bluuebunny/crossref_metadata_2025 huggingface dataset:
Down-regulation of Mammalian Sterile 20–Like Kinase 1 by Heat Shock Protein 70 Mediates Cisplatin Resistance in Prostate Cancer Cells
Authors: Ren Aixia , Yan Guijun , You Bei , Sun Jianxin
Published: April 2008

Mammalian sterile 20–like kinase 1 (Mst1) is an ubiquitously expressed serine/threonine kinase, and its activation results in cell apoptosis. Recent studies suggest that Mst1 may function as a tumor suppressor. Here, we reported that heat shock protein 70 (Hsp70), which is thought to protect cells against cellular stress, has been identified as an Mst1-interacting protein, in a yeast two-hybrid screen of human adult prostate cDNA library with a dominant-negative Mst1 (K59R) as bait. The interaction of Mst1 with Hsp70 was confirmed by coimmunoprecipitation in both cotransfected HEK293 cells and prostate cancer cells. Hsp70 colocalized with Mst1 in the cytoplasm of LNCaP cells. The interaction sites with Mst1 consisted of NH2-terminal ATPase domain in Hsp70, whereas the inhibitory domain of Mst1 mediates the binding of Hsp70 in Mst1. Overexpression of Hsp70 mediates proteasomal degradation of Mst1 in a Hsp70 interacting protein (CHIP)-dependent manner. Furthermore, the proapoptotic effect of Mst1 was markedly inhibited by overexpression of Hsp70 or CHIP. Most strikingly, in response to the treatment of anticancer drug cisplatin, the induction of Hsp70 expression is higher in the androgen-independent DU145 cells compared with the androgen-dependent LNCaP cells. The higher levels of Hsp70 induction and subsequent Mst1 degradation mediate cisplatin resistance in prostate cancer DU145 cells. Moreover, overexpression of Mst1 sensitizes prostate cancer cells to cisplatin treatment. These findings implicate that Mst1, a downstream target of Hsp70, may be developed as a target for sensitizing hormone-refractory prostate cancers to chemotherapy. [Cancer Res 2008;68(7):2266–74]
A Novel Lung Cancer Signature Mediates Metastatic Bone Colonization by a Dual Mechanism
Authors: Vicent Silvestre , Luis-Ravelo Diego , Antón Iker , García-Tuñón Ignacio , Borrás-Cuesta Francisco , Dotor Javier , De Las Rivas Javier , Lecanda Fernando
Published: April 2008

Bone is a frequent target of lung cancer metastasis, which is associated with significant morbidity and a dismal prognosis. To identify and functionally characterize genes involved in the mechanisms of osseous metastasis, we developed a murine lung cancer model. Comparative transcriptomic analysis identified genes encoding signaling molecules (such as TCF4 and PRKD3) and cell anchorage–related proteins (MCAM and SUSD5), some of which were basally modulated by transforming growth factor-β (TGF-β) in tumor cells and in conditions mimicking tumor-stromal interactions. Triple gene combinations induced not only high osteoclastogenic activity but also a marked enhancement of global metalloproteolytic activities in vitro. These effects were strongly associated with robust bone colonization in vivo, whereas this gene subset was ineffective in promoting local tumor growth and cell homing activity to bone. Interestingly, global inhibition of metalloproteolytic activities and simultaneous TGF-β blockade in vivo led to increased survival and a remarkable attenuation of bone tumor burden and osteolytic metastasis. Thus, this metastatic gene signature mediates bone matrix degradation by a dual mechanism of induction of TGF-β–dependent osteoclastogenic bone resorption and enhancement of stroma-dependent metalloproteolytic activities. Our findings suggest the cooperative contribution of host-derived and cell autonomous effects directed by a small subset of genes in mediating aggressive osseous colonization. [Cancer Res 2008;68(7):2275–85]
Enhancing Poxvirus Oncolytic Effects through Increased Spread and Immune Evasion
Authors: Kirn David H. , Wang Yaohe , Liang Wenchung , Contag Christopher H. , Thorne Stephen H.
Published: April 2008

The antitumoral effects of oncolytic viruses have generally been limited by inefficient spread of the viruses within infected tumors and by inefficient systemic delivery, particularly in preimmunized hosts. Tumor-selective poxviruses have biological characteristics that may overcome these limitations. Nevertheless, physical barriers within the tumor microenvironment, including the extracellular matrix, can still limit intratumoral spread, and neutralizing antibodies can impede systemic delivery. To counter these limitations, we sought to take advantage of a naturally occurring poxvirus form known as extracellular enveloped virus (EEV). The EEV is shrouded by a host cell–derived lipid bilayer containing anticomplement proteins and is typically released from infected cells early during the infection cycle. Therefore, the EEV form evolved for rapid systemic spread within the host and for evasion of immune-mediated clearance. We compared the oncolytic potential of low versus high EEV-producing strains of vaccinia. EEV-enhanced vaccinia strains displayed improved spread within tumors after systemic delivery, resulting in significantly improved antitumor effects. The EEV-enhanced strains also displayed a greater ability to spread between injected and noninjected distant tumors through the blood and, importantly, displayed reduced clearance by neutralizing antibody. Safety was unaffected. The incorporation of EEV-enhancing mutations into next generation oncolytic vaccinia strains may improve the potency of these viruses without sacrificing safety. [Cancer Res 2008;68(7):2071–5]
Bisphenol A Induces a Profile of Tumor Aggressiveness in High-Risk Cells from Breast Cancer Patients
Authors: Dairkee Shanaz H. , Seok Junhee , Champion Stacey , Sayeed Aejaz , Mindrinos Michael , Xiao Wenzhong , Davis Ronald W. , Goodson William H.
Published: April 2008

Breast cancer outcome is highly variable. Whether inadvertent exposure to environmental xenobiotics evokes a biological response promoting cancer aggressiveness and a higher probability of tumor recurrence remains unknown. To determine specific molecular alterations which arise in high-risk breast tissue in the presence of the ubiquitous xenoestrogen, bisphenol A (BPA), we used nonmalignant random periareolar fine-needle aspirates in a novel functional assay. Early events induced by BPA in epithelial-stromal cocultures derived from the contralateral tissue of patients with breast cancer included gene expression patterns which facilitate apoptosis evasion, endurance of microenvironmental stress, and cell cycle deregulation without a detectable increase in cell numbers. This BPA response profile was significantly associated with breast tumors characterized by high histologic grade (P < 0.001) and large tumor size (P = 0.002), resulting in decreased recurrence-free patient survival (P < 0.001). Our assays show a biological “fingerprint” of probable prior exposure to endocrine-disrupting agents, and suggest a scenario in which their presence in the microenvironmental milieu of high-risk breast tissue could play a deterministic role in establishing and maintaining tumor aggressiveness and poor patient outcome. [Cancer Res 2008;68(7):2076–80]
Development of Novel Tetravalent Anti-CD20 Antibodies with Potent Antitumor Activity
Authors: Li Bohua , Shi Shu , Qian Weizhu , Zhao Lei , Zhang Dapeng , Hou Sheng , Zheng Lei , Dai Jianxin , Zhao Jian , Wang Hao , Guo Yajun
Published: April 2008

Despite the effectiveness of the anti-CD20 monoclonal antibody (mAb) Rituximab (C2B8) in the treatment of B-cell lymphoma, its efficacy remains variable and often modest. It seems likely that a combination of multiple mechanisms, such as complement-dependent cytotoxicity (CDC) and apoptotic signaling, underlies the therapeutic success of anti-CD20 mAbs. Unfortunately, all the current anti-CD20 mAbs effective in CDC are relatively inactive in signaling cell death and vice versa. In this study, we developed two genetically engineered tetravalent antibodies (TetraMcAb) respectively derived from the anti-CD20 mAbs C2B8 and 2F2. TetraMcAbs, with a molecular mass only 25 kDa higher than native divalent antibodies (DiMcAb), were shown not only to be as effective in mediating CDC and antibody-dependent cellular cytotoxicity against B-lymphoma cells as DiMcAbs but also to have antiproliferative and apoptosis-inducing activity markedly superior to that of DiMcAbs. Interestingly, whereas 2F2 and C2B8 were equally effective in inducing cell growth arrest and apoptosis, the functions of their tetravalent versions, 2F2(ScFvHL)4-Fc and C2B8(ScFvHL)4-Fc, were significantly different. 2F2(ScFvHL)4-Fc exhibited exceptionally more potent antiproliferative and apoptosis-inducing activity than that of C2B8(ScFvHL)4-Fc. Immunotherapeutic studies further showed that 2F2(ScFvHL)4-Fc was far more effective in prolonging the survival of severe combined immunodeficient mice bearing systemic Daudi or Raji tumors than C2B8, 2F2, and C2B8(ScFvHL)4-Fc, suggesting that it might be a promising therapeutic agent for B-cell lymphoma. [Cancer Res 2008;68(7):2400–8]
Treatment of Human Colon Cancer Xenografts with TRA-8 Anti-death Receptor 5 Antibody Alone or in Combination with CPT-11
Authors: Oliver Patsy G. , LoBuglio Albert F. , Zinn Kurt R. , Kim Hyunki , Nan Li , Zhou Tong , Wang Wenquan , Buchsbaum Donald J.
Published: April 2008

Purpose: This study was designed to evaluate the in vitro cytotoxicity and in vivo efficacy of TRA-8, a mouse monoclonal antibody that binds to the DR5 death receptor for tumor necrosis factor–related apoptosis-inducing ligand (also called Apo2L), alone and in combination with CPT-11, against human colon cancer cells and xenografts.

Experimental Design: DR5 expression was assessed on human colon cancer cell lines using flow cytometry, and cellular cytotoxicity after TRA-8 treatment, alone and in combination with SN-38, was determined by measuring cellular ATP levels. Tumor growth inhibition and regression rates of well-established subcutaneous COLO 205, SW948, HCT116, and HT-29 colon cancer xenografts in athymic nude mice treated with TRA-8 or CPT-11 alone and in combination were determined. 99mTc-TRA-8 was used to examine tumor localization of TRA-8 in animals bearing each of the four xenografts. In addition, whole-body biodistribution and imaging was carried out in COLO 205–bearing animals using in vivo single-photon emission computed tomography imaging and tissue counting.

Results: DR5 expression was highest on HCT116, intermediate on SW948 and COLO 205 cells, and lowest on HT-29 cells. COLO 205 cells were the most sensitive to TRA-8–induced cytotoxicity in vitro, SW948 and HCT116 cell lines were moderately sensitive, and HT-29 cells were resistant. Combination treatment with TRA-8 and SN-38 produced additive to synergistic cytotoxicity against all cell lines compared with either single agent. The levels of apoptosis in all cell lines, including HT-29, were increased by combination treatment with SN-38. In vivo, combination therapy with TRA-8 and CPT-11 was superior to either single-agent regimen for three of the xenografts: COLO 205, SW948, and HCT116. COLO 205 tumors were most responsive to therapy with 73% complete regressions after combination therapy. HT-29 cells derived no antitumor efficacy from TRA-8 therapy. Tumor xenografts established from the four colon cancer cell lines had comparable specific localization of 99mTc-TRA-8.

Conclusions: In vitro and in vivo effects of TRA-8 anti-DR5 monoclonal antibody on four different colon cancer cell lines and xenografts were quite variable. The HT-29 cell line had low surface DR5 expression and was resistant to TRA-8 both in vitro and in vivo. Three cell lines (COLO 205, SW948, and HCT116) exhibited moderate to high sensitivity to TRA-8–mediated cytotoxicity which was further enhanced by the addition of SN-38, the active metabolite of CPT-11. In vivo, the combination of TRA-8 and CPT-11 treatment produced the highest antitumor efficacy against xenografts established from the three TRA-8–sensitive tumor cell lines. All four colon cancer xenografts had comparable localization of 99mTc-TRA-8. These studies support the strategy of TRA-8/CPT-11 combined treatment in human colon cancer clinical trials.
Tissue Transglutaminase Regulates Focal Adhesion Kinase/AKT Activation by Modulating PTEN Expression in Pancreatic Cancer Cells
Authors: Verma Amit , Guha Sushovan , Wang Huamin , Fok Jansina Y. , Koul Dimpy , Abbruzzese James , Mehta Kapil
Published: April 2008

Purpose: Pancreatic ductal adenocarcinoma (PDAC) progresses rapidly and exhibits profound resistance to treatment. We recently reported that a great majority of PDAC tumors and tumor cell lines express elevated levels of tissue transglutaminase (TG2). Here, we provide first evidence that TG2 expression in PDAC cells results in constitutive activation of focal adhesion kinase/AKT by modulating the expression of the tumor suppressor phosphatase PTEN.

Experimental Design: Using PDAC cell lines, we determined the effect of TG2 overexpression on PTEN stability and functions. We confirmed the correlation between TG2 expression and PTEN levels in a few (n = 51) PDAC tumor samples.

Results: We observed that expression of TG2 is inversely correlated with PTEN expression in PDAC cells. Ectopic expression of TG2 inhibited PTEN phosphorylation and promoted its degradation by ubiquitin-proteasomal pathway. Conversely, down-regulation of TG2 by small interfering RNA up-regulated PTEN expression. Clinical relevance of these results was evident in an athymic nude mouse model where down-regulation of endogenous TG2 caused a significant retardation in PDAC xenograft growth. Importantly, the analysis of 51 tumor samples from patients with stage II PDAC revealed that overexpression of TG2 was associated with loss of PTEN expression (P = 0.023; odds ratio, 4.1). In multivariate analysis, TG2-mediated loss of PTEN was a prognostic factor for overall survival in patients with stage II pancreatic ductal carcinoma independent of tumor stage/lymph node status and tumor differentiation (P = 0.01).

Conclusion: TG2 expression in PDAC promotes degradation of PTEN by ubiquitin-proteasomal pathway and results in constitutive activation of focal adhesion kinase/AKT cell survival signaling.
Stress, Immunity, and Cervical Cancer: Biobehavioral Outcomes of a Randomized Clinical Trail
Authors: Nelson Edward L. , Wenzel Lari B. , Osann Kathryn , Dogan-Ates Aysun , Chantana Nissa , Reina-Patton Astrid , Laust Amanda K. , Nishimoto Kevin P. , Chicz-DeMet Alexandra , du Pont Nefertiti , Monk Bradley J.
Published: April 2008

Purpose: Cancer diagnosis and treatment imparts chronic stressors affecting quality of life (QOL) and basic physiology. However, the capacity to increase survival by improving QOL is controversial. Patients with cervical cancer, in particular, have severely compromised QOL, providing a population well-suited for the evaluation of novel psychosocial interventions and the exploration of mechanisms by which modulation of the psychoneuroimmune axis might result in improved clinical outcomes.

Experimental Design: A randomized clinical trial was conducted in cervical cancer survivors that were enrolled at ≥13 and <22 months after diagnosis (n = 50), comparing a unique psychosocial telephone counseling (PTC) intervention to usual care. QOL and biological specimens (saliva and blood) were collected at baseline and 4 months post-enrollment.

Results: The PTC intervention yielded significantly improved QOL (P = 0.011). Changes in QOL were significantly associated with a shift of immune system T helper type 1 and 2 (Th1/Th2) bias, as measured by IFN-γ/interleukin-5 ELISpot T lymphocyte precursor frequency; improved QOL being associated with increased Th1 bias (P = 0.012). Serum interleukin-10 and the neuroendocrine variables of cortisol and dehydroepiandrosterone revealed trends supporting this shift in immunologic stance and suggested a PTC-mediated decrease of the subject's chronic stress response.

Conclusions: This study documents the utility of a unique PTC intervention and an association between changes in QOL and adaptive immunity (T helper class). These data support the integration of the chronic stress response into biobehavioral models of cancer survivorship and suggests a novel mechanistic hypotheses by which interventions leading to enhanced QOL could result in improved clinical outcome including survival.
Combination of Vascular Endothelial Growth Factor Receptor/Platelet-Derived Growth Factor Receptor Inhibition Markedly Improves Radiation Tumor Therapy
Authors: Timke Carmen , Zieher Heike , Roth Alexandra , Hauser Kai , Lipson Kenneth E. , Weber Klaus J. , Debus Jürgen , Abdollahi Amir , Huber Peter E.
Published: April 2008

Purpose: Investigations on the combination of radiotherapy with vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) antiangiogenic agents, which has the potential to improve the clinical outcome in cancer patients.

Experimental Design: Here, we analyze the combined VEGF (SU5416) and PDGF (SU6668) receptor tyrosine kinase inhibition with irradiation in human endothelium (HUVEC), prostate cancer (PC3), and glioblastoma (U87) in vitro and in vivo.

Results: Combined inhibition of VEGF and PDGF signaling resulted in enhanced apoptosis, reduced cell proliferation, and clonogenic survival as well as reduced endothelial cell migration and tube formation compared with single pathway inhibition. These effects were further enhanced by additional irradiation. Likewise, in PC3 and U87 tumors growing s.c. on BALB/c nu/nu mice, dual inhibition of VEGF and PDGF signaling significantly increased tumor growth delay versus each monotherapy. Interestingly, radiation at ∼20% of the dose necessary to induce local tumor control exerts similar tumor growth-inhibitory effects as the antiangiogenic drugs given at their maximum effective dose. Addition of radiotherapy to both mono- as well as dual-antiangiogenic treatment markedly increased tumor growth delay. With respect to tumor angiogenesis, radiation further decreased microvessel density (CD31 count) and tumor cell proliferation (Ki-67 index) in all drug-treated groups. Of note, the slowly growing PC3 tumor responded better to the antiangiogenic drug treatments than the faster-growing U87 tumor. In addition to the beneficial effect of abrogating VEGF survival signaling when combined with radiation, we identified here a novel mechanism for the tumor escape from radiation damage. We found that radiation induced up-regulation of all four isoforms of PDGF (A-D) in endothelial cells supporting adjacent smooth muscle cells resulting in a prosurvival effect of radiation. The addition of SU6668 attenuated this undesirable paracrine radiation effect, which may rationalize the combined application of radiation with PDGF signaling inhibition to increase antitumor effects.

Conclusion: A relative low radiation dose markedly enhances local antitumor effects of combined VEGF and PDGF signaling inhibition, suggesting a promising combination regimen for local tumor treatment with radiotherapy remaining an essential element.
Fibroblast Growth Factor 2 and Estrogen Control the Balance of Histone 3 Modifications Targeting MAGE-A3 in Pituitary Neoplasia
Authors: Zhu Xuegong , Asa Sylvia L. , Ezzat Shereen
Published: April 2008

Purpose: Four members of the fibroblast growth factor receptor (FGFR) family transduce signals of a diverse group of FGF ligands. The FGFR2-IIIb isoform is abundantly present in the normal pituitary gland with contrasting down-regulation in neoplastic pituitary cells. cDNA profiling identified the cancer-testis antigen melanoma-associated antigen A3 (MAGE-A3) as a putative target negatively regulated by FGFR2.

Experimental Design: Comparisons were made between normal and neoplastic human and mouse pituitary cells. Gene expression was examined by reverse transcription-PCR, DNA methylation was determined by methylation-specific PCR and combined bisulfite restriction analysis, and histone modification marks were identified by chromatin immunoprecipitation.

Results: Normal human pituitary tissue that expresses FGFR2-IIIb does not express MAGE-A3; in contrast, pituitary tumors that are FGFR2 negative show abundant MAGE-A3 mRNA expression. MAGE-A3 expression correlates with the presence and extent of DNA promoter methylation; more frequent and higher-degree methylation is present in the normal gland compared with pituitary tumors. Conversely, pituitary tumors are hypomethylated, particularly in females where MAGE-A3 expression is nearly thrice higher than in males. Estradiol treatment induces MAGE-A3 through enhanced histone 3 acetylation and diminished methylation. The effects of estradiol are directly opposed by FGF7/FGFR2-IIIb. Down-regulation of MAGE-A3 results in p53 transcriptional induction, also through reciprocal histone acetylation and methylation modifications.

Conclusions: These findings highlight MAGE-A3 as a target of FGFR2-IIIb and estrogen action and provide evidence for a common histone-modifying network in the control of the balance between opposing signals.
Toxigenic Helicobacter pylori Infection Precedes Gastric Hypochlorhydria in Cancer Relatives, and H. pylori Virulence Evolves in These Families
Authors: Argent Richard H. , Thomas Rachael J. , Aviles-Jimenez Francisco , Letley Darren P. , Limb Marie C. , El-Omar Emad M. , Atherton John C.
Published: April 2008

Purpose: Helicobacter pylori infection by virulent strains is associated with gastric adenocarcinoma. We aimed to determine whether infection with virulent H. pylori preceeded precancerous gastric hypochlorhydria and atrophy in gastric cancer relatives and quantify the extent of virulence factor evolution.

Experimental Design: H. pylori strains from 51 Scottish gastric cancer relatives were characterized by genetic fingerprinting and typing the vacuolating cytotoxin gene (vacA), the cytotoxin-associated gene (cagA), and housekeeping genes. We phenotyped strains by coculture with gastric epithelial cells and assessing vacuolation (microscopy), CagA tyrosine phosphorylation (immunoblot), and interleukin-8 secretion (ELISA).

Results: Toxigenic (vacA type s1/m1) H. pylori was associated with precancerous gastric hypochlorhydria (P < 0.01). Adult family members with this type of H. pylori had the same strain as currently noncohabiting adult family members in 68% cases, implying acquisition during childhood from each other or a common source. We analyzed different isolates of the same strain within families and showed that H. pylori commonly microevolved to change virulence: this occurred in 22% individuals and a striking 44% cases where the strain was shared within families. Microevolution in vacA occurred by extragenomic recombination and in cagA by this or duplication/deletion. Microevolution led to phenotypic changes in virulence. Passage of microevolved strains could be tracked within families.

Conclusions: Toxigenic H. pylori infection precedes and so likely causes gastric hypochlorhydria, suggesting that virulent H. pylori increases cancer risk by causing this condition. Microevolution of virulence genes is common within families of gastric cancer patients and changes H. pylori virulence.
Loss of Retinoblastoma Tumor Suppressor Protein Makes Human Breast Cancer Cells More Sensitive to Antimetabolite Exposure
Authors: Derenzini Massimo , Donati Giulio , Mazzini Giuliano , Montanaro Lorenzo , Vici Manuela , Ceccarelli Claudio , Santini Donatella , Taffurelli Mario , Treré Davide
Published: April 2008

Purpose: The RB tumor-suppressor activity may influence the therapeutic response in human breast cancers. The effect of adjuvant therapy on clinical outcome of breast cancer patients was analyzed, and the sensitivity to 5-fluorouracil (5-FU) and methotrexate was investigated in MCF-7 and HCT-116 human cancer cells, according to their RB status.

Experimental Design: RB protein (pRB) expression was prospectively evaluated by immunocytochemistry in 518 consecutive patients and its predictive value was determined according to the adjuvant therapeutic treatments. MCF-7 and HCT-116 human cancer cells silenced for RB1 expression were treated with 5-FU and methotrexate, at the same concentrations and time exposures as determined in the interstitium of breast cancers of patients treated with adjuvant chemotherapy.

Results: Multivariate analysis of disease-free survival, including all the established clinical and histopathologic prognostic variables, indicated that the absence of pRB expression was the only predictive factor of good clinical outcome in patients treated with standard systemic chemotherapy (cyclophosphamide, methotrexate, and 5-FU) but not in patients treated with endocrine therapy alone. 5-FU and methotrexate significantly reduced the growth rate of RB1-silenced but not of control MCF-7 and HCT-116 cells. This was likely due to the absence of a DNA damage checkpoint with accumulation of DNA double-strand breaks in RB1-silenced but not in control cells.

Conclusions: The absence of pRB expression renders human breast cancer cells more sensitive to 5-FU and methotrexate and predicts a good clinical outcome for patients treated with adjuvant chemotherapy. We suggest that patients with RB-negative breast cancers should be treated with systemic chemotherapy.
The Potential Predictive Value of Cyclooxygenase-2 Expression and Increased Risk of Gastrointestinal Hemorrhage in Advanced Non–Small Cell Lung Cancer Patients Treated with Erlotinib and Celecoxib
Authors: Fidler Mary J. , Argiris Athanassios , Patel Jyoti D. , Johnson David H. , Sandler Alan , Villaflor Victoria M. , Coon John , Buckingham Lela , Kaiser Kelly , Basu Sanjib , Bonomi Philip
Published: April 2008

Purpose: Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, potentiates antitumor effects of erlotinib in preclinical studies, and COX-2 is frequently expressed in non–small cell lung cancer (NSCLC). With these observations, we designed a phase II trial to evaluate the efficacy and safety of erlotinib plus celecoxib in advanced NSCLC.

Experimental Design: Previously treated stage IIIB/IV NSCLC patients were given celecoxib at 400 mg orally twice daily and erlotinib at 150 mg orally daily until disease progression. Planned accrual was 40 patients. Tissue was collected for epidermal growth factor receptor (EGFR) analysis and COX-2 immunohistochemistry.

Results: Twenty-six patients were enrolled (17 men, 9 women; median age, 66 years). Eighteen and 21 patients had tissue available for EGFR analysis and COX-2 immunohistochemistry, respectively. The median progression-free survival (PFS) and overall survival were 2.0 and 9.2 months, respectively. Eleven of 21 patients tested had increased tumor COX-2 expression, which was strongly associated with prolonged PFS (P = 0.048). Four patients on anticoagulation or with a history of peptic ulcer disease had grade 3/grade 4 upper gastrointestinal bleeding (GIB), prompting early study closure. Three patients with GIB had endoscopy that found peptic ulcers.

Conclusions: The combination of erlotinib and celecoxib does not seem superior to erlotinib alone in unselected patients. However, longer PFS with high-tumor COX-2 expression suggests that trials of EGFR and COX-2 inhibitors may be warranted in this patient subset. GIB observed in our trial supports excluding patients with a history of peptic ulcer disease or those requiring therapeutic anticoagulation from future EGFR and COX-2 inhibitor studies.
Heterogeneity of Breast Cancer Metastases: Comparison of Therapeutic Target Expression and Promoter Methylation Between Primary Tumors and Their Multifocal Metastases
Authors: Wu Julie M. , Fackler Mary Jo , Halushka Marc K. , Molavi Diana W. , Taylor M. Evangeline , Teo Wei Wen , Griffin Constance , Fetting John , Davidson Nancy E. , De Marzo Angelo M. , Hicks Jessica L. , Chitale Dhananjay , Ladanyi Marc , Sukumar Saraswati , Argani Pedram
Published: April 2008

Purpose: A comprehensive comparison of biomarker expression between patients' primary breast carcinoma (PBC) and their metastatic breast carcinomas (MBC) has not been done.

Experimental Design: We did rapid autopsies (postmortem intervals, 1-4 hours) on 10 consenting patients who died of MBC. We constructed single-patient tissue microarrays from the patients' archived PBC and multiple different MBCs harvested at autopsy, which were immunohistochemically labeled for multiple biomarkers. Methylation of multiple gene promoters was assessed quantitatively on dissected PBC and MBC samples.

Results: Extensive heterogeneity was observed between PBC and their paired MBC, as well as among multiple MBC from the same patient. Estrogen and progesterone receptors tended to be uniformly down-regulated in metastases. E-cadherin was down-regulated in a subset of the MBC of one case. Variable overexpression in MBC compared with the PBC was observed for cyclooxygenase-2 (five cases), epidermal growth factor receptor (EGFR; four cases), MET (four cases), and mesothelin (four cases). No case strongly overexpressed HER-2/neu by immunohistochemistry, but eight cases showed variable protein expression ranging from negative to equivocal (2+) in different MBC. In one case, variable low-level HER-2/neu gene amplification was found. EGFR and MET overexpression were restricted to the four basal-type cancers. EGFR protein overexpression did not correlate with EGFR gene amplification. Multigene promoter hypermethylation of RASSF1a, HIN1, cyclin D2, Twist, estrogen receptor α, APC1, and RARβ was overall very similar in the PBC and all MBCs in all cases.

Conclusions: Therapeutic targets identified in the PBC or even some MBC may not reflect targets present in all metastatic sites.
Familial Breast Cancers without Mutations in BRCA1 or BRCA2 Have Low Cyclin E and High Cyclin D1 in Contrast to Cancers in BRCA Mutation Carriers
Authors: Aaltonen Kirsimari , Blomqvist Carl , Amini Rose-Marie , Eerola Hannaleena , Aittomäki Kristiina , Heikkilä Päivi , Nevanlinna Heli
Published: April 2008

Purpose: We analyzed the expression of critical cell cycle regulators cyclin E and cyclin D1 in familial breast cancer, focusing on BRCA mutation–negative tumors. Cyclin E expression in tumors of BRCA1 or BRCA2 carriers is higher, and cyclin D1 expression lower, than in sporadic tumors. In familial non-BRCA1/2 tumors, cyclin E and cyclin D1 expression has not been studied.

Experimental Design: Cyclin E and cyclin D1 immunohistochemical expression was studied in tissue microarrays consisting of 53 BRCA1, 58 BRCA2, 798 familial non-BRCA1/2, and 439 sporadic breast tumors.

Results: In univariate analysis, BRCA1 tumors had significantly more frequently high cyclin E (88%) and low cyclin D1 (84%) expression than sporadic (54% and 49%, respectively) or familial non-BRCA1/2 (38% and 45%, respectively) tumors. BRCA2 tumors had significantly more frequently low cyclin D1 expression (68%) than sporadic or familial non-BRCA1/2 tumors and significantly more frequently high cyclin E expression than familial non-BRCA1/2 tumors. In a logistic regression model, cyclin expression, early age of onset, and estrogen receptor (ER) and human epidermal growth factor receptor-2 (HER2) status were the independent factors most clearly distinguishing tumors of BRCA1 mutation carriers from other familial breast cancers. High cyclin E and low cyclin D1 expression were also independent predictors of BRCA2 mutation when compared with familial non-BRCA1/2 tumors. Most interestingly, lower frequency of high cyclin E expression independently distinguished familial non-BRCA1/2 tumors also from sporadic ones.

Conclusions: Cyclin E and cyclin D1 expression distinguishes non-BRCA1/2 tumors from both sporadic and BRCA1- and BRCA2-associated tumors and may reflect different predisposition and pathogenesis in these groups.
Histogenesis of Clear Cell Adenocarcinoma in the Urinary Tract: Evidence of Urothelial Origin
Authors: Sung Ming-Tse , Zhang Shaobo , MacLennan Gregory T. , Lopez-Beltran Antonio , Montironi Rodolfo , Wang Mingsheng , Tan Puay-Hoon , Cheng Liang
Published: April 2008

Purpose: Clear cell adenocarcinoma in the urinary tract is a rare entity with an appearance resembling its counterpart in the female genital tract. Although several theories have been proposed about its origin, its exact histogenesis has remained uncertain.

Experimental Design: We integrated molecular genetic evaluation by fluorescence in situ hybridization and X-chromosome inactivation with conventional morphologic and immunohistochemical analyses in 12 patients with clear cell adenocarcinomas in the urinary tract.

Results: Concurrent urothelial carcinoma or urothelial carcinoma in situ was present in six cases (50%) and foci of cystitis glandularis were observed in four cases (33%). Neither intestinal metaplasia nor Müllerian component was identified in any case. Cytoplasmic expression of α-methylacyl-CoA racemase was demonstrable in 10 of 12 tumors (83%). Moderate to diffuse immunostaining for cytokeratin 7 was identified in all 12 tumors (100%), whereas only 3 of 12 (25%) tumors showed positive immunostaining for cytokeratin 20. Focal uroplakin III staining was seen in 6 of 12 tumors (50%). In five cases (42%), focal to moderate CD10 immunoreactivity was observed. Immunostains for OCT4 and CDX2 were completely negative in all tumors. In UroVysion fluorescence in situ hybridization assays, all tumors displayed chromosomal alterations similar to those commonly found in urothelial carcinoma. Identical patterns of nonrandom X-chromosome inactivation in concurrent clear cell adenocarcinoma and urothelial neoplasia were identified in two informative female cases.

Conclusions: Our findings support an urothelial origin for most clear cell adenocarcinomas of the urinary tract, despite their morphologic resemblance to certain Müllerian-derived tumors of the female genital tract.
Lupeol Inhibits Growth of Highly Aggressive Human Metastatic Melanoma Cells In vitro and In vivo by Inducing Apoptosis
Authors: Saleem Mohammad , Maddodi Nityanand , Abu Zaid Mohammad , Khan Naghma , bin Hafeez Bilal , Asim Mohammad , Suh Yewseok , Yun Jung-Mi , Setaluri Vijayasaradhi , Mukhtar Hasan
Published: April 2008

Purpose: Poor prognosis of metastatic melanoma mandates the development of novel strategies for its treatment and prevention. In this study, the effect of lupeol, a diet-based triterpene, was determined on the growth and tumorigenicity of human melanoma cells in vitro and in vivo.

Experimental Design: Normal human melanocytes, and human metastatic (451Lu) and nonmetastatic (WM35) cells were treated with lupeol; its effect on growth, proliferation, and apoptosis were evaluated. Further athymic nude mice bearing 451Lu cell–originated tumors were administered with lupeol thrice a week, and its effect on tumor growth and surrogate biomarkers was evaluated.

Results: Lupeol significantly decreased the viability of 451Lu and WM35 melanoma cells but had only a marginal effect on normal human melanocyte cells at similar doses. Lupeol treatment of 451Lu cells caused (a) G1-S phase cell cycle arrest and apoptosis; (b) down-regulation of Bcl2 and up-regulation of Bax; (c) activation of caspase-3 and induction of poly(ADP)ribose polymerase cleavage; (d) decreased expression of cyclin D1, cyclin D2, and cdk2; and (e) increased expression of p21 protein. Next, lupeol significantly reduced 451Lu tumor growth in athymic nude mice and modulated the expression of proliferation markers, apoptotic markers, and cell cycle regulatory molecules in tumor xenografts.

Conclusion: Our findings showed the anticancer efficacy of lupeol with mechanistic rationale against metastatic human melanoma cells. We suggest that lupeol, alone or as an adjuvant to current therapies, could be useful for the management of human melanoma.
In vivoOptical Coherence Tomography Imaging of Preinvasive Bronchial Lesions
Authors: Lam Stephen , Standish Beau , Baldwin Corisande , McWilliams Annette , leRiche Jean , Gazdar Adi , Vitkin Alex I. , Yang Victor , Ikeda Norihiko , MacAulay Calum
Published: April 2008

Purpose: Optical coherence tomography (OCT) is an optical imaging method that can visualize cellular and extracellular structures at and below tissue surface. The objective of the study was to determine if OCT could characterize preneoplastic changes in the bronchial epithelium identified by autofluorescence bronchoscopy.

Experimental Design: A 1.5-mm fiberoptic probe was inserted via a bronchoscope into the airways of 138 volunteer heavy smokers participating in a chemoprevention trial and 10 patients with lung cancer to evaluate areas that were found to be normal or abnormal on autofluorescence bronchoscopy. Radial scanning of the airways was done to generate OCT images in real time. Following OCT imaging, the same sites were biopsied for pathologic correlation.

Results: A total of 281 OCT images and the corresponding bronchial biopsies were obtained. The histopathology of these areas includes 145 normal/hyperplasia, 61 metaplasia, 39 mild dysplasia, 10 moderate dysplasia, 6 severe dysplasia, 7 carcinoma in situ, and 13 invasive carcinomas. Quantitative measurement of the epithelial thickness showed that invasive carcinoma was significantly different than carcinoma in situ (P = 0.004) and dysplasia was significantly different than metaplasia or hyperplasia (P = 0.002). In addition, nuclei of the cells corresponding to histologic results became more discernible in lesions that were moderate dysplasia or worse compared with lower-grade lesions.

Conclusion: Preliminary data suggest that autofluorescence bronchoscopy–guided OCT imaging of bronchial lesions is technically feasible. OCT may be a promising nonbiopsy tool for in vivo imaging of preneoplastic bronchial lesions to study their natural history and the effect of chemopreventive intervention.
Direct Visualization of Heterogeneous Extravascular Distribution of Trastuzumab in Human Epidermal Growth Factor Receptor Type 2 Overexpressing Xenografts
Authors: Baker Jennifer H.E. , Lindquist Kirstin E. , Huxham Lynsey A. , Kyle Alastair H. , Sy Jonathan T. , Minchinton Andrew I.
Published: April 2008

Purpose: The high molecular weight and binding affinity of trastuzumab, a monoclonal antibody in use for treatment of breast cancers overexpressing human epidermal growth factor receptor type 2 (HER2), in combination with microenvironmental factors, may limit its distribution and efficacy. We assessed and mapped the distribution of systemically given, unlabeled trastuzumab at micrometer resolution in tumor xenografts using immunohistochemistry.

Experimental Design: Mice bearing MDA-435/LCC6HER2 xenografts were given single doses of 4 or 20 mg/kg unlabeled trastuzumab with tumor harvest at various time points thereafter; bound trastuzumab was imaged directly in tumor cryosections using fluorescently tagged antihuman secondary antibodies. Combinations of additional markers, including HER2, 5-bromo-2-deoxyuridine, CD31, DioC7(3), desmin, and collagen IV were also mapped on the same tumor sections.

Results: Distribution of trastuzumab in MDA-435/LCC6HER2 tumors is found to be heterogeneous, with tumor margins saturating more thoroughly in doses and times analyzed. Considerable intervessel heterogeneity is also seen. For example, in unsaturated tissues, there remain perfused vessels without any trastuzumab in addition to vessels with a few layers of positively stained perivascular cells, in addition to vessels with bound drug up to 150 μm away. This heterogeneity is independent of HER2 expression, microvessel density, and perfusion. A slightly greater proportion of vessels were associated with pericytes in sections with greater trastuzumab saturation, but this would not adequately account for observed heterogeneous trastuzumab distribution.

Conclusions: Complete penetration of trastuzumab in tumor tissue was not seen in our study, leaving the possibility that inadequate distribution may represent a mechanism for resistance to trastuzumab.
Endoglin (CD105): A Marker of Tumor Vasculature and Potential Target for Therapy
Authors: Dallas Nikolaos A. , Samuel Shaija , Xia Ling , Fan Fan , Gray Michael J. , Lim Sherry J. , Ellis Lee M.
Published: April 2008

Endoglin (CD105) is an accessory protein of the transforming growth factor-β receptor system expressed on vascular endothelial cells. Mutation of the endoglin gene is associated with hereditary hemorrhagic telangiectasias, or Osler-Weber-Rendu syndrome, and has been studied extensively in the context of this disease. The expression of endoglin is elevated on the endothelial cells of healing wounds, developing embryos, inflammatory tissues, and solid tumors. Endoglin is a marker of activated endothelium, and its vascular expression is limited to proliferating cells. Recent studies identified endoglin expression in several solid tumor types, with the level of expression correlating with various clinicopathologic factors including decreased survival and presence of metastases. Attempts to target endoglin and the cells that express this protein in tumor-bearing mice have yielded promising results.
Intratumoral Epiregulin Is a Marker of Advanced Disease in Non–Small Cell Lung Cancer Patients and Confers Invasive Properties onEGFR-Mutant Cells
Authors: Zhang Jie , Iwanaga Kentaro , Choi Kuicheon C. , Wislez Marie , Raso Maria Gabriela , Wei Wei , Wistuba Ignacio I. , Kurie Jonathan M.
Published: April 2008

Non–small cell lung cancer (NSCLC) cells with activating epidermal growth factor receptor (EGFR) somatic mutations have unique biological properties, including high expression of the ErbB ligand epiregulin; however, the biological role of epiregulin in these cells has not been elucidated. To examine its role, we used an immunohistochemical approach to detect epiregulin expression in NSCLC biopsy samples and pharmacologic and genetic approaches to inhibit epiregulin in cultured NSCLC cells. In NSCLC biopsy samples, epiregulin was detected in 237 of 366 (64.7%) tumors, which correlated with nodal metastasis and a shorter duration of survival. In EGFR-mutant NSCLC cell lines, treatment with a small-molecule EGFR tyrosine kinase inhibitor diminished mRNA levels of the gene encoding epiregulin (EREG). The ability of EGFR-mutant NSCLC cells to invade through Matrigel in vitro was inhibited by treatment with an anti-epiregulin neutralizing antibody or by transfection with an EREG short hairpin RNA. Collectively, these findings show that epiregulin expression correlated with advanced disease, was EGFR dependent, and conferred invasive properties on NSCLC cells. Additional studies are warranted in NSCLC patients to evaluate whether epiregulin expression predicts the metastatic potential of primary tumors and whether anti-epiregulin treatment strategies are efficacious in the prevention of metastasis.
Targeting the Activator Protein 1 Transcription Factor for the Prevention of Estrogen Receptor–Negative Mammary Tumors
Authors: Shen Qiang , Uray Ivan P. , Li Yuxin , Zhang Yun , Hill Jamal , Xu Xiao-Chun , Young Matthew R. , Gunther Edward J. , Hilsenbeck Susan G. , Colburn Nancy H. , Chodosh Lewis A. , Brown Powel H.
Published: April 2008

The oncogene erbB2 is overexpressed in 20% to 30% human breast cancers and is most commonly overexpressed in estrogen receptor (ER)–negative breast cancers. Transgenic mice expressing erbB2 develop ER-negative mammary tumors, mimicking human breast carcinogenesis. Previously, we have shown that activator protein 1 (AP-1) regulates proliferation of ER-negative breast cancer cells. We hypothesized that blockade of AP-1 in mouse mammary epithelial cells will suppress ER-negative tumorigenesis induced by erbB2. Trigenic erbB2 mice were generated by crossing a bigenic pUHD-Tam67/MMTV-rtTA mouse to a MMTV-erbB2 mouse. The resulting trigenic mice develop tumors and express a doxycycline-inducible c-Jun dominant negative mutant (Tam67) in the mammary glands. In vivo AP-1 blockade by Tam67 expression started delayed mammary tumor formation in MMTV-erbB2 mice by more than 11 weeks. By 52 weeks of age, 100% (18 of 18) of the untreated animals had developed mammary tumors, whereas 56% (9 of 16) of the doxycycline-treated trigenic mice developed tumors. In addition, the tumors that arose in the AP-1–blocked erbB2 mice failed to express Tam67. Twenty-five percent of the doxycycline-treated MMTV-erbB2 mice survived more than 72 weeks of age without developing mammary tumors. Examination of normal-appearing mammary glands from these mice showed that AP-1 blockade by Tam67 also significantly prevents the development of premalignant lesions in these glands. The expression of erbB2 either in normal mammary tissue or in mammary tumors was not altered. Our results show that blocking the AP-1 signaling in mammary cells suppresses erbB2-induced transformation, and show that the AP-1 transcription factor is a critical transducer of erbB2. These results provide a scientific rationale to develop targeted drugs that inhibit AP-1 to prevent the development of ER-negative breast cancer.
Diabetes Patients Requiring Glucose-Lowering Therapy and Nondiabetics With a Prior Myocardial Infarction Carry the Same Cardiovascular Risk
Authors: Schramm Tina Ken , Gislason Gunnar H. , Køber Lars , Rasmussen Søren , Rasmussen Jeppe N. , Abildstrøm Steen Z. , Hansen Morten Lock , Folke Fredrik , Buch Pernille , Madsen Mette , Vaag Allan , Torp-Pedersen Christian
Published: April 2008

Background—

Previous studies reveal major differences in the estimated cardiovascular risk in diabetes mellitus, including uncertainty about the risk in young patients. Therefore, large studies of well-defined populations are needed.

Methods and Results—

All residents in Denmark ≥30 years of age were followed up for 5 years (1997 to 2002) by individual-level linkage of nationwide registers. Diabetes patients receiving glucose-lowering medications and nondiabetics with and without a prior myocardial infarction were compared. At baseline, 71 801 (2.2%) had diabetes mellitus and 79 575 (2.4%) had a prior myocardial infarction. Regardless of age, age-adjusted Cox proportional-hazard ratios for cardiovascular death were 2.42 (95% confidence interval [CI], 2.35 to 2.49) in men with diabetes mellitus without a prior myocardial infarction and 2.44 (95% CI, 2.39 to 2.49) in nondiabetic men with a prior myocardial infarction (
P
=0.60), with nondiabetics without a prior myocardial infarction as the reference. Results for women were 2.45 (95% CI, 2.38 to 2.51) and 2.62 (95% CI, 2.55 to 2.69) (
P
=0.001), respectively. For the composite of myocardial infarction, stroke, and cardiovascular death, the hazard ratios in men with diabetes only were 2.32 (95% CI, 2.27 to 2.38) and 2.48 (95% CI, 2.43 to 2.54) in those with a prior myocardial infarction only (
P
=0.001). Results for women were 2.48 (95% CI, 2.43 to 2.54) and 2.71 (95% CI, 2.65 to 2.78) (
P
=0.001), respectively. Risks were similar for both diabetes types. Analyses with adjustments for comorbidity, socioeconomic status, and prophylactic medical treatment showed similar results, and propensity score–based matched-pair analyses supported these findings.

Conclusions—

Patients requiring glucose-lowering therapy who were ≥30 years of age exhibited a cardiovascular risk comparable to nondiabetics with a prior myocardial infarction, regardless of sex and diabetes type. Therefore, requirement for glucose-lowering therapy should prompt intensive prophylactic treatment for cardiovascular diseases.