing to ErbB2-mediated metastasis, we found that ErbB2-activated breast cancer cells that had higher metastatic potentials also had increased Src activity compared with ErbB2 low-expressing cells. The increased Src activity in ErbB2-activated cells paralleled higher Src protein levels, whereas Src RNA levels were not significantly altered. Our studies revealed two novel mechanisms that are involved in Src protein up-regulation and activation by ErbB2: (a) ErbB2 increased Src translation through activation of the Akt/mammalian target of rapamycin/4E-BP1 pathway and (b) ErbB2 increased Src stability most likely through the inhibition of the calpain protease. Furthermore, inhibition of Src activity by a Src-specific inhibitor, PP2, or a Src dominant-negative mutant dramatically reduced ErbB2-mediated cancer cell invasion in vitro and metastasis in an experimental metastasis animal model. Together, activation of ErbB2 and downstream signaling pathways can lead to increased Src protein synthesis and decreased Src protein degradation resulting in Src up-regulation and activation, which play critical roles in ErbB2-mediated breast cancer invasion and metastasis.
Adaptor Proteins, Signal Transducing↗
Expression of receptor tyrosine kinases epidermal growth factor receptor and HER-2/neu in synovial sarcoma.
BACKGROUND: Synovial sarcomas are high-grade soft tissue neoplasms often characterized by a biphasic spindle and epithelioid cell morphology. The majority of synovial sarcomas harbor a specific chromosomal translocation in which the proximal portion of the SYT gene at chromosome 18q11 is fused to the distal portion of one of several duplicated SSX genes (most notably SSX1 and SSX2) at chromosome Xp11. SYT/SSX1 translocations are seen in nearly three times as many synovial sarcomas as SYT/SSX2 translocations. Although the SYT/SSX2 fusion is usually associated with the monophasic disease pattern, the SYT/SSX1 fusion is present in tumors with biphasic or monophasic patterns. The SYT/SSX1 fusion gene is associated with more aggressive tumor growth and poor outcome. Despite advances in the therapy of local disease, distant metastasis remains the predominant cause of death. Accordingly, there is a need for alternate therapies, such as those recently developed against the receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and HER-2/neu. METHODS: Archival specimens of synovial sarcoma (n=38) representing 30 patients were assessed for EGFR and HER-2/neu protein expression by standard immunohistochemical techniques. To validate the immunohistochemistry results, quantitative real-time polymerase chain reaction (Q-PCR) assays using either fresh and/or archival material was performed. The presence of gene amplification was determined by chromogenic in-situ hybridization. RESULTS: EGFR and HER-2/neu protein were detected by immunohistochemistry in 21 of 38 (55.3%) and 20 of 38 (52.6%) synovial specimens, respectively. EGFR immunoreactivity showed a granular and membranous pattern, whereas HER-2/neu immunoreactivity demonstrated only a membrane pattern. Coexpression was observed in 13 of 38 specimens (34.2%). HER-2/neu expression by immunohistochemistry in synovial sarcomas was restricted to tumors with the SYT/SSX1 translocations. Of 6 specimens with SSX2 translocation, none (0%) showed HER-2/neu immunoreactivity and 1 (17%) demonstrated EGFR expression. Q-PCR demonstrated the presence of mRNA for EGFR and HER-2/neu in 19 of 30 specimens (63.3%) and 22 of 30 specimens (73.3%), respectively. EGFR and HER-2/neu were expressed at low concentrations compared with the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). No evidence of gene amplification was observed. CONCLUSIONS: EGFR and HER-2/neu are expressed in the majority of patients with SYT/SSX1 synovial sarcomas, albeit at low levels. Treatment with tyrosine kinase inhibitors may represent appropriate alternate therapy for these patients.
Adolescent↗
A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas.
PURPOSE AND EXPERIMENTAL DESIGN: Telomeres of tumor cells may be maintained by telomerase or by alternative lengthening of telomeres (ALT). The standard ALT assay requires Southern analysis of high molecular weight genomic DNA. We aimed to establish and validate an ALT assay suitable for archived paraffin-embedded tumors and to use it to examine the prevalence and clinical significance of ALT in various types of tumors that are often telomerase negative. RESULTS: To assay for ALT, we detected ALT-associated promyelocytic leukemia (PML) bodies (APBs) by combined PML immunofluorescence and telomere fluorescence in situ hybridization. APBs are PML nuclear domains containing telomeric DNA and are a known hallmark of ALT in cell lines. The APB assay concurred with the standard ALT assay in 62 of 62 tumors and showed that 35% of 101 soft tissue sarcomas (STS), 47% of 58 osteosarcomas (especially younger patients), 34% of 50 astrocytomas, and 0% of 17 papillary thyroid carcinomas were ALT positive (ALT+). The prevalence of ALT varied greatly among different STS subtypes: malignant fibrous histiocytomas, 77%; leiomyosarcomas, 62%; liposarcomas, 33%; synovial sarcomas, 9%; and rhabdomyosarcomas, 6%. ALT correlated with survival in glioblastoma multiforme and occurred more often in lower-grade astrocytomas, but ALT+ and ALT- sarcomas were equally aggressive in terms of grade and clinical outcome. CONCLUSION: The APB assay for ALT is suitable for paraffin-embedded tumors. It showed that a substantial proportion of STS, osteosarcomas, and astrocytomas, but not papillary thyroid carcinomas use ALT. APB positivity correlated strongly with survival of patients with astrocytomas.
Adult↗
A knotty turnabout?: Akt1 as a metastasis suppressor.
Akt is well known to enhance malignancy and is recognized as a key target for antineoplastic therapies. However, intriguing findings reported by Yoeli-Lerner et al. in the November 23, 2005 issue of Molecular Cell, suggest a novel, antimetastasis function of Akt: activation of Akt1 inhibited invasion in some cancer cells. One possible mechanism for this surprising phenotype was that Akt activated the E3 ubiquitin ligase HDM2, causing ubiquitination and degradation of NFAT, an invasion-promoting factor. These findings clearly justify further investigations and, if validated in vivo, call for reevaluation of some Akt-targeting therapeutic strategies currently under development.
Animals↗
Mechanisms of trastuzumab resistance and their clinical implications.
Trastuzumab (Herceptin) is an excellent model of rationally designed targeted cancer treatment. However, less than 35% of patients with ErbB2-positive breast tumors respond to trastuzumab as a single agent, and 2-5% of trastuzumab-treated patients suffer from severe side effects, including cardiac dysfunction. Recent progress in understanding the mechanisms of trastuzumab antitumor function and cellular defects leading to trastuzumab resistance is summarized. Also explored is the potential of combination therapies for reversing trastuzumab resistance.
Animals↗
Transcriptional repression of protein kinase Calpha via Sp1 by wild type p53 is involved in inhibition of multidrug resistance 1 P-glycoprotein phosphorylation.
The protein kinase C (PKC) family consists of serine/threonine protein kinases that play important roles in signal transduction, cell proliferation, and tumor formation. Recent studies found that PKCs are commonly overexpressed in human tumors, including soft tissue sarcoma (STS). Overexpression of PKCs contributes to invasion and migration of tumor cells and induction of angiogenesis. PKC can also phosphorylate the multidrug resistance (MDR) gene-encoded P-glycoprotein and induce MDR phenotype. Our previous studies showed that mutation of p53 enhanced STS metastasis and mediated the MDR phenotype. Restoring wild type (WT) p53 in STS cells containing mutant p53 sensitized the cells to chemotherapy. In the present study, we found that PKCalpha protein expression is inhibited by WT p53 partly due to reduced PKCalpha mRNA expression in STS cells, but p53 does not affect PKCalpha mRNA stability. Deletion and mutation analysis of the PKCalpha promoter fused to the luciferase reporter gene identified a Sp1 binding site (-244/-234) in the PKCalpha promoter that is required for p53-mediated inhibition of PKCalpha promoter activity. More importantly, PKCalpha phosphorylates and activates MDR1 P-glycoprotein, whereas inhibition of PKCalpha by p53 leads to decreased MDR1 phosphorylation in STS cells, which sensitizes STS cells to chemotherapeutic agents. These data indicate that WT p53 may resensitize STS to chemotherapeutic agents by reducing MDR1 phosphorylation via transcriptional repression of PKCalpha expression. Thus, molecular-based therapies targeting mutant p53 and PKCalpha may be an effective new strategy to improve chemotherapeutic efficacy in STS.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Activation of the Akt/mammalian target of rapamycin/4E-BP1 pathway by ErbB2 overexpression predicts tumor progression in breast cancers.
The Akt/mammalian target of rapamycin (mTOR)/4E-BP1 pathway is considered to be a central regulator of protein synthesis, involving the regulation of cell proliferation, differentiation, and survival. The inhibitors of mTOR as anticancer reagents are undergoing active evaluation in various malignancies including breast cancer. However, the activation status of the Akt/mTOR/4E-BP1 pathway and its potential roles in breast cancers remain unknown. Thus, we examined 165 invasive breast cancers with specific antibodies for the phosphorylation of Akt, mTOR, and 4E-BP1 by immunohistochemistry and compared them with normal breast epithelium, fibroadenoma, intraductal hyperplasia, and ductal carcinoma in situ. We discovered that the phosphorylation of Akt, mTOR, and 4E-BP1 increased progressively from normal breast epithelium to hyperplasia and abnormal hyperplasia to tumor invasion. Phosphorylated Akt, mTOR, and 4E-BP1 were positively associated with ErbB2 overexpression. Survival analysis showed that phosphorylation of each of these three markers was associated with poor disease-free survival independently. In vitro, we further confirmed the causal relationship between ErbB2 overexpression and mTOR activation, which was associated with enhanced invasive ability and sensitivity to a mTOR inhibitor, rapamycin. Our results, for the first time, demonstrate the following: (a) high levels of phosphorylation of Akt, mTOR, and 4E-BP1 in breast cancers, indicating activation of the Akt/mTOR/4E-BP1 pathway in breast cancer development and progression; (b) a link between ErbB2 and the Akt/mTOR/4E-BP1 pathway in breast cancers in vitro and in vivo, indicating the possible role of Akt/mTOR activation in ErbB2-mediated breast cancer progression; and (c) a potential role for this pathway in predicting the prognosis of patients with breast cancer, especially those treated with mTOR inhibitors.
Adaptor Proteins, Signal Transducing↗
PI3K: Missense Mutation Motivates Malignancy.
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Biomarkers, Tumor↗