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Breast cancer is one of the leading causes of death in women worldwide. To develop novel therapeutic approaches for the refractory cases, the mouse models which recapitulate the tumor tissues biologically and pathologically similar to human breast cancer are required. Although xenograft models of established cell lines in immune-deficient mice are frequently used for preclinical experiments, such xenograft models are not sufficient because the heterogenous structure based on the microenvironment and the intrinsic characteristics of cancer cells is not correctly formed. In this study, we have established induced cancer stem cells (iCSC) from normal mouse mammary stem/progenitor cells through minimal required genetic manipulations and generated mouse breast cancer models by inoculating the iCSCs in the mammary fat pads. Initially, we established iCSC by introducing the H-RasV12 into lnk4a/Arf-knockout mammary stem/progenitor cells and this iCSC formed tumor similar to human triple negative breast cancer in mouse. This finding suggested that two genetic events, an activation of oncogenic signal and a tumor suppressor inactivation, are required for generating the breast cancer iCSC. Anaplastic Lymphoma Kinase (ALK) gene, which encodes a receptor tyrosine kinase, was reported to be amplified and/or overexpressed up to 86% of patients in inflammatory breast cancer, and pleiotrophin (PTN), which is a physiological ligand for ALK, was also shown to be highly expressed in about 60% of human breast cancers. Therefore, we hypothesized that ALK pathway is involved in tumorigenesis of breast cancers and, then, attempted to generate iCSC by using ALK gene. Interestingly, we found that one of the naturally occurring mutations of ALK is sufficient for generating iCSC and tumor formation in vivo without any prior tumor suppressor inactivation. The ALK-induced iCSCs developed highly aggressive breast cancers in mice. Furthermore, the tumor formation was significantly suppressed when the ALK-induced iCSCs were generated by using mammary stem/progenitor cells derived from mouse deficient in CD44 which is a CSC marker. We have recently revealed a role of CD44, in particular that of a variant isoform (CD44v), in the protection of CSCs from high levels of oxidative stress derived from both tumor cells and their microenvironment (Cancer Cell 19: 387-400, 2011; Cancer Res 72: 1438-1448, 2012; Nat Commun 3: 883, 2012). We will discuss the underlying mechanism of ALK-induced tumorigenesis and a role of CD44 in the CSC functions. [ABSTRACT FROM AUTHOR]

Excess stroma and cancer-associated fibroblasts (CAF) enhance cancer progression and facilitate immune evasion. Insights into the mechanisms by which the stroma manipulates the immune microenvironment could help improve cancer treatment. Here, we aimed to elucidate potential approaches for stromal reprogramming and improved cancer immunotherapy. Platelet-derived growth factor C (PDGFC) and D expression were significantly associated with a poor prognosis in patients with gastric cancer, and PDGF receptor beta (PDGFRβ) was predominantly expressed in diffuse-type gastric cancer stroma. CAFs stimulated with PDGFs exhibited markedly increased expression of CXCL1, CXCL3, CXCL5, and CXCL8, which are involved in polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) recruitment. Fibrotic gastric cancer xenograft tumors exhibited increased PMN-MDSC accumulation and decreased lymphocyte infiltration, as well as resistance to anti-PD-1. Single-cell RNA sequencing and spatial transcriptomics revealed that PDGFRα/β blockade reversed the immunosuppressive microenvironment through stromal modification. Finally, combining PDGFRα/β blockade and anti-PD-1 treatment synergistically suppressed the growth of fibrotic tumors. These findings highlight the impact of stromal reprogramming on immune reactivation and the potential for combined immunotherapy for patients with fibrotic cancer., Significance: Stromal targeting with PDGFRα/β dual blockade reverses the immunosuppressive microenvironment and enhances the efficacy of immune checkpoint inhibitors in fibrotic cancer. See related commentary by Tauriello, p. 655., (©2022 American Association for Cancer Research.)

Invasive lung adenocarcinoma (LADC) can be classified histologically as lepidic, acinar, papillary, micropapillary, or solid. Most LADC tumors manifest several of these histological subtypes, with heterogeneity being related to therapeutic resistance. We report here that in immunodeficient mice, human LADC cells form tumors with distinct histological features, MUC5AC-expressing solid-type or cytokeratin 7 (CK7)-expressing acinar-type tumors, depending on the site of development, and that a solid-to-acinar transition (SAT) could be induced by the tumor microenvironment. The TGFβ-Smad signaling pathway was activated in both tumor and stromal cells of acinar-type tumors. Immortalized cancer-associated fibroblasts (CAF) derived from acinar-type tumors induced SAT in 3D cocultures with LADC cells. Exogenous TGFβ1 or overexpression of an active form of TGFβ1 increased CK7 expression and reduced MUC5AC expression in LADC cells, and knockdown of Tgfb1 mRNA in CAFs attenuated SAT induction. RNA-sequencing analysis suggested that angiogenesis and neutrophil recruitment are associated with SAT in vivo . Our data indicate that CAF-mediated paracrine TGFβ signaling induces remodeling of tumor tissue and determines the histological pattern of LADC, thereby contributing to tumor heterogeneity. SIGNIFICANCE: CAFs secrete TGFβ to induce a solid-to-acinar transition in lung cancer cells, demonstrating how the tumor microenvironment influences histological patterns and tumor heterogeneity in lung adenocarcinoma., (©2021 The Authors; Published by the American Association for Cancer Research.)

Death receptor Fas-mediated apoptosis not only eliminates nonspecific and autoreactive B cells but also plays a major role in antitumor immunity. However, the possible mechanisms underlying impairment of Fas-mediated induction of apoptosis during lymphomagenesis remain unknown. In this study, we employed our developed syngeneic lymphoma model to demonstrate that downregulation of Fas is required for both lymphoma development and lymphoma cell survival to evade immune cytotoxicity. CD40 signal activation significantly restored Fas expression and thereby induced apoptosis after Fas ligand treatment in both mouse and human lymphoma cells. Nevertheless, certain human lymphoma cell lines were found to be resistant to Fas-mediated apoptosis, with Livin (melanoma inhibitor of apoptosis protein; ML-IAP) identified as a driver of such resistance. High expression of Livin and low expression of Fas were associated with poor prognosis in patients with aggressive non-Hodgkin's lymphoma. Livin expression was tightly driven by bromodomain and extraterminal (BET) proteins BRD4 and BRD2, suggesting that Livin expression is epigenetically regulated in refractory lymphoma cells to protect them from Fas-mediated apoptosis. Accordingly, the combination of CD40-mediated Fas restoration with targeting of the BET proteins-Livin axis may serve as a promising immunotherapeutic strategy for refractory B-cell lymphoma. SIGNIFICANCE: These findings yield insights into identifying risk factors in refractory lymphoma and provide a promising therapy for tumors resistant to Fas-mediated antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/20/4439/F1.large.jpg., (©2020 American Association for Cancer Research.)

Tumors comprise heterogeneous cell types including cancer stem cells (CSC), progenitor cells, and differentiated cells. Chemoresistance is a potential cause of relapse and a key characteristic of CSC, but the development of novel therapeutic approaches for targeting these cells has been limited. We previously established osteosarcoma-initiating (OSi) cells by introducing the gene for c-Myc into bone marrow stromal cells of Ink4a/Arf knockout mice. These OSi cells are composed of two distinct clones: highly tumorigenic cells (AX cells), similar to bipotent committed osteochondral progenitor cells, and tripotent cells of low tumorigenicity (AO cells), similar to mesenchymal stem cells. Here we show that depolymerization of the actin cytoskeleton induces terminal adipocyte differentiation and suppresses tumorigenesis in chemoresistant OSi cells. In contrast to AX cells, AO cells were highly resistant to conventional chemotherapeutic agents such as doxorubicin and were thus identified as chemoresistant cells. Inhibition of Rho-associated coiled-coil containing protein kinase (ROCK) elicited terminal adipocyte differentiation in chemoresistant AO cells through negative regulation of the transcriptional coactivator megakaryoblastic leukemia 1 associated with actin depolymerization. The clinically administered ROCK inhibitor fasudil significantly suppressed growth in vitro and tumorigenicity in vivo of chemoresistant AO cells as well as of OSi cells. Our findings thus suggest a new therapeutic strategy based on the induction of trans-terminal differentiation via modulation of actin cytoskeleton dynamics for therapy-resistant osteosarcoma stem cells. SIGNIFICANCE: These findings suggest that induction of trans-terminal differentiation through regulation of actin dynamics is a potential novel therapeutic approach for targeting chemoresistant stem-like tumor cells., (©2019 American Association for Cancer Research.)

Extracellular free amino acids contribute to the interaction between a tumor and its microenvironment through effects on cellular metabolism and malignant behavior. System xc(-) is composed of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. Here, we show that the EGFR interacts with xCT and thereby promotes its cell surface expression and function in human glioma cells. EGFR-expressing glioma cells manifested both enhanced antioxidant capacity as a result of increased cystine uptake, as well as increased glutamate, which promotes matrix invasion. Imaging mass spectrometry also revealed that brain tumors formed in mice by human glioma cells stably overexpressing EGFR contained higher levels of reduced glutathione compared with those formed by parental cells. Targeted inhibition of xCT suppressed the EGFR-dependent enhancement of antioxidant capacity in glioma cells, as well as tumor growth and invasiveness. Our findings establish a new functional role for EGFR in promoting the malignant potential of glioma cells through interaction with xCT at the cell surface. Cancer Res; 76(10); 2954-63. ©2016 AACR., (©2016 American Association for Cancer Research.)

Tumor-initiating cells thought to drive brain cancer are embedded in a complex heterogeneous histology. In this study, we isolated primary cells from 21 human brain tumor specimens to establish cell lines with high tumorigenic potential and to identify the molecules enabling this capability. The morphology, sphere-forming ability upon expansion, and differentiation potential of all cell lines were indistinguishable in vitro However, testing for tumorigenicity revealed two distinct cell types, brain tumor-initiating cells (BTIC) and non-BTIC. We found that macrophage migration inhibitory factor (MIF) was highly expressed in BTIC compared with non-BTIC. MIF bound directly to both wild-type and mutant p53 but regulated p53-dependent cell growth by different mechanisms, depending on glioma cell line and p53 status. MIF physically interacted with wild-type p53 in the nucleus and inhibited its transcription-dependent functions. In contrast, MIF bound to mutant p53 in the cytoplasm and abrogated transcription-independent induction of apoptosis. Furthermore, MIF knockdown inhibited BTIC-induced tumor formation in a mouse xenograft model, leading to increased overall survival. Collectively, our findings suggest that MIF regulates BTIC function through direct, intracellular inhibition of p53, shedding light on the molecular mechanisms underlying the tumorigenicity of certain malignant brain cells. Cancer Res; 76(9); 2813-23. ©2016 AACR., (©2016 American Association for Cancer Research.)

Osteosarcoma is a malignant bone tumor in children and adolescents characterized by intrinsic therapeutic resistance. The IGF2 is expressed at elevated levels in osteosarcoma after treatment with chemotherapy, prompting an examination of its functional contributions to resistance. We found that continuous exposure to IGF2 or insulin in the absence of serum created a dormant growth state in osteosarcoma cells that conferred resistance to various chemotherapeutic drugs in vitro. Mechanistic investigations revealed that this dormant state correlated with downregulation of downstream signaling by the IGF1 receptor, heightened cell survival, enhanced autophagy, and the presence of extracellular glutamine. Notably, inhibiting autophagy or depleting glutamine was sufficient to increase chemotherapeutic sensitivity in osteosarcoma xenografts in mice. Clinically, we confirmed that IGF expression levels were elevated in human osteosarcoma specimens from patients who received chemotherapy. Together, our results suggest that activation of IGF or insulin signaling preserves the survival of osteosarcoma cells under chemotherapeutic stress, providing a drug-resistant population that may engender minimal residual disease. Attenuating this survival mechanism may help overcome therapeutic resistance in osteosarcoma., (©2014 American Association for Cancer Research.)

NFATc1 is a transcription factor that regulates T-cell development, osteoclastogenesis, and macrophage function. Given that T cells, osteoclasts, and macrophages in the tumor microenvironment are thought to modulate tumor progression, tumor cells may acquire NFATc1 expression through fusion with these NFATc1-expressing normal cells. We here revealed that a small proportion of tumor cells in human carcinoma specimens expressed NFATc1. To investigate the consequences of NFATc1 acquisition by tumor cells, we established A549 and MCF7 cell lines expressing a constitutively active form of NFATc1 (NFATc1CA) in an inducible manner. The expression of NFATc1CA promoted cancer cell invasion in association with changes in cell morphology. Analysis of gene expression and RNA interference experiments revealed that NFATc1CA suppressed E-cadherin expression by upregulating the transcriptional repressors Snail and Zeb1 in a manner independent of TGF-β signaling. Induced expression of NFATc1CA also downregulated E-cadherin expression and increased invasive activity in tumor xenografts in vivo. Our results thus suggest that the acquisition of NFATc1 expression contributes to tumor progression.

The targeting of antioxidant systems that allow stem-like cancer cells to avoid the adverse consequences of oxidative stress might be expected to improve the efficacy of cancer treatment. Here, we show that head and neck squamous cell carcinoma (HNSCC) cells that express variant isoforms of CD44 (CD44v) rely on the activity of the cystine transporter subunit xCT for control of their redox status. xCT inhibition selectively induces apoptosis in CD44v-expressing tumor cells without affecting CD44v-negative differentiated cells in the same tumor. In contrast to CD44v-expressing undifferentiated cells, CD44v-negative differentiated cells manifest EGF receptor (EGFR) activation and rely on EGFR activity for their survival. Combined treatment with inhibitors of xCT-dependent cystine transport and of EGFR resulted in a synergistic reduction of EGFR-expressing HNSCC tumor growth. Thus, xCT-targeted therapy may deplete CD44v-expressing undifferentiated HNSCC cells and concurrently sensitize the remaining differentiating cells to available treatments including EGFR-targeted therapy.

The prognosis for individuals diagnosed with hepatocellular carcinoma (HCC) remains poor because of the high frequency of invasive tumor growth, intrahepatic spread, and extrahepatic metastasis. Here, we investigated the role of the standard isoform of CD44 (CD44s), a major adhesion molecule of the extracellular matrix and a cancer stem cell marker, in the TGF-β-mediated mesenchymal phenotype of HCC. We found that CD44s was the dominant form of CD44 mRNA expressed in HCC cells. Overexpression of CD44s promoted tumor invasiveness and increased the expression of vimentin, a mesenchymal marker, in HCC cells. Loss of CD44s abrogated these changes. Also in the setting of CD44s overexpression, treatment with TGF-β1 induced the mesenchymal phenotype of HCC cells, which was characterized by low E-cadherin and high vimentin expression. Loss of CD44s inhibited TGF-β-mediated vimentin expression, mesenchymal spindle-like morphology, and tumor invasiveness. Clinically, overexpression of CD44s was associated with low expression of E-cadherin, high expression of vimentin, a high percentage of phospho-Smad2-positive nuclei, and poor prognosis in HCC patients, including reduced disease-free and overall survival. Together, our findings suggest that CD44s plays a critical role in the TGF-β-mediated mesenchymal phenotype and therefore represents a potential therapeutic target for HCC., (©2012 AACR.)

An increased glycolytic flux accompanied by activation of the pentose phosphate pathway (PPP) is implicated in chemoresistance of cancer cells. In this study, we found that CD44, a cell surface marker for cancer stem cells, interacts with pyruvate kinase M2 (PKM2) and thereby enhances the glycolytic phenotype of cancer cells that are either deficient in p53 or exposed to hypoxia. CD44 ablation by RNA interference increased metabolic flux to mitochondrial respiration and concomitantly inhibited entry into glycolysis and the PPP. Such metabolic changes induced by CD44 ablation resulted in marked depletion of cellular reduced glutathione (GSH) and increased the intracellular level of reactive oxygen species in glycolytic cancer cells. Furthermore, CD44 ablation enhanced the effect of chemotherapeutic drugs in p53-deficient or hypoxic cancer cells. Taken together, our findings suggest that metabolic modulation by CD44 is a potential therapeutic target for glycolytic cancer cells that manifest drug resistance.

Glioblastoma (GBM) is the most aggressive and destructive form of brain cancer. Animal models that can unravel the mechanisms underlying its progression are needed to develop rational and effective molecular therapeutic approaches. In this study, we report the development of mouse models for spontaneous gliomas representing distinct progressive stages of disease that are governed by defined genetic alterations. Neural stem/progenitor cell (NPC)-specific constitutive Ras activation in vivo plus p53 deficiency led to development of primarily anaplastic astrocytoma (grade III), whereas combined loss of p53 plus p16(Ink4a)/p19(Arf) led to development of GBM (grade IV) at 100% penetrance within 6 weeks. These glioma models showed enhanced stem cell properties (stemness) accompanied by malignant progression. Notably, we determined that, in our models and in human specimens, downregulation of the homeodomain transcription factor NKX2.2, which is essential for oligodendroglial differentiation, was correlated with increased tumor malignancy. NKX2.2 overexpression by GBM-derived glioma-initiating cells (GIC) induced oligodendroglial differentiation and suppressed self-renewal capacity. By contrast, Nkx2.2 downregulation in mouse NPCs accelerated GBM formation. Importantly, the inhibitory effects of NXK2.2 on GIC self-renewal were conserved in human cells. Thus, our mouse models offer pathobiologically significant advantages to investigate the nature of brain tumors, with improved opportunities to develop novel mechanism-based therapeutic approaches.

PSF1 (partner of sld five 1) is an evolutionarily conserved DNA replication factor implicated in DNA replication in lower species that is strongly expressed in a wide range of normal stem cell populations and progenitor cell populations. Because stem and progenitor cells possess high proliferative capacity, we hypothesized that PSF1 may play an important role in tumor growth. To begin to investigate PSF1 function in cancer cells, we cloned the mouse PSF1 promoter and generated lung and colon carcinoma cells that stably express a PSF1 promoter-reporter gene. Reporter expression in cells correlated with endogenous PSF1 mRNA expression. In a tumor cell xenograft model, high levels of reporter expression correlated with high proliferative activity, serial transplantation potential, and metastatic capability. Notably, cancer cells expressing reporter levels localized to perivascular regions in tumors and displayed expression signatures related to embryonic stem cells. RNAi-mediated silencing of endogenous PSF1 inhibited cancer cell growth by disrupting DNA synthesis and chromosomal segregation. These findings implicate PSF1 in tumorigenesis and offer initial evidence of its potential as a theranostic target.

The aim of this study was to investigate the mechanism of inhibition of Eg5 (kinesin spindle protein), a mitotic kinesin that plays an essential role in establishing mitotic spindle bipolarity, by the novel small molecule inhibitor K858. K858 was selected in a phenotype-based forward chemical genetics screen as an antimitotic agent, and subsequently characterized as an inhibitor of Eg5. K858 blocked centrosome separation, activated the spindle checkpoint, and induced mitotic arrest in cells accompanied by the formation of monopolar spindles. Long-term continuous treatment of cancer cells with K858 resulted in antiproliferative effects through the induction of mitotic cell death, and polyploidization followed by senescence. In contrast, treatment of nontransformed cells with K858 resulted in mitotic slippage without cell death, and cell cycle arrest in G(1) phase in a tetraploid state. In contrast to paclitaxel, K858 did not induce the formation of micronuclei in either cancer or nontransformed cells, suggesting that K858 has minimal effects on abnormalities in the number and structure of chromosomes. K858 exhibited potent antitumor activity in xenograft models of cancer, and induced the accumulation of mitotic cells with monopolar spindles in tumor tissues. Importantly, K858, unlike antimicrotubule agents, had no effect on microtubule polymerization in cell-free and cell-based assays, and was not neurotoxic in a motor coordination test in mice. Taken together, the Eg5 inhibitor K858 represents an important compound for further investigation as a novel anticancer therapeutic.

The retinoblastoma tumor suppressor protein (Rb) is mutated or expressed at very low levels in several tumor types, including retinoblastoma and osteosarcoma, as well as small cell lung, colon, prostate, bladder, and breast carcinomas. Loss or reduction of Rb expression is seen most commonly in high-grade breast adenocarcinomas, suggesting that a relationship may exist between loss of Rb function and a less-differentiated state, increased proliferation, and high metastatic potential. In this study, we found that knockdown of Rb by small interfering RNA in MCF7 breast cancer cells disrupts cell-cell adhesion and induces a mesenchymal-like phenotype. The epithelial-to-mesenchymal transition (EMT), a key event in embryonic morphogenesis, is implicated in the metastasis of primary tumors. Additionally, Rb is decreased during growth factor- and cytokine-induced EMT and overexpression of Rb inhibits the EMT in MCF10A human mammary epithelial cells. Ectopic expression and knockdown of Rb resulted in increased or reduced expression of E-cadherin, which is specifically involved in epithelial cell-cell adhesion. Other EMT-related transcriptional factors, including Slug and Zeb-1, are also induced by Rb depletion. Furthermore, we confirmed that Rb binds to an E-cadherin promoter sequence in association with the transcription factor activator protein-2alpha. Finally, in breast cancer specimens, we observed a concurrent down-regulation of Rb and E-cadherin expression in mesenchymal-like invasive cancers. These findings suggest that Rb inactivation contributes to tumor progression due to not only loss of cell proliferation control but also conversion to an invasive phenotype and that the inhibition of EMT is a novel tumor suppressor function of Rb.

The centrosome plays a fundamental role in cell division, cell polarity, and cell cycle progression. Centrosome duplication is mainly controlled by cyclin-dependent kinase 2 (CDK2)/cyclin E and cyclin A complexes, which are inhibited by the CDK inhibitors p21Cip1 and p27Kip1. It is thought that abnormal activation of CDK2 induces centrosome amplification that is frequently observed in a wide range of aggressive tumors. We previously reported that overexpression of the oncogene MYCN leads to centrosome amplification after DNA damage in neuroblastoma cells. We here show that centrosome amplification after gamma-irradiation was caused by suppression of p27 expression in MYCN-overexpressing cells. We further show that p27-/- and p27+/- mouse embryonic fibroblasts and p27-silenced human cells exhibited a significant increase in centrosome amplification after DNA damage. Moreover, abnormal mitotic cells with amplified centrosomes were frequently observed in p27-silenced cells. In response to DNA damage, the level of p27 gradually increased in normal cells independently of the ataxia telangiectasia mutated/p53 pathway, whereas Skp2, an F-box protein component of an SCF ubiquitin ligase complex that targets p27, was reduced. Additionally, p27 levels in MYCN-overexpressing cells were restored by treatment with Skp2 small interfering RNA, indicating that down-regulation of p27 by MYCN was due to high expression of Skp2. These results suggest that the accumulation of p27 after DNA damage is required for suppression of centrosome amplification, thereby preventing chromosomal instability.

AURKA/STK15/BTAK, the gene encoding Aurora A kinase that is involved in the regulation of centrosomes and segregation of chromosomes, is frequently amplified and overexpressed in various kinds of human cancers, including pancreatic cancer. To address its possibility as a therapeutic target for pancreatic cancer, we employed the RNA interference technique to knockdown AURKA expression and analyzed its phenotypes. We found that the specific knockdown of AURKA in cultured pancreatic cancer cells strongly suppressed in vitro cell growth and in vivo tumorigenicity. The knockdown induced the accumulation of cells in the G(2)-M phase and eventual apoptosis. Furthermore, we observed a synergistic enhancement of the cytotoxicity of taxanes, a group of chemotherapeutic agents impairing G(2)-M transition, by the RNA interference-mediated knockdown of AURKA. These results indicate that inhibition of AURKA expression can result in potent antitumor activity and chemosensitizing activity to taxanes in human pancreatic cancer.

Paclitaxel stabilizes microtubules, causing mitotic arrest and activating the spindle assembly checkpoint. We determined whether suppression of the checkpoint genes Mad2 and BubR1 affects paclitaxel resistance and whether overexpression of Mad2 protein in checkpoint-defective cells enhances paclitaxel sensitivity. Suppression of Mad2 and BubR1 in paclitaxel-treated cancer cells abolished checkpoint function, resulting in paclitaxel resistance that correlated with suppression of cyclin-dependent kinase-1 activity. In contrast, overexpression of Mad2 in cells with a checkpoint defect attributable to low Mad2 expression restored checkpoint function, resulting in enhanced paclitaxel sensitivity that correlated with enhanced cyclin-dependent kinase-1 activity. However, overexpression of Mad2 failed to enhance paclitaxel sensitivity via checkpoint activation in Mad2-independent checkpoint-defective and -intact cells. Thus, checkpoint function is required for paclitaxel sensitivity. These findings show that any molecules that could interfere with the spindle assembly checkpoint could generate paclitaxel resistance in any patient.

Glioblastoma multiforme (GBM) frequently involves amplification and alteration of the epidermal growth factor receptor (EGFR) gene, resulting in overexpression of varied mutations, including the most common mutation, EGFRvIII, as well as wild-type EGFR (EGFRwt). To test the prognostic value of EGFR, we retrospectively analyzed the relationship between treatment outcomes and the EGFR gene in 87 newly diagnosed adult patients with supratentorial GBM enrolled in clinical trials. The EGFR gene status was assessed by Southern blots and EGFR expression by immunohistochemistry using three monoclonal antibodies (EGFR.25 for EGFR, EGFR.113 for EGFRwt, and DH8.3 for EGFRvIII). EGFR amplification was detected in 40 (46%) of the 87 GBM patients; in 39 (97.5%) of these, EGFR was overexpressed. On the other hand, in 46 of 47 patients without EGFR amplification (97.9%), no EGFR overexpression was present. There was a close correlation between EGFR amplification and EGFR overexpression (P < 0.0001). EGFRwt was overexpressed in 27 of the 40 (67.5%) patients with, and in none without, EGFR amplification (P < 0.0001). Similarly, EGFRvIII was overexpressed in 18 (45.0%) of 40 patients with and in 4 (8.5%) of 47 patients without EGFR amplification (P < 0.0001). The finding that 8 (20%) of the patients with EGFR amplification/EGFR overexpression manifested overexpression of neither EGFRwt nor EGFRvIII indicates that they overexpressed other types of EGFR. Multivariate analysis demonstrated that EGFR amplification was an independent, significant, unfavorable predictor for overall survival (OS) in all patients (P = 0.038, HR = 1.67). With respect to the relationship of age to EGFR prognostication, the EGFR gene status was a more significant prognosticator in younger patients, particularly in those <60 years (P = 0.0003, HR = 3.15), whereas not so in older patients. EGFRvIII overexpression, on the other hand, was not predictive for OS. However, in patients with EGFR amplification, multivariate analysis revealed that EGFRvIII overexpression was an independent, significant, poor prognostic factor for OS (P = 0.0044, HR = 2.71). This finding indicates that EGFRvIII overexpression in the presence of EGFR amplification is the strongest indicator of a poor survival prognosis. In GBM patients, EGFR is of significant prognostic value for predicting survival, and the overexpression of EGFRvIII with amplification plays an important role in enhanced tumorigenicity.

A novel method for high-efficiency and region- controlled in vivo gene transfer was developed by combining in vivo electroporation and intraarterial plasmid DNA injection. A mammalian expression plasmid for the Escherichia coli lacZ gene (driven with a SV40 early promoter) was injected into the internal carotid artery of rats whose brain tumors (from prior inoculation) had been electroporated between two electrodes. The lacZ gene was efficiently transferred and expressed in the tumor cell 3 days after plasmid injection. However, neither any gene transfers nor any elevated lacZ activity was detected in tissues outside the electrodes. The plasmid was not transferred without electroporation. Human monocyte chemoattractant protein-1 cDNA was also transferred by this method, and its long-lasting (3 weeks) expression was confirmed by using the Epstein-Barr virus episomal replicon system. The expressed monocyte chemoattractant protein-1 protein was functional, as evident by the presence of a large number of monocytes in tumor tissue. This method, electrogene therapy, which does not require viral genes or particles, allows genes to be transferred and expressed in desired organs or tissues, and it may lead to the development of a new type of highly effective gene therapy.

The expression and phosphotyrosine activity of pp60v-src were measured in the B31 avian sarcoma virus-transformed rat cell line by flow cytometry using monoclonal antibodies against pp60v-src (EB7) and phosphotyrosine (1G2). Although the immunocytochemical staining was markedly heterogeneous, binding of both antibodies was significantly greater to B31 cells than to untransformed Rat 1 cells. Binding of 1G2 to phosphotyrosine residues was specific; it was entirely inhibited by adding excess phenylphosphate but was not affected by phosphoserine or phosphothreonine. The relationship between the amount of phosphorylated tyrosine measured by our FCM technique and total cellular phosphotyrosine measured by phosphoamino acid analysis was linear in vanadate-treated BALB/c 3T3 cells. Treatment of B31 cells for 48 h with herbimycin A, a benzenoid ansamycin antibiotic, to decrease the expression and tyrosine kinase activity of pp60v-src caused reductions of 42% in anti-pp60v-src and 58% in anti-phosphotyrosine antibody immunofluorescence. DNA staining with the fluorescent dye propidium iodide showed no cell cycle specificity in the binding of either antibody. Herbimycin A also caused the transformed cell line to revert to the morphology, actin configuration, and growth behavior of untransformed cells; these changes were reversed within 12 h after removal of the drug. Flow cytometric evaluation of tyrosine kinase expression and activity was fast and easy, and the results correlated well with other measures of cell phenotype. This technique can be used to quantitate the effects of drugs on oncogenic proteins such as pp60v-src and their associated tyrosine kinase activity.

We investigated the effect of retinoic acid (RA) and herbimycin A (herb-A) on cell growth, cell differentiation, and colony formation of human neuroblastoma cell lines. The neuroblastoma line SK-N-SH expressed both neuroblast and nonneuronal phenotypes, whereas its subclone SH-SY5Y and the Kelly cell line were predominantly neuroblastic. Both herb-A and RA, given alone, moderately reduced cell growth and colony formation of the neuroblastic cell lines. Growth curve analyses with SK-N-SH suggested that herb-A greatly reduced the number of initially growing cells, whereas RA slightly enhanced initial cell growth. Morphological changes were determined with the use of rhodaminephalloidin staining of actin. Retinoic acid caused an increase in the fraction of neuroblast cell in SK-N-SH, and conversely of nonneuronal cells in SH-SY5Y and Kelly cell lines. Both drugs also caused partial differentiation towards a neuronal phenotype, and herb-A induced selective lysis of nonneuronal cells of SK-N-SH. Because of their discrepant effects, RA (10 microM) and herb-A (236 nM) were tested in combination at a concentration that had only moderate effects when given alone. The combination further reduced cell growth and colony formation and dramatically enhanced differentiation towards a neuronal morphology. The Kelly cell line with amplified N-myc genome, which correlates with clinical progression of neuroblastoma, was also sensitive to RA plus herb-A. These results recommend the combination of RA and herb-A for differentiation therapy of neuroblastoma.