bluuebunny/crossref_metadata_2025 huggingface dataset:
High Cancer-Specific Expression of Mesothelin (MSLN) Is Attributable to an Upstream Enhancer Containing a Transcription Enhancer Factor–Dependent MCAT Motif
Authors: Hucl Tomas , Brody Jonathan R. , Gallmeier Eike , Iacobuzio-Donahue Christine A. , Farrance Iain K. , Kern Scott E.
Published: October 2007

Identification of genes with cancer-specific overexpression offers the potential to efficiently discover cancer-specific activities in an unbiased manner. We apply this paradigm to study mesothelin (MSLN) overexpression, a nearly ubiquitous, diagnostically and therapeutically useful characteristic of pancreatic cancer. We identified an 18-bp upstream enhancer, termed CanScript, strongly activating transcription from an otherwise weak tissue-nonspecific promoter and operating selectively in cells having aberrantly elevated cancer-specific MSLN transcription. Introducing mutations into CanScript showed two functionally distinct sites: an Sp1-like site and an MCAT element. Gel retardation and chromatin immunoprecipitation assays showed the MCAT element to be bound by transcription enhancer factor (TEF)-1 (TEAD1) in vitro and in vivo. The presence of TEF-1 was required for MSLN protein overexpression as determined by TEF-1 knockdown experiments. The cancer specificity seemed to be provided by a putative limiting cofactor of TEF-1 that could be outcompeted by exogenous TEF-1 only in a MSLN-overexpressing cell line. A CanScript concatemer offered enhanced activity. These results identify a TEF family member as a major regulator of MSLN overexpression, a fundamental characteristic of pancreatic and other cancers, perhaps due to an upstream and highly frequent aberrant cellular activity. The CanScript sequence represents a modular element for cancer-specific targeting, potentially suitable for nearly a third of human malignancies. [Cancer Res 2007;67(19):9055–65]
Tenascin-W Is a Novel Marker for Activated Tumor Stroma in Low-grade Human Breast Cancer and Influences Cell Behavior
Authors: Degen Martin , Brellier Florence , Kain Renate , Ruiz Christian , Terracciano Luigi , Orend Gertraud , Chiquet-Ehrismann Ruth
Published: October 2007

This is the first report about human tenascin-W, the fourth and final member of the extracellular matrix protein family of tenascins. Sixty-three human breast tumor extracts were analyzed by Western blotting for the presence of tenascin-W and compared with tenascin-C, an established marker of tumor stroma. Interestingly, we found tenascin-W expression in the majority of the tumor tissues, but no detectable expression in the normal mammary parenchyma. Eighty-one percent of the breast tumor samples were tenascin-W positive and 86% showed expression of tenascin-C. However, tenascin-W and tenascin-C amounts varied greatly between tumors and some contained either tenascin-W or tenascin-C exclusively, indicating independent mechanisms regulating their expression. Although there was no difference between high- or low-grade tumors with respect to the presence of tenascin-C, tenascin-W was more prominent in low-grade tumors. For 42 of the breast cancer tissues, a frozen tumor microarray was available to confirm the Western blot data by immunohistochemistry. Similar to tenascin-C, tenascin-W was detected in the tumor stroma. Fibroblasts adhered to tenascin-W in a β1 integrin–dependent manner and spread with a distinctive morphology under conditions where they remained round on tenascin-C. CHOB2 cells expressing αvβ1 or α4β1 integrins were able to spread on tenascin-W. Furthermore, addition of tenascin-W to the culture medium increased migration of breast cancer cells toward a fibronectin substratum in vitro. These data imply that tenascin-W expression in the activated tumor stroma facilitates tumorigenesis by supporting the migratory behavior of breast cancer cells. [Cancer Res 2007;67(19):9169–79]
Systemic Therapy of Spontaneous Prostate Cancer in Transgenic Mice with Oncolytic Herpes Simplex Viruses
Authors: Varghese Susan , Rabkin Samuel D. , Nielsen G. Petur , MacGarvey Usha , Liu Renbin , Martuza Robert L.
Published: October 2007

Oncolytic viruses are an innovative therapeutic strategy for cancer, wherein viral replication and cytotoxicity are selective for tumor cells. Here we show the efficacy of systemically administered oncolytic viruses for the treatment of spontaneously arising tumors, specifically the use of oncolytic herpes simplex viruses (HSV) administered i.v. to treat spontaneously developing primary and metastatic prostate cancer in the transgenic TRAMP mouse, which recapitulates human prostate cancer progression. Four administrations of systemically delivered NV1023 virus, an HSV-1/HSV-2 oncolytic recombinant, to TRAMP mice at 12 or 18 weeks of age (presence of prostate adenocarcinoma or metastatic disease, respectively) inhibited primary tumor growth and metastases to lymph nodes. Expression of interleukin 12 (IL-12) from NV1042 virus, a derivative of NV1023, was additionally effective, significantly reducing the frequency of development of prostate cancer and lung metastases, even when the mice were treated after the onset of metastasis at 18 weeks of age. NV1042-infected cells, as detected by 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside staining for Lac Z expressed by the virus, were present in prostate tumors 1 week after the final virus injection and viral DNA was detected at 2 weeks after final virus injection by real-time PCR in primary and metastatic tumors but not in liver or blood. No toxicity was observed in any of the treated mice. The efficacy of the IL-12–expressing NV1042 virus in this aggressive prostate cancer model using a clinically relevant treatment paradigm merits its consideration for clinical studies. [Cancer Res 2007;67(19):9371–9]
Roles for the Stem Cell–Associated Intermediate Filament Nestin in Prostate Cancer Migration and Metastasis
Authors: Kleeberger Wolfram , Bova G. Steven , Nielsen Matthew E. , Herawi Mehsati , Chuang Ai-Ying , Epstein Jonathan I. , Berman David M.
Published: October 2007

The intermediate filament protein Nestin identifies stem/progenitor cells in adult tissues, but the function of Nestin is poorly understood. We investigated Nestin expression and function in common lethal cancers. Nestin mRNA was detected in cell lines from small cell lung, and breast cancers, and particularly elevated in cell lines derived from prostate cancer metastases. Whereas the androgen-independent lines PC3, 22RV1, and DU145 all expressed Nestin transcripts under standard culture conditions, the androgen-dependent line LnCaP expressed Nestin only on androgen withdrawal. We confirmed associations of Nestin expression, androgen withdrawal, and metastatic potential by immunohistochemical analysis of samples from 254 prostate cancer patients. Cytoplasmic Nestin protein was readily identifiable in prostate cancer cells from 75% of patients with lethal androgen-independent disease, even in cancer sampled from the prostate itself. However, Nestin expression was undetectable in localized androgen-deprived tumors and in metastases without prior androgen deprivation. To address its function, we reduced Nestin levels with short hairpin RNAs, markedly inhibiting in vitro migration and invasion in prostate cancer cells but leaving cell growth intact. Nestin knockdown also diminished metastases 5-fold compared with controls despite uncompromised tumorigenicity at the site of inoculation. These results specify a function for Nestin in cell motility and identify a novel pathway for prostate cancer metastasis. Activity of this pathway may be selected by the extraprostatic environment or, as supported by our data, may originate within the prostate after androgen deprivation. Further dissection of this novel Nestin migration pathway may lead to strategies to prevent and neutralize metastatic spread. [Cancer Res 2007;67(19):9199–206]
Epigenetic Inactivation of a Cluster of Genes Flanking MLH1 in Microsatellite-Unstable Colorectal Cancer
Authors: Hitchins Megan P. , Lin Vita Ap , Buckle Andrew , Cheong Kayfong , Halani Nimita , Ku Su , Kwok Chau-To , Packham Deborah , Suter Catherine M. , Meagher Alan , Stirzaker Clare , Clark Susan , Hawkins Nicholas J. , Ward Robyn L.
Published: October 2007

Biallelic promoter methylation and transcriptional silencing of the MLH1 gene occurs in the majority of sporadic colorectal cancers exhibiting microsatellite instability due to defective DNA mismatch repair. Long-range epigenetic silencing of contiguous genes has been found on chromosome 2q14 in colorectal cancer. We hypothesized that epigenetic silencing of MLH1 could occur on a regional scale affecting additional genes within 3p22, rather than as a focal event. We studied the levels of CpG island methylation and expression of multiple contiguous genes across a 4 Mb segment of 3p22 including MLH1 in microsatellite-unstable and -stable cancers, and their paired normal colonic mucosa. We found concordant CpG island hypermethylation, H3-K9 dimethylation and transcriptional silencing of MLH1 and multiple flanking genes spanning up to 2.4 Mb in microsatellite-unstable colorectal cancers. This region was interspersed with unmethylated genes, which were also transcriptionally repressed. Expression of both methylated and unmethylated genes was reactivated by methyltransferase and histone deacetylase inhibitors in a microsatellite-unstable colorectal carcinoma cell line. Two genes at the telomeric end of the region were also hypermethylated in microsatellite-stable cancers, adenomas, and at low levels in normal colonic mucosa from older individuals. Thus, the cluster of genes flanking MLH1 that was specifically methylated in the microsatellite-unstable group of cancers extended across 1.1 Mb. Our results show that coordinate epigenetic silencing extends across a large chromosomal region encompassing MLH1 in microsatellite-unstable colorectal cancers. Simultaneous epigenetic silencing of this cluster of 3p22 genes may contribute to the development or progression of this type of cancer. [Cancer Res 2007;67(19):9107–16]
ABCG2/BCRP Expression Modulates d-Luciferin–Based Bioluminescence Imaging
Authors: Zhang Yimao , Bressler Joseph P. , Neal Jeff , Lal Bachchu , Bhang Hyo-Eun C. , Laterra John , Pomper Martin G.
Published: October 2007

Bioluminescence imaging (BLI) is becoming indispensable to the study of transgene expression during development and, in many in vivo models of disease such as cancer, for high throughput drug screening in vitro. Because reaction of d-luciferin with firefly luciferase (fLuc) produces photons of sufficiently long wavelength to permit imaging in intact animals, use of this substrate and enzyme pair has become the method of choice for performing BLI in vivo. We now show that expression of the ATP-binding cassette (ABC) family transporter ABCG2/BCRP affects BLI signal output from the substrate d-luciferin. In vitro studies show that d-luciferin is a substrate for ABCG2/BCRP but not for the MDR1 P-glycoprotein (ABCB1/Pgp), multidrug resistance protein 1 (MRP1/ABCC1), or multidrug resistance protein 2 (MRP2/ABCC2). d-Luciferin uptake within cells is shown to be modulated by ABC transporter inhibitors, including the potent and selective ABCG2/BCRP inhibitor fumitremorgin C. Images of xenografts engineered to express transgenic ABCG2/BCRP, as well as xenografts derived from the human prostate cancer cell line 22Rv1 that naturally express ABCG2/BCRP, show that ABCG2/BCRP expression and function within regions of interest substantially influence d-luciferin–dependent bioluminescent output in vivo. These findings highlight the need to consider ABCG2/BCRP effects during d-luciferin–based BLI and suggest novel high throughput methods for identifying new ABCG2/BCRP inhibitors. [Cancer Res 2007;67(19):9389–97]
Accelerated Bone Resorption, Due to Dietary Calcium Deficiency, Promotes Breast Cancer Tumor Growth in Bone
Authors: Zheng Yu , Zhou Hong , Modzelewski James R.K. , Kalak Robert , Blair Julie M. , Seibel Markus J. , Dunstan Colin R.
Published: October 2007

The skeleton is a major site of breast cancer metastases. High bone turnover increases risk of disease progression and death. However, there is no direct evidence that high bone turnover is causally associated with the establishment and progression of metastases. In this study, we investigate the effects of high bone turnover in a model of breast cancer growth in bone. Female nude mice commenced a diet containing normal (0.6%; ‘Normal-Ca’) or low (0.1%; ‘Low-Ca’) calcium content. Mice were concurrently treated with vehicle or osteoprotegerin (1 mg/kg/d s.c; n = 16 per group). Three days later (day 0), 50,000 Tx-SA cells (variant of MDA-MB-231 cells) were implanted by intratibial injection. On day 0, mice receiving Low-Ca had increased serum parathyroid hormone (PTH) and tartrate-resistant acid phosphatase 5b levels, indicating secondary hyperparathyroidism and high bone turnover, which was maintained until day 17. Osteoprotegerin increased serum PTH but profoundly reduced bone resorption. On day 17, in mice receiving Low-Ca alone, lytic lesion area, tumor area, and cancer cell proliferation increased by 43%, 24%, and 24%, respectively, compared with mice receiving Normal Ca (P < 0.01). Osteoprotegerin treatment completely inhibited lytic lesions, reduced tumor area, decreased cancer cell proliferation, and increased cancer cell apoptosis. Increased bone turnover, due to dietary calcium deficiency, promotes tumor growth in bone, independent of the action of PTH. Breast cancer patients frequently have low dietary calcium intake and high bone turnover. Treatment to correct calcium insufficiency and/or treatment with antiresorptive agents, such as osteoprotegerin, may be of benefit in the adjuvant as well as palliative setting. [Cancer Res 2007;67(17):9542–8]
β-Catenin Regulates Multiple Steps of RNA Metabolism as Revealed by the RNA Aptamer in Colon Cancer Cells
Authors: Lee Hee Kyu , Kwak Ho Yoon , Hur Jung , Kim In Ae , Yang Ji Sun , Park Min Woo , Yu Jaehoon , Jeong Sunjoo
Published: October 2007

Nuclear β-catenin forms a transcription complex with TCF-4, which is implicated in colon cancer development and progression. Recently, we and others have shown that β-catenin could be a regulator of RNA splicing and it also stabilizes the cyclooxygenase-2 (COX-2) mRNA. Here, we further explored the role of β-catenin in the RNA metabolism in colon cancer cells. To specifically modulate the subcellular functions of β-catenin, we expressed the RNA aptamer in the form of RNA intramers with unique cellular localizations. The nucleus-expressed RNA intramer proved to be effective in reducing the protein-protein interaction between β-catenin and TCF-4, thus shown to be a specific regulator of β-catenin–activated transcription. It could also regulate the alternative splicing of E1A minigene in diverse colon cancer cell lines. In addition, we tested whether β-catenin could stabilize any other mRNAs and found that cyclin D1 mRNA was also bound and stabilized by β-catenin. Significantly, the cytoplasm-expressed RNA intramer reverted the β-catenin–induced COX-2 and cyclin D1 mRNA stabilization. We show here that β-catenin regulated multiple steps of RNA metabolism in colon cancer cells and might be the protein factor coordinating RNA metabolism. We suggest that the RNA intramers could provide useful ways for inhibiting β-catenin–mediated transcription and RNA metabolism, which might further enhance the antitumorigenic effects of these molecules in colon cancer cells. [Cancer Res 2007;67(19):9315–20]
Cisplatin Depletes TREX2 and Causes Robertsonian Translocations as Seen in TREX2 Knockout Cells
Authors: Chen Ming-Jiu , Dumitrache Lavinia C. , Wangsa Danny , Ma Sheng-Mei , Padilla-Nash Hesed , Ried Thomas , Hasty Paul
Published: October 2007

Cisplatin, an anticancer drug, forms DNA interstrand cross-links (ICL) that interfere with replication, whereas TREX2 is a 3′→5′ exonuclease that removes 3′ mismatched nucleotides and promotes cellular proliferation. Here, we show that TREX2 is depleted in human cells derived from cancer after exposure to cisplatin but not other genotoxins including another cross-linking agent, mitomycin C (MMC), indicating a potential role for TREX2 depletion in cisplatin-induced cytotoxicity. To better understand TREX2 cellular function, we deleted TREX2 in mouse embryonic stem (ES) cells by gene targeting and find these cells exhibit reduced proliferation and gross chromosomal rearrangements including Robertsonian translocations (RbT). Quite interestingly, ES cells exposed to cisplatin also exhibit RbTs. By contrast, RbTs are not observed for ES cells exposed to MMC, indicating that RbTs are not caused by ICLs but instead TREX2 depletion by either cisplatin exposure or mutation. Taken together, our results show that cisplatin depletes TREX2 and causes genomic instability that is similarly observed in TREX2-mutant cells. Thus, cisplatin has two potential cytotoxic activities: (a) the generation of ICLs and (b) the depletion of TREX2. [Cancer Res 2007;67(19):9077–83]
c-Fos as a Proapoptotic Agent in TRAIL-Induced Apoptosis in Prostate Cancer Cells
Authors: Zhang Xiaoping , Zhang Liang , Yang Hongmei , Huang Xu , Otu Hasan , Libermann Towia A. , DeWolf William C. , Khosravi-Far Roya , Olumi Aria F.
Published: October 2007

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)/Apo-2L promotes apoptosis in cancer cells while sparing normal cells. Although many cancers are sensitive to TRAIL-induced apoptosis, some evade the proapoptotic effects of TRAIL. Therefore, differentiating molecular mechanisms that distinguish between TRAIL-sensitive and TRAIL-resistant tumors are essential for effective cancer therapies. Here, we show that c-Fos functions as a proapoptotic agent by repressing the antiapoptotic molecule c-FLIP(L). c-Fos binds the c-FLIP(L) promoter, represses its transcriptional activity, and reduces c-FLIP(L) mRNA and protein levels. Therefore, c-Fos is a key regulator of c-FLIP(L), and activation of c-Fos determines whether a cancer cell will undergo cell death after TRAIL treatment. Strategies to activate c-Fos or inhibit c-FLIP(L) may potentiate TRAIL-based proapoptotic therapies. [Cancer Res 2007;67(19):9425–34]
A Placenta-Specific Gene Ectopically Activated in Many Human Cancers Is Essentially Involved in Malignant Cell Processes
Authors: Koslowski Michael , Sahin Ugur , Mitnacht-Kraus Rita , Seitz Gerhard , Huber Christoph , Türeci Özlem
Published: October 2007

The identification and functional characterization of tumor-specific genes is a prerequisite for the development of targeted cancer therapies. Using an integrated data mining and experimental validation approach for the discovery of new targets for antibody therapy of cancer, we identified PLAC1. PLAC1 is a placenta-specific gene with no detectable expression in any other normal human tissue. However, it is frequently aberrantly activated and highly expressed in a variety of tumor types, in particular breast cancer. RNAi-mediated silencing of PLAC1 in MCF-7 and BT-549 breast cancer cells profoundly impairs motility, migration, and invasion and induces a G1-S cell cycle block with nearly complete abrogation of proliferation. Knockdown of PLAC1 is associated with decreased expression of cyclin D1 and reduced phosphorylation of AKT kinase. Moreover, PLAC1 is localized on the surface of cancer cells and is accessible for antibodies which antagonize biological functions of this molecule. These features, in summary, make PLAC1 an attractive candidate for targeted immunotherapeutic approaches. [Cancer Res 2007;67(19):9528–34]
Cell Cycle–Dependent and Schedule-Dependent Antitumor Effects of Sorafenib Combined with Radiation
Authors: Plastaras John P. , Kim Seok-Hyun , Liu Yingqiu Y. , Dicker David T. , Dorsey Jay F. , McDonough James , Cerniglia George , Rajendran Ramji R. , Gupta Anjali , Rustgi Anil K. , Diehl J. Alan , Smith Charles D. , Flaherty Keith T. , El-Deiry Wafik S.
Published: October 2007

The antineoplastic drug sorafenib (BAY 43-9006) is a multikinase inhibitor that targets the serine-threonine kinase B-Raf as well as several tyrosine kinases. Given the numerous molecular targets of sorafenib, there are several potential anticancer mechanisms of action, including induction of apoptosis, cytostasis, and antiangiogenesis. We observed that sorafenib has broad activity in viability assays in several human tumor cell lines but selectively induces apoptosis in only some lines. Sorafenib was found to decrease Mcl-1 levels in most cell lines tested, but this decrease did not correlate with apoptotic sensitivity. Sorafenib slows cell cycle progression and prevents irradiated cells from reaching and accumulating at G2-M. In synchronized cells, sorafenib causes a reversible G1 delay, which is associated with decreased levels of cyclin D1, Rb, and phosphorylation of Rb. Although sorafenib does not affect intrinsic radiosensitivity using in vitro colony formation assays, it significantly reduces colony size. In HCT116 xenograft tumor growth delay experiments in mice, sorafenib alters radiation response in a schedule-dependent manner. Radiation treatment followed sequentially by sorafenib was found to be associated with the greatest tumor growth delay. This study establishes a foundation for clinical testing of sequential fractionated radiation followed by sorafenib in gastrointestinal and other malignancies. [Cancer Res 2007;67(19):9443–54]
The Cooked Meat–Derived Genotoxic Carcinogen 2-Amino-3-Methylimidazo[4,5-b]Pyridine Has Potent Hormone-Like Activity: Mechanistic Support for a Role in Breast Cancer
Authors: Lauber Sandra N. , Gooderham Nigel J.
Published: October 2007

The cooked meat–derived heterocyclic amine 2-amino-3-methylimidazo[4,5-b]pyridine (PhIP) is activated by CYP1A2 to the N-hydroxy metabolite, then esterified by acetyl transferase and sulfur transferase into unstable DNA-reactive products that can lead to mutation. The genotoxicity of PhIP has been implicated in its carcinogenicity. Yet, CYP1A2-null mice are still prone to PhIP-mediated cancer, inferring that alternative mechanisms must be operative in tumor induction. PhIP induces tumors of the breast, prostate, and colon in rats and lymphoma in mice. This profile of carcinogenicity is indicative of hormonal involvement. We recently reported that PhIP has potent estrogenic activity inducing transcription of estrogen (E2)-regulated genes, proliferation of E2-dependent cells, up-regulation of progesterone receptor, and stimulation of mitogen-activated protein kinase signaling. In this report, we show for the first time that PhIP at doses as low as of 10−11 mol/L has direct effects on a rat pituitary lactotroph model (GH3 cells) and is able to induce cell proliferation and the synthesis and secretion of prolactin. This PhIP-induced pituitary cell proliferation and synthesis and secretion of prolactin can be attenuated by an estrogen receptor (ER) inhibitor, implying that PhIP effects on lactotroph responses are ERα mediated. In view of the strong association between estrogen, progesterone, prolactin, and breast cancer, the PhIP repertoire of hormone-like activities provides further mechanistic support for the tissue-specific carcinogenicity of the chemical. Furthermore, the recent epidemiology studies that report an association between consumption of cooked red meat and premenopausal and postmenopausal human breast cancer are consonant with these observations. [Cancer Res 2007;67(19):9597–602]
Inhibition of the Proteasome Activity by Gallium(III) Complexes Contributes to Their Anti–Prostate Tumor Effects
Authors: Chen Di , Frezza Michael , Shakya Rajendra , Cui Qiuzhi Cindy , Milacic Vesna , Verani Claudio N. , Dou Q. Ping
Published: October 2007

The investigation of metal-based complexes with potential antitumor activity has been of paramount importance in recent years due to the successful use of cisplatin against various cancers. Gallium(III) and subsequently developed gallium(III)-containing complexes have shown promising antineoplastic effects when tested in a host of malignancies, specifically in lymphomas and bladder cancer. However, the molecular mechanism responsible for their anticancer effect is yet to be fully understood. We report here for the first time that the proteasome is a molecular target for gallium complexes in a variety of prostate cancer cell lines and in human prostate cancer xenografts. We tested five gallium complexes (1–5) in which the gallium ion is bound to an NN′O asymmetrical ligand containing pyridine and substituted phenolate moieties in a 1:2 (M/L) ratio. We found that complex 5 showed superior proteasome inhibitory activity against both 26S proteasome (IC50, 17 μmol/L) and purified 20S (IC50, 16 μmol/L) proteasome. Consistently, this effect was associated with apoptosis induction in prostate cancer cells. Additionally, complex 5 was able to exert the same effect in vivo by inhibiting growth of PC-3 xenografts in mice (66%), which was associated with proteasome inhibition and apoptosis induction. Our results strongly suggest that gallium complexes, acting as potent proteasome inhibitors, have a great potential to be developed into novel anticancer drugs. [Cancer Res 2007;67(19):9258–65]
Insulin Receptor Substrate-1 Is an Important Mediator of Ovarian Cancer Cell Growth Suppression by All-trans Retinoic Acid
Authors: Ravikumar Sharada , Perez-Liz Georgina , Del Vale Luis , Soprano Dianne Robert , Soprano Kenneth J.
Published: October 2007

There is a need to identify more effective drugs for the treatment of ovarian cancer as it is the leading cause of death among gynecologic tumors. All-trans retinoic acid (ATRA), a natural retinoid, arrests the growth of CA-OV3 ovarian carcinoma cells in G0-G1. Because the insulin-like growth factor-I receptor has been implicated in the proliferation of various tumors, we investigated its potential role in the suppression of ovarian cancer cell growth by ATRA. Our studies revealed that insulin receptor substrate-1 (IRS-1) protein levels decrease in CA-OV3 cells on ATRA treatment, whereas no differences in IRS-1 levels were seen in the ATRA-resistant SK-OV3 cells. Moreover, CA-OV3 clones overexpressing IRS-1 were growth inhibited less by ATRA, whereas SK-OV3 clones in which levels of IRS-1 were reduced by expression of antisense IRS-1 became sensitive to growth inhibition by ATRA treatment. Studies to determine the mechanism by which ATRA reduced IRS-1 expression showed that ATRA altered steady-state levels of IRS-1 mRNA and the stability of IRS-1 protein. Finally, the role of IRS-1 as a potential molecular target of ATRA in ovarian tumors was assessed by immunohistochemistry in an ovarian cancer tissue array. Compared with normal ovary, the majority of malignant epithelial ovarian tumors overexpressed IRS-1. Thus, there seems to be a correlation between IRS-1 expression and malignancy in ovarian tumors. Our results suggest that IRS-1 is in fact an important growth-regulatory molecule that can be a potential effective target for chemotherapeutic intervention with growth-suppressive agents, including retinoids. [Cancer Res 2007;67(19):9266–75]
Association of Wwox with ErbB4 in Breast Cancer
Authors: Aqeilan Rami I. , Donati Valentina , Gaudio Eugenio , Nicoloso Milena S. , Sundvall Maria , Korhonen Anna , Lundin Johan , Isola Jorma , Sudol Marius , Joensuu Heikki , Croce Carlo M. , Elenius Klaus
Published: October 2007

WWOX, WW domain-containing oxidoreductase, is a tumor suppressor that is altered in many human cancers, including breast cancer. Wwox interacts with the ErbB4 receptor, reduces nuclear translocation of the cleaved intracellular domain of ErbB4, and inhibits its transactivation function mediated through Yes-associated protein. Here, we assessed the clinical significance of the Wwox-ErbB4 association. We determined Wwox protein expression by immunohistochemistry in a series of 556 breast cancers. Wwox expression was absent in 36% of the cancers, and loss of Wwox expression was associated with unfavorable outcome (P = 0.02). Membranous location of ErbB4 was associated with favorable survival compared with women whose cancer lacked such ErbB4 expression (P = 0.02). Wwox expression was strongly associated with membranous ErbB4 localization (P = 0.0003) and with overall ErbB4 expression (P = 0.0002). Coexpression of membranous ErbB4 and Wwox was associated with favorable outcome compared with cases with membranous ErbB4 and no Wwox immunoreactivity (P = 0.002). In vitro, Wwox associated with the two ErbB4 isoforms, JM-a CYT-1 and JM-a CYT-2, expressed in breast cancer. Moreover, expression of Wwox both in vitro and in vivo led to accumulation of total full-length membrane-associated ErbB4. These results suggest that expression of Wwox is associated with ErbB4 expression and that their coexpression has prognostic significance in breast cancer. [Cancer Res 2007;67(19):9330–6]
Prolonged Exposure to Reduced Levels of Androgen Accelerates Prostate Cancer Progression in Nkx3.1; Pten Mutant Mice
Authors: Banach-Petrosky Whitney , Jessen Walter J. , Ouyang Xuesong , Gao Hui , Rao Jayashree , Quinn John , Aronow Bruce J. , Abate-Shen Cory
Published: October 2007

In this report, we have investigated the relationship between androgen levels and prostate tumorigenesis in Nkx3.1; Pten mutant mice, a genetically engineered mouse model of human prostate cancer. By experimentally manipulating serum levels of testosterone in these mice for an extended period (i.e., 7 months), we have found that prolonged exposure of Nkx3.1; Pten mutant mice to androgen levels that are 10-fold lower than normal (the “Low-T” group) resulted in a marked acceleration of prostate tumorigenesis compared with those exposed to androgen levels within the reference range (the “Normal-T” group). We found that prostate tumors from the Low-T mutant mice share a similar gene expression profile as androgen-independent prostate tumors from these mutant mice, which includes the deregulated expression of several genes that are up-regulated in human hormone-refractory prostate cancer, such as Vav3 and Runx1. We propose that exposure to reduced androgens may promote prostate tumorigenesis by selecting for molecular events that promote more aggressive, hormone-refractory tumors. [Cancer Res 2007;67(19):9089–96]
The AA Genotype of the Regulatory BCL2 Promoter Polymorphism (−938C>A) Is Associated with a Favorable Outcome in Lymph Node–Negative Invasive Breast Cancer Patients
Authors: Bachmann Hagen S. , Otterbach Friedrich , Callies Rainer , Nückel Holger , Bau Maja , Schmid Kurt W. , Siffert Winfried , Kimmig Rainer
Published: October 2007

Purpose: Expression of the antiapoptotic and antiproliferative protein Bcl-2 has been repeatedly shown to be associated with better clinical outcome in breast cancer. We recently showed a novel regulatory (−938C>A) single-nucleotide polymorphism (SNP) in the inhibitory P2 BCL2 gene promoter generating significantly different BCL2 promoter activities.

Experimental Design: Paraffin-embedded neoplastic and nonneoplastic tissues from 274 patients (161 still alive after a follow-up period of at least 80 months) with primary unilateral invasive breast carcinoma were investigated. Bcl-2 expression of tumor cells was shown by immunohistochemistry; nonneoplastic tissues were used for genotyping. Both the Bcl-2 expression and the (−938C>A) genotypes were correlated with the patients' survival.

Results: Kaplan-Meier curves revealed a significant association of the AA genotype with increased survival (P = 0.030) in lymph node–negative breast cancer patients, whereas no genotype effect could be observed in lymph node–positive cases. Ten-year survival rates were 88.6% for the AA genotype, 78.4% for the AC genotype, and 65.8% for the CC genotype. Multivariable Cox regression identified the BCL2 (−938CC) genotype as an independent prognostic factor for cancer-related death in lymph node–negative breast carcinoma patients (hazard ratio, 3.59; P = 0.032). Immunohistochemical Bcl-2 expression was significantly associated with the clinical outcome of lymph node–positive but not of lymph node–negative breast cancer patients. In lymph node–negative cases, the (−938C>A) SNP was both significantly related with the immunohistochemically determined level of Bcl-2 expression (P = 0.044) and the survival of patients with Bcl-2–expressing carcinomas (P = 0.006).

Conclusions: These results suggest the (−938C>A) polymorphism as a survival prognosticator as well as indicator of a high-risk group within patients with lymph node–negative breast cancer.
Preclinical Testing of Clinically Applicable Strategies for Overcoming Trastuzumab Resistance Caused by PTEN Deficiency
Authors: Lu Chien-Hsing , Wyszomierski Shannon L. , Tseng Ling-Ming , Sun Meng-Hong , Lan Keng-Hsueh , Neal Christopher L. , Mills Gordon B. , Hortobagyi Gabriel N. , Esteva Francisco J. , Yu Dihua
Published: October 2007

Purpose: We have previously shown that PTEN loss confers trastuzumab resistance in ErbB2-overexpressing breast cancer using cell culture, xenograft models, and patient samples. This is a critical clinical problem because trastuzumab is used in a variety of therapeutic regimens, and at the current time, there are no established clinical strategies to overcome trastuzumab resistance. Here, we did preclinical studies on the efficacy of clinically applicable inhibitors of the Akt/mammalian target of rapamycin (mTOR) pathway to restore trastuzumab sensitivity to PTEN-deficient cells.

Experimental Design: Cell culture and xenograft models were used to test a panel of clinically applicable, small-molecule inhibitors of the Akt/mTOR signal transduction pathway, a critical pathway downstream of ErbB2, and identify compounds with the ability to restore trastuzumab sensitivity to PTEN-deficient cells.

Results: When trastuzumab was combined with the Akt inhibitor triciribine, breast cancer cell growth was inhibited and apoptosis was induced. In a xenograft model, combination therapy with trastuzumab and triciribine dramatically inhibited tumor growth. The combination of trastuzumab and the mTOR inhibitor RAD001 also slowed breast cancer cell growth in vitro and in vivo.

Conclusions: Combining trastuzumab with inhibitors of the Akt/mTOR pathway is a clinically applicable strategy and combinations of trastuzumab with triciribine or RAD001 are promising regimens for rescue of trastuzumab resistance caused by PTEN loss.
CMTM5 Exhibits Tumor Suppressor Activities and Is Frequently Silenced by Methylation in Carcinoma Cell Lines
Authors: Shao Luning , Cui Yan , Li Hongyu , Liu Yanan , Zhao Hongshan , Wang Yu , Zhang Yingmei , Ng Ka Man , Han Wenling , Ma Dalong , Tao Qian
Published: October 2007

Purpose: CMTM5 (CKLF-like MARVEL transmembrane domain containing member 5) is located at 14q11.2, a locus associated with multiple cancers. It has six RNA splicing variants with CMTM5-v1 as the major one. We explored its expression pattern in normal tissues and tumor cell lines, as well as its functions in carcinoma cells.

Experimental Design: We evaluated CMTM5 expression by semiquantitative reverse transcription-PCR (RT-PCR) in normal tissues and carcinoma cell lines of cervical, breast, nasopharyngeal, lung, hepatocellular, esophageal, gastric, colon, and prostate. We further examined CMTM5 promoter methylation in these cell lines. We also analyzed CMTM5 expression after 5-aza-2′-deoxycytidine treatment and genetic demethylation and the functional consequences of restoring CMTM5 in HeLa and PC-3 cells.

Results: CMTM5-v1 is broadly expressed in human normal adult and fetal tissues, but undetectable or down-regulated in most carcinoma cell lines. Its promoter methylation was detected in virtually all the silenced or down-regulated cell lines. The silencing of CMTM5 could be reversed by pharmacologic demethylation or genetic double-knockout of DNMT1 and DNMT3B, indicating methylation-mediated mechanism. Restoration of CMTM5-v1 suppressed carcinoma cell proliferation, migration, and invasion.

Conclusions: These results indicate that CMTM5 exhibits tumor suppressor activities, but with frequent epigenetic inactivation in carcinoma cell lines.
Imaging and Modulating Antisense Microdistribution in Solid Human Xenograft Tumor Models
Authors: Mocanu Joseph D. , Yip Kenneth W. , Skliarenko Julia , Shi Wei , Busson Pierre , Lo Kwok-Wai , Bastianutto Carlo , Liu Fei-Fei
Published: October 2007

Purpose: The tumor microenvironment is complex and heterogeneous, populated by tortuous irregular vasculature, hypoxic cells, and necrotic regions. These factors can all contribute to the biodistribution difficulties encountered by most cancer therapeutic agents. Antisense oligodeoxynucleotides (ASO) are a class of therapeutics where limited information is available about their distribution within a solid tumor environment.

Experimental Design: To assess ASO distribution, a fluorescein-labeled phosphorothionated ASO based on the G3139 mismatch control was injected systemically (i.v.) into tumor-bearing severe combined immunodeficient mice. Hoechst 33342 was injected i.v. to visualize active vasculature. Unstained sections were imaged through tiled fluorescence stereomicroscopy and then quantitated using novel algorithms. Tumor sections from four human tumor models were examined (CaSki, DU-145, C666-1, and C15) for hypoxia, apoptosis/necrosis, and morphology.

Results: For all four tumors, ASO accumulated within regions of hypoxia, necrosis, and apoptosis. Scatter plots of ASO versus active vasculature generated for each individual tumor revealed a consistent pattern of distribution of the ASO within each model. In C666-1 xenografts, the slopes of these scatter plots were significantly reduced from 0.41 to 0.16 when pretreated with the antivascular agent ZD6126 48 h before ASO injection. This was accompanied by the formation of large disseminated necrotic regions in the tumor, along with a 13.1 mmHg reduction in interstitial fluid pressure.

Conclusions: These data suggest the possibility that these algorithms might offer a generalizable and objective methodology to describe the distribution of molecular therapeutic agents within a tumor microenvironment and to quantitatively assess distribution changes in response to combination therapies.
Short Telomeres: A Novel Potential Predictor of Relapse in Ewing Sarcoma
Authors: Avigad Smadar , Naumov Inna , Ohali Anat , Jeison Marta , Berco Gili Halevy , Mardoukh Jacques , Stark Batia , Ash Shifra , Cohen Ian J. , Meller Isaac , Kollender Yehuda , Issakov Josephine , Yaniv Isaac
Published: October 2007

Purpose: Despite advances in therapy, >50% of patients with Ewing sarcoma will relapse. The current prognostic factors are not optimal for risk prediction. Studies have shown that telomere length could predict outcome in different malignancies. Our aim was to evaluate whether telomere length could be a better prognostic factor in Ewing sarcoma and correlate the results with clinical variables, outcome, and chromosomal instability.

Experimental Design: Telomere length was determined in the primary tumor and peripheral blood of 32 patients with Ewing sarcoma. Chromosomal instability was evaluated by combining classical cytogenetics, comparative genomic hybridization and random aneuploidy. Telomere length was correlated to clinical variables, chromosomal instability, and outcome.

Results: In 75% of the tumors, changes in telomere length, when compared with the corresponding peripheral blood lymphocytes, were noted. The majority of changes consisted of a reduction in telomere length. Patients harboring shorter telomeres had a significantly adverse outcome (P = 0.015). Chromosomal instability was identified in 65% of tumors, significantly correlating with short telomeres (P = 0.0094). Using multivariate analysis, telomere length remained the only significant prognostic variable (P = 0.034). Patients with short telomeres had a 5.3-fold risk of relapse as compared to those with unchanged or longer telomeres.

Conclusion: We have shown that tumors with telomere length reduction result in genomic instability. In addition, telomere length reduction was the only significant predictor of outcome. We suggest that reduction of telomere length in tumor cells at diagnosis could serve as a prognostic marker in Ewing sarcoma.
Caspase-3 Activity Predicts Local Recurrence in Rectal Cancer
Authors: de Heer Pieter , de Bruin Elza C. , Klein-Kranenbarg Elma , Aalbers Remco I.J.M. , Marijnen Corrie A.M. , Putter Hein , de Bont Hans J. , Nagelkerke J. Fred , van Krieken J. Han J.M. , Verspaget Hein W. , van de Velde Cornelis J.H. , Kuppen Peter J.K. ,   for the Dutch Colorectal Cancer Group
Published: October 2007

Purpose: Radiotherapy followed by total mesorectal excision surgery has been shown to significantly reduce local recurrence rates in rectal cancer patients. Radiotherapy, however, is associated with considerable morbidity. The present study evaluated the use of biochemical detection of enzymatic caspase-3 activity as preoperative marker for apoptosis to preselect patients that are unlikely to develop a local recurrence to spare these patients from overtreatment and the negative side effects of radiotherapy.

Experimental Design: Nonirradiated freshly frozen tissue samples from 117 stage III rectal cancer patients were collected from a randomized clinical trial that evaluated preoperative radiotherapy in total mesorectal excision surgery. Additional frozen archival tissues from 47 preoperative biopsies and corresponding resected colorectal tumors were collected. Level of apoptosis was determined by measuring the enzymatic activity of caspase-3 in a biochemical assay.

Results: In tumor tissue, caspase-3 activity lower than the median was predictive of 5-year local recurrence (hazard ratio, 7.4; 95% confidence interval, 1.7-32.8; P = 0.008), which was unaffected by adjustment for type of resection, tumor location, and T status (adjusted hazard ratio, 7.5; 95% confidence interval, 1.7-34.1; P = 0.009). Caspase-3 activity in preoperative biopsies was significantly correlated with caspase-3 activity in corresponding resected tumors (r = 0.56; P < 0.0001).

Conclusion: Detection of tumor apoptosis levels by measuring caspase-3 activity, for which a preoperative biopsy can be used, accurately predicted local recurrence in rectal cancer patients. These findings indicate that caspase-3 activity is an important denominator of local recurrence and should be evaluated prospectively to be added to the criteria to select rectal cancer patients in which radiotherapy is redundant.
Inhibition of Jun NH2-Terminal Kinases Suppresses the Growth of Experimental Head and Neck Squamous Cell Carcinoma
Authors: Gross Neil D. , Boyle Jay O. , Du Baoheng , Kekatpure Vikram D. , Lantowski Agnieszka , Thaler Howard T. , Weksler Babette B. , Subbaramaiah Kotha , Dannenberg Andrew J.
Published: October 2007

Purpose: This study was carried out to investigate whether c-Jun NH2-terminal kinases (JNK) are potential targets for treating head and neck squamous cell carcinoma (HNSCC).

Experimental Design: JNK activity was first evaluated in 20 paired samples of human HNSCC. The antitumor activity of SP600125, a reversible nonselective ATP-competitive inhibitor of JNKs, was then investigated both in an HNSCC xenograft model and in vitro using immunohistochemistry, immunoblotting, enzyme immunoassay, flow cytometry, and a Matrigel assay of capillary tube formation. Complementary studies were carried out using small interfering RNA to JNK1/2.

Results: JNK activity was increased in human HNSCC compared with normal-appearing epithelium. Treatment of mice bearing HNSCC xenografts with SP600125 resulted in >60% inhibition of tumor growth relative to vehicle-treated animals. Inhibition of tumor growth was associated with significant reductions in both cell proliferation and microvessel density. SP600125 inhibited tumor cell proliferation by causing delays in both the S and G2-M phases of the cell cycle. Inhibition of angiogenesis seemed to reflect effects on both tumor and endothelial cells. The JNK inhibitor suppressed the production of vascular endothelial growth factor and interleukin-8 by tumor cells and also inhibited endothelial cell proliferation and capillary tube formation. Reduced amounts and phosphorylation of epidermal growth factor receptor were found in tumor cells after treatment with SP600125. Small interfering RNA–mediated suppression of JNK1/2 led to reduced tumor cell proliferation and decreased levels of epidermal growth factor receptor, vascular endothelial growth factor, and interleukin-8.

Conclusions: JNK activity is commonly increased in HNSCC. Our preclinical results provide a rationale for evaluating JNK inhibition as an approach to treating HNSCC.
A Novel Nuclear Factor-κB Gene Signature Is Differentially Expressed in Head and Neck Squamous Cell Carcinomas in Association with TP53 Status
Authors: Lee Tin Lap , Yang Xin Ping , Yan Bin , Friedman Jay , Duggal Praveen , Bagain Lorena , Dong Gang , Yeh Ning T. , Wang Jie , Zhou Jian , Elkahloun Abdel , Van Waes Carter , Chen Zhong
Published: October 2007

Purpose: To determine if gene signatures differentially expressed in head and neck squamous cell carcinomas (HNSCC) are related to alterations in transcription factors nuclear factor-κB (NF-κB) and TP53 previously associated with decreased cell death, response to therapy, and worse prognosis.

Experimental Design: Unique gene signatures expressed by HNSCC lines were identified by cDNA microarray, principal components, and cluster analyses and validated by quantitative reverse transcription-PCR (RT-PCR) and in situ hybridization. Bioinformatic analysis of the promoters and ontogeny of these clustered genes was done. Expression of proteins encoded by genes of a putative NF-κB signature, NF-κB p65, and TP53 were examined in HNSCC tissue specimens by immunostaining. Predicted promoter binding and modulation of expression of candidate NF-κB genes and cell survival were evaluated by p65 chromatin immunoprecipitation (ChIP) and small interfering RNA (siRNA) knockdown.

Results: Two groups of HNSCC exhibiting distinct gene signatures were identified: cluster A enriched for histone genes, with a higher prevalence of TP53 promoter binding motifs; and cluster B enriched for injury response genes with NF-κB regulatory motifs. Coexpression of cluster B proteins was observed with strong NF-κB phospho-p65 and weak TP53 staining, and NF-κB phospho-p65 was inversely associated with TP53 (P = 0.02). Promoter binding of the NF-κB signature genes was confirmed by p65 ChIP, and down-modulation of their expression and cell death were induced by p65 siRNA.

Conclusion: NF-κB promotes expression of a novel NF-κB–related gene signature and cell survival in HNSCC that weakly express TP53, a subset previously associated with inactivated wild-type TP53, greater resistance to chemoradiotherapy, and worse prognosis.