bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Interactions of the Hdm2/p53 and Proteasome Pathways May Enhance the Antitumor Activity of Bortezomib
Authors: Ooi Melissa G. , Hayden Patrick J. , Kotoula Vassiliki , McMillin Douglas W. , Charalambous Elpida , Daskalaki Emily , Raje Noopur S. , Munshi Nikhil C. , Chauhan Dharminder , Hideshima Teru , Buon Leutz , Clynes Martin , O'Gorman Peter , Richardson Paul G. , Mitsiades Constantine S. , Anderson Kenneth C. , Mitsiades Nicholas
Published: April 2023

Abstract
Purpose: p53 is inactivated in many human malignancies through missense mutations or overexpression of the human homologue of Mdm2 (Hdm2), an E3 ubiquitin ligase that ubiquitinates p53, thereby promoting its proteasomal degradation. The cis-imidazoline nutlin-3 can disrupt the p53-Hdm2 interaction and activate p53, inducing apoptosis in vitro in many malignancies, including multiple myeloma (MM).

Experimental Design: We hypothesized that suppression of Hdm2-mediated p53 ubiquitination may augment sequelae of p53 accumulation caused by proteasomal inhibition. We compared the response of MM cells versus several epithelial cancer models to the proteasome inhibitor bortezomib in combination with nutlin-3.

Results: The combination of sublethal concentrations of bortezomib plus nutlin-3 induced additive cytotoxicity against bortezomib-sensitive MM cell lines. Importantly, however, in breast, prostate, colon, and thyroid (papillary, follicular, anaplastic, and medullary) carcinoma cell lines, this combination triggered synergistic cytotoxicity, and increased expression of p53, p21, Hdm2, Bax, Noxa, PUMA, and cleavage of caspase-3 and poly ADP ribose polymerase. Coculture with bone marrow stromal cells attenuated MM cell sensitivity to nutlin-3 monotherapy and was associated with evidence of suppression of p53 activity in MM cells, whereas combined bortezomib-nutlin-3 treatment maintained cytotoxicity even in the presence of bone marrow stromal cells.

Conclusions: This differential response of MM versus epithelial carcinomas to combination of nutlin-3 with bortezomib sheds new light on the role of p53 in bortezomib-induced apoptosis. Concurrent Hdm2 inhibition with bortezomib may extend the spectrum of bortezomib applications to malignancies with currently limited sensitivity to single-agent bortezomib or, in the future, to MM patients with decreased clinical responsiveness to bortezomib-based therapy. (Clin Cancer Res 2009;15(23):7153–60)
Data from Regulation of Breast Cancer Metastasis by Atypical Chemokine Receptors
Authors: Cheng Xiaoyun , Hung Mien-Chie
Published: April 2023

Abstract
The interaction between chemokines and their G-protein-coupled receptors plays an important role in promoting metastasis of different kinds of human cancers. However, the expression of an atypical chemokine receptor, CCX-CKR, which serves as a decoy receptor to attract chemokines, inhibits the growth and metastasis of breast cancer by sequestration of chemokines.
Data from ERBB2 Induces an Antiapoptotic Expression Pattern of Bcl-2 Family Members in Node-Negative Breast Cancer
Authors: Petry Ilka Brigitte , Fieber Esther , Schmidt Marcus , Gehrmann Mathias , Gebhard Susanne , Hermes Matthias , Schormann Wiebke , Selinski Silvia , Freis Evgenia , Schwender Holger , Brulport Marc , Ickstadt Katja , Rahnenführer Jörg , Maccoux Lindsey , West Jonathan , Kölbl Heinz , Schuler Martin , Hengstler Jan Georg
Published: April 2023

Abstract
Purpose: Members of the Bcl-2 family act as master regulators of mitochondrial homeostasis and apoptosis. We analyzed whether ERBB2 influences the prognosis of breast cancer by influencing the proapoptotic versus antiapoptotic balance of Bcl-2 family members.

Experimental Design: ERBB2-regulated Bcl-2 family members were identified by inducible expression of ERBB2 in MCF-7 breast cancer cells and by correlation analysis with ERBB2 expression in breast carcinomas. The prognostic relevance of ERBB2-regulated and all additional Bcl-2 family members was determined in 782 patients with untreated node-negative breast cancer. The biological relevance of ERBB2-induced inhibition of apoptosis was validated in a murine tumor model allowing conditional ERBB2 expression.

Results: ERBB2 caused an antiapoptotic phenotype by upregulation of MCL-1, TEGT, BAG1, BNIP1, and BECN1 as well as downregulation of BAX, BMF, BNIPL, CLU, and BCL2L13. Upregulation of the antiapoptotic MCL-1 [P = 0.001, hazard ratio (HR) 1.5] and BNIP3 (P = 0.024; HR, 1.4) was associated with worse prognosis considering metastasis-free interval, whereas clusterin (P = 0.008; HR, 0.88) and the proapoptotic BCL2L13 (P = 0.019; HR, 0.45) were associated with better prognosis. This indicates that ERBB2 alters the expression of Bcl-2 family members in a way that leads to adverse prognosis. Analysis of apoptosis and tumor remission in a murine tumor model confirmed that the prototypic Bcl-2 family member Bcl-xL could partially substitute for ERBB2 to antagonize tumor remission.

Conclusions: Our results support the concept that ERBB2 influences the expression of Bcl-2 family members to induce an antiapoptotic phenotype. Antagonization of antiapoptotic Bcl-2 family members might improve breast cancer therapy, whereby MCL-1 and BNIP3 represent promising targets. Clin Cancer Res; 16(2); 451–60
Data from LYRIC/AEG-1 Is Targeted to Different Subcellular Compartments by Ubiquitinylation and Intrinsic Nuclear Localization Signals
Authors: Thirkettle Hayley J. , Girling Joanne , Warren Anne Y. , Mills Ian G. , Sahadevan Kanagasabai , Leung Hing , Hamdy Freddie , Whitaker Hayley C. , Neal David E.
Published: April 2023

Abstract
Purpose: LYRIC/AEG-1 has been reported to influence breast cancer survival and metastases, and its altered expression has been found in a number of cancers. The cellular function of LYRIC/AEG-1 has previously been related to its subcellular distribution in cell lines. LYRIC/AEG-1 contains three uncharacterized nuclear localization signals (NLS), which may regulate its distribution and, ultimately, function in cells.

Experimental Design: Immunohistochemistry of a human prostate tissue microarray composed of 179 prostate cancer and 24 benign samples was used to assess LYRIC/AEG-1 distribution. Green fluorescent protein-NLS fusion proteins and deletion constructs were used to show the ability of LYRIC/AEG-1 NLS to target green fluorescent protein from the cytoplasm to the nucleus. Immunoprecipitation and Western blotting were used to show posttranslational modification of LYRIC/AEG-1 NLS regions.

Results: Using a prostate tissue microarray, significant changes in the distribution of LYRIC/AEG-1 were observed in prostate cancer as an increased cytoplasmic distribution in tumors compared with benign tissue. These differences were most marked in high grade and aggressive prostate cancers and were associated with decreased survival. The COOH-terminal extended NLS-3 (amino acids 546-582) is the predominant regulator of nuclear localization, whereas extended NLS-1 (amino acids 78-130) regulates its nucleolar localization. Within the extended NLS-2 region (amino acids 415-486), LYRIC/AEG-1 can be modified by ubiquitin almost exclusively within the cytoplasm.

Conclusions: Changes in LYRIC/AEG-1 subcellular distribution can predict Gleason grade and survival. Two lysine-rich regions (NLS-1 and NLS-3) can target LYRIC/AEG-1 to subcellular compartments whereas NLS-2 is modified by ubiquitin in the cytoplasm.
Data from Spectrum of Phosphatidylinositol 3-Kinase Pathway Gene Alterations in Bladder Cancer
Authors: Platt Fiona M. , Hurst Carolyn D. , Taylor Claire F. , Gregory Walter M. , Harnden Patricia , Knowles Margaret A.
Published: April 2023

Abstract
Purpose: The phosphatidylinositol 3-kinase (PI3K) pathway can be activated by alterations affecting several pathway components. For rational application of targeted therapies, detailed understanding of tumor biology and approaches to predict efficacy in individual tumors are required. Our aim was to assess the frequency and distribution of pathway alterations in bladder cancer.

Experimental Design: We examined the pathway components (PIK3CA, PTEN, TSC1, RHEB, and LKB1) and putative upstream regulators (FGFR3 and RAS genes) for mutation, allelic loss, copy number alteration, and expression in bladder tumors and cell lines.

Results: No mutations were found in RHEB and only a single mutation in LKB1. PIK3CA mutations were detected in 25% of tumors and 26% of cell lines with a significant excess of helical domain mutations (E542K and E545K). There was over-representation but not amplification of the gene. Loss of heterozygosity of the PTEN region and homozygous deletion were found in 12% and 1.4% of tumors, and reduced expression in 49%. Forty-six percent of cell lines showed alterations that implicated PTEN. Sixteen percent of tumors and 11% of cell lines showed TSC1 mutation, and 9q loss of heterozygosity was common (57%). Pathway alterations were independently distributed, suggesting that the mutation of two pathway members may have additive or synergistic effects through noncanonical functions.

Conclusions: PI3K pathway alterations are common in bladder cancer. The lack of redundancy of alterations suggests that single-agent PI3K-targeted therapy may not be successful in these cancers. This study provides a well-characterized series of cell lines for use in preclinical studies of targeted agents. (Clin Cancer Res 2009;15(19):6008–17)
Data from Differentiation Therapy Exerts Antitumor Effects on Stem-like Glioma Cells
Authors: Campos Benito , Wan Feng , Farhadi Mohammad , Ernst Aurélie , Zeppernick Felix , Tagscherer Katrin E. , Ahmadi Rezvan , Lohr Jennifer , Dictus Christine , Gdynia Georg , Combs Stephanie E. , Goidts Violaine , Helmke Burkhard M. , Eckstein Volker , Roth Wilfried , Beckhove Philipp , Lichter Peter , Unterberg Andreas , Radlwimmer Bernhard , Herold-Mende Christel
Published: April 2023

Abstract
Purpose: Stem-like tumor cells comprise a highly tumorigenic and therapy-resistant tumor subpopulation, which is believed to substantially influence tumor initiation and therapy resistance in glioma. Currently, therapeutic, drug-induced differentiation is considered as a promising approach to eradicate this tumor-driving cell population; retinoic acid is well known as a potent modulator of differentiation and proliferation in normal stem cells. In glioma, knowledge about the efficacy of retinoic acid–induced differentiation to target the stem-like tumor cell pool could have therapeutic implications.

Experimental Design: Stem-like glioma cells (SLGC) were differentiated with all-trans retinoic acid–containing medium to study the effect of differentiation on angiogenesis, invasive growth, as well as radioresistance and chemoresistance of SLGCs. In vivo effects were studied using live microscopy in a cranial window model.

Results: Our data suggest that in vitro differentiation of SLGCs induces therapy-sensitizing effects, impairs the secretion of angiogenic cytokines, and disrupts SLGCs motility. Further, ex vivo differentiation reduces tumorigenicity of SLGCs. Finally, we show that all-trans retinoic acid treatment alone can induce antitumor effects in vivo.

Conclusions: Altogether, these results highlight the potential of differentiation treatment to target the stem-like cell population in glioblastoma. Clin Cancer Res; 16(10); 2715–28. ©2010 AACR.
Data from Overexpression of Smoothened Activates the Sonic Hedgehog Signaling Pathway in Pancreatic Cancer–Associated Fibroblasts
Authors: Walter Kimberly , Omura Noriyuki , Hong Seung-Mo , Griffith Margaret , Vincent Audrey , Borges Michael , Goggins Michael
Published: April 2023

Abstract
Purpose: Accumulating evidence suggests that cancer-associated stromal fibroblasts (CAF) contribute to tumor growth by actively communicating with cancer cells. Our aim is to identify signaling pathways involved in tumor-stromal cell interactions in human pancreatic cancer.

Experimental Design: We established primary fibroblast cultures from human pancreatic adenocarcinomas and nonneoplastic pancreas tissues. To identify differentially expressed genes in CAFs, we did gene expression profiling of human pancreatic CAFs and nonneoplastic pancreatic fibroblasts.

Results: The Hedgehog receptor Smoothened (SMO) was upregulated in CAFs relative to control fibroblasts. CAFs expressing SMO could transduce the Sonic hedgehog signal to activate Gli1 expression, and small interfering RNA knockdown of SMO blocked the induction of Gli1 in these cells. Stromal fibroblasts of human primary pancreatic adenocarcinomas overexpressed Smo compared with normal pancreatic fibroblasts.

Conclusions: These findings implicate overexpression of Smo as a mechanism for the activation of Hedgehog signaling in human pancreatic CAFs and suggest that stromal cells may be a therapeutic target for Smo antagonists in pancreatic cancer. Clin Cancer Res; 16(6); 1781–9
Data from Gene Expression Profiling of Paraffin-Embedded Primary Melanoma Using the DASL Assay Identifies Increased Osteopontin Expression as Predictive of Reduced Relapse-Free Survival
Authors: Conway Caroline , Mitra Angana , Jewell Rosalyn , Randerson-Moor Juliette , Lobo Samira , Nsengimana Jrmie , Edward Sara , Sanders D. Scott , Cook Martin , Powell Barry , Boon Andy , Elliott Faye , de Kort Floor , Knowles Margaret A. , Bishop D. Timothy , Newton-Bishop Julia
Published: April 2023

Abstract
Purpose: Gene expression studies in melanoma have been few because tumors are small and cryopreservation is rarely possible. The purpose of this study was to evaluate the Illumina DASL Array Human Cancer Panel for gene expression studies in formalin-fixed melanoma primary tumors and to identify prognostic biomarkers.

Experimental Design: Primary tumors from two studies were sampled using a tissue microarray needle. Study 1: 254 tumors from a melanoma cohort recruited from 2000 to 2006. Study 2: 218 tumors from a case-control study of patients undergoing sentinel node biopsy.

Results: RNA was obtained from 76 of blocks; 1.4 of samples failed analysis (transcripts from <250 of the 502 genes on the DASL chip detected). Increasing age of the block and increased melanin in the tumor were associated with reduced number of genes detected. The gene whose expression was most differentially expressed in association with relapse-free survival in study 1 was osteopontin (SPP1; P = 2.11 106) and supportive evidence for this was obtained in study 2 used as a validation set (P = 0.006; unadjusted data). Osteopontin level in study 1 remained a significant predictor of relapse-free survival when data were adjusted for age, sex, tumor site, and histologic predictors of relapse. Genes whose expression correlated most strongly with osteopontin were PBX1, BIRC5 (survivin), and HLF.

Conclusion: Expression data were obtained from 74 of primary melanomas and provided confirmatory evidence that osteopontin expression is a prognostic biomarker. These results suggest that predictive biomarker studies may be possible using stored blocks from mature clinical trials. (Clin Cancer Res 2009;15(22):693946)
Data from Safety, Pharmacokinetics, and Efficacy of CPX-1 Liposome Injection in Patients with Advanced Solid Tumors
Authors: Batist Gerald , Gelmon Karen A. , Chi Kim N. , Miller Wilson H. , Chia Stephen K.L. , Mayer Lawrence D. , Swenson Christine E. , Janoff Andrew S. , Louie Arthur C.
Published: April 2023

Abstract
Purpose: CPX-1 is a novel, liposome-encapsulated formulation of irinotecan and floxuridine designed to prolong in vitro optimized synergistic molar ratios of both drugs postinfusion. This open-label, single-arm, dose-escalating phase I study was designed to determine the maximum tolerated dose and pharmacokinetics of CPX-1 in patients with advanced solid tumors.

Experimental Design: Patients received CPX-1 at 30, 60, 100, 150, 210, or 270 units/m2 (1 unit = 1 mg irinotecan + 0.36 mg floxuridine) infused over 90 minutes every 14 days in 28-day cycles. Pharmacokinetic samples were collected on days 1 and 15 of cycle 1.

Results: Thirty-three patients were enrolled, treated, and evaluated for safety; 30 patients were evaluated for response. A 1:1 plasma irinotecan to floxuridine molar ratio was maintained for 8 to 12 hours. Grade 3/4 toxicities included diarrhea (24.2%), neutropenia (12.1%), and hypokalemia (12.1%); 1 patient (270 units/m2) died of persistent diarrhea, which led to dehydration and renal failure (grade 5). Partial response occurred in 3 (12%) of the 25 subjects evaluated through Response Evaluation Criteria in Solid Tumors. Progression-free survival lasting >6 months occurred in 9 patients, 6 with colorectal cancer. Among 15 colorectal cancer patients (10 with prior irinotecan), the calculated median progression-free survival was 5.4 months; 11 patients (72.7%) achieved disease control and 2 patients (13%) had partial response.

Conclusions: Outpatient CPX-1 was well tolerated and antitumor activity was shown in patients with advanced solid tumors. The recommended dose for future studies is 210 units/m2. This is the first clinical evaluation of fixed drug ratio dosing designed to maintain synergistic molar ratios for enhanced therapeutic benefit.
Data from Evaluation of T- and NK-Cell-Targeted Therapies: Is There a Role for Rituximab Prophylaxis?
Authors: Ambinder Richard F.
Published: April 2023

Abstract
Splizimomab, an antibody that targets CD2, was studied in the treatment of T and natural killer cell lymphoma and was found to be associated with Epstein-Barr virus lymphoproliferative disease. B cell depletion may provide a platform for further evaluation of this and other promising antibody approaches that result in T cell depletion.
Data from Retinoblastoma Tumor Suppressor Gene Expression Determines the Response to Sequential Flavopiridol and Doxorubicin Treatment in Small-Cell Lung Carcinoma
Authors: Budak-Alpdogan Tulin , Chen Bobin , Warrier Anjali , Medina Daniel J. , Moore Dirk , Bertino Joseph R.
Published: April 2023

Abstract
Purpose: Small-cell lung cancers (SCLC) are defective in many regulatory mechanisms that control cell cycle progression, i.e., functional retinoblastoma protein (pRb). Flavopiridol inhibits proliferation and induces apoptosis in SCLC cell lines. We hypothesized that the sequence flavopiridol followed by doxorubicin would be synergistic in pRb-deficient SCLC cells.

Experimental Design: A H69 pRb-deficient SCLC cell line, H865, with functional pRb and H865 pRb small interfering RNA (siRNA) knockdown cells were used for in vitro and in vivo experiments. The in vivo efficiencies of various sequential combinations were tested using nude/nude athymic mice and human SCLC xenograft models.

Results: Flavopiridol then doxorubicin sequential treatment was synergistic in the pRB-negative H69 cell line. By knocking down pRb with specific siRNA, H865 clones with complete pRb knockdown became sensitive to flavopiridol and doxorubicin combinations. pRb-deficient SCLC cell lines were highly sensitive to flavopiridol-induced apoptosis. pRb-positive H865 cells arrested in G0-G1 with flavopiridol exposure, whereas doxorubicin and all flavopiridol/doxorubicin combinations caused a G2-M block. In contrast, pRb-negative SCLC cells did not arrest in G0-G1 with flavopiridol exposure. Flavopiridol treatment alone did not have an in vivo antitumor effect, but sequential flavopiridol followed by doxorubicin treatment provided tumor growth control and a survival advantage in Rb-negative xenograft models, compared with the other sequential treatments.

Conclusions: Flavopiridol and doxorubicin sequential treatment induces potent in vitro and in vivo synergism in pRb-negative SCLC cells and should be clinically tested in tumors lacking functional pRB.
Data from Enhancing Cancer Vaccine Efficacy via Modulation of the Tumor Microenvironment
Authors: Disis Mary L.
Published: April 2023

Abstract
The development of therapeutic cancer vaccines is impacted by immunosuppressive elements in the tumor microenvironment. Most immunogenic cancer proteins are “self,” therefore, peripheral tolerance contributes substantially to tumor immune escape. Transforming growth factor β (TGFβ) actively modulates both inflammation and tolerance induction. Combining vaccination with agents that disarm TGFβ will enhance vaccine efficacy. (Clin Cancer Res 2009;15(21):1–3) (Clin Cancer Res 2009;15(21):6476–8)
Data from Phase I/II Trial of Bevacizumab and Radiotherapy for Locally Advanced Inoperable Colorectal Cancer: Vasculature-Independent Radiosensitizing Effect of Bevacizumab
Authors: Koukourakis Michael I. , Giatromanolaki Alexandra , Sheldon Helen , Buffa Francesca M. , Kouklakis George , Ragoussis Ioannis , Sivridis Efthimios , Harris Adrian L.
Published: April 2023

Abstract
Purpose: Antivascular endothelial growth factor therapy enhances the activity of radiotherapy in experimental models, and bevacizumab has therapeutic activity in patients with metastatic colorectal cancer.

Experimental Design: Twenty-two patients with locally advanced inoperable colorectal carcinomas (LA/I-CRC) were treated with conformal hypofractionated (3.4 Gy/fraction 15) split-course accelerated radiotherapy (biological equivalent dose, 67.2 Gy) supported with amifostine, capecitabine (600 mg/m2 daily, 5 days/week), and bevacizumab (5 mg/kg every 2 weeks, five cycles). Biopsies from nine patients, performed before and 1 week after bevacizumab administration, were analyzed for changes in mRNA expression with Illumina gene arrays.

Results: No serious grade 3 chemotherapy-related side effects were recorded. There was low acute toxicity, with moist perineal desquamation noted in 2 of 22 patients, diarrhea grade 2 to 3 in 5 of 22 patients, and severe proctalgia in 2 of 22 patients. One patient died from Fournier's gangrene before treatment completion. Within a median follow-up of 18 months, two patients with preradiotheraphy direct involvement of adjacent organs expressed recto-vaginal/perineal fistula. Out of 19 evaluable cases, 13 (68.5) showed complete response and 4 showed (21.1) partial response. Fourteen patients are alive with no evidence of loco-regional relapse. In the gene array analysis, 30 known genes associated with transcription factors, DNA repair, and proliferation were downregulated by bevacizumab. DUSP1 gene was the most consistently downregulated transcript.

Conclusions: The combination of radiotherapy with bevacizumab is feasible and results in a high rate of durable complete responses in patients with LA/I-CRC. Radiosensitization may occur through a direct effect on tumor cells followed by a wide scale suppression of transcription factors and genes involved in DNA repair and proliferation. (Clin Cancer Res 2009;15(22):706976)
Data from Ambulatory Monitoring Detects Sorafenib-Induced Blood Pressure Elevations on the First Day of Treatment
Authors: Maitland Michael L. , Kasza Kristen E. , Karrison Theodore , Moshier Kristin , Sit Laura , Black Henry R. , Undevia Samir D. , Stadler Walter M. , Elliott William J. , Ratain Mark J.
Published: April 2023

Abstract
Purpose: Hypertension is a mechanism-based toxicity of sorafenib and other cancer therapeutics that inhibit the vascular endothelial growth factor (VEGF) signaling pathway. This prospective, single-center, cohort study characterized ambulatory blood pressure monitoring as an early pharmacodynamic biomarker of VEGF signaling pathway inhibition by sorafenib.

Experimental Design: Fifty-four normotensive advanced cancer patients underwent 24-hour ambulatory blood pressure monitoring before and between days 6 and 10 of sorafenib therapy. After blood pressure changes were detected among the first cohort within 10 days, ambulatory blood pressure monitoring was done during the first 24 hours of treatment for the second cohort.

Results: For the entire patient population, the blood pressure increase [mean systolic, +10.8 mm Hg; 95% confidence interval (95% CI), 8.6-13.0; range, −5.2 to +28.7 mm Hg; mean diastolic, +8.0 mm Hg; 95% CI, 6.3-9.7; range, −4.4 to +27.1 mm Hg] was detected between days 6 and 10 (P < 0.0001 for both) and plateaued thereafter. Variability in blood pressure change did not associate with: age, body size, sex, self-reported race, baseline blood pressure, or steady-state sorafenib plasma concentrations. In the second cohort, the blood pressure elevation was detected during the first 24 hours (mean systolic, +8.2 mm Hg; 95% CI, 5.0-11.3; mean diastolic, +6.5 mm Hg; 95% CI, 4.7-8.3; P < 0.0001 for both).

Conclusions: Ambulatory blood pressure monitoring detects the blood pressure response to VEGF signaling pathway inhibition by sorafenib during the first 24 hours of treatment. The magnitude of blood pressure elevation is highly variable and unpredictable but could be important in optimizing the therapeutic index of VEGF signaling pathway inhibitor therapy. (Clin Cancer Res 2009;15(19):6250–7)
Data from Expression of a BTB/POZ Protein, NAC1, Is Essential for the Proliferation of Normal Cyclic Endometrial Glandular Cells and Is Up-regulated by Estrogen
Authors: Ishibashi Masako , Nakayama Kentaro , Yeasmin Shamima , Katagiri Atsuko , Iida Kouji , Nakayama Naomi , Miyazaki Kohji
Published: April 2023

Abstract
Purpose: The purpose of this study was to investigate the expression and localization of NAC1, a member of the BTB/POZ gene family in the human cyclic endometrium.

Experimental Design: NAC1 expression in normal cyclic endometrium was assessed by immunohistochemistry, and data on clinical variables were collected by retrospective chart review. To elucidate the molecular mechanisms of NAC1 expression in the normal endometrium endometrial carcinoma cell lines (Ishikawa, HHUA; ER+, PR+) and primary cultured normal endometria were tested in a sex steroid induction assay and a NAC1 knockdown assay using siRNA.

Results: Expression of NAC1 in glandular cells was significantly higher in the early and mid proliferative phases than in the other menstrual phases. Both NAC1 RNA and protein expression were up-regulated by treatment with 10 nmol/L 17β-Estradiol (E2) in Ishikawa, HHUA and primary cultured normal endometrial cells. The estrogen receptor antagonist ICI 182,780 significantly attenuated E2-induced NAC1 expression. NAC1 gene knockdown inhibited cell growth and induced apoptosis in Ishikawa, HHUA, and normal endometria, all of which expressed NAC1. Furthermore, NAC1 siRNA significantly abrogated estrogen-driven cellular proliferation in Ishikawa, HHUA, and primary cultured normal endometrial cells, whereas the control siRNA had no effect on cell growth in any of these cells.

Conclusions: These findings suggest that NAC1 is functionally involved in E2-induced cell growth of the normal endometrial glandular cells. Because NAC1 is thought to have oncogenic potential, the current findings may provide new insight into the mechanism of estrogen induced endometrial carcinogenesis.
Data from Tumor-Localized Ligation of CD3 and CD28 with Systemic Regulatory T-Cell Depletion Induces Potent Innate and Adaptive Antitumor Responses
Authors: Lee Chien-Hsin , Chiang Yi-Hsuan , Chang Shih-En , Chong Chin-Liew , Cheng Bing-Mae , Roffler Steve R.
Published: April 2023

Abstract
Purpose: Tumor-localized activation of immune cells by membrane-tethered anti-CD3 antibodies (CD3L) is under investigation to treat poorly immunogenic tumors. Here we sought to elucidate the mechanism of antitumor immunity elicited by CD3L.

Experimental Design: CD3L and CD86 were expressed on poorly immunogenic B16 melanoma cells (B16/3L86 cells) and the effect of various lymphocytes, including CD4+ and CD8+ T cells, natural killer T (NKT) cells, and regulatory T cells, on antitumor activity was investigated.

Results: B16/3L86 cells activated naïve T cells; suppressed tumor growth in subcutaneous, peritoneal, and metastasis models; and protected mice from rechallenge with B16 melanoma cells. However, in vivo antitumor activity against primary B16/3L86 tumors unexpectedly depended on NKT cells rather than CD4+ or CD8+ T cells. Treatment of mice with low-dose cyclophosphamide or anti-CD25 antibody to deplete regulatory T cells unmasked latent T-cell antitumor activity; the number of activated CD8+ T cells in tumors increased and B16/3L86 tumors were completely rejected in a CD8+ and CD4+ T-cell–dependent fashion. Furthermore, fibroblasts expressing CD3L and CD86 suppressed the growth of neighboring B16 cancer cells in vivo, and direct intratumoral injection of adenoviral vectors expressing CD3L and CD86 or CD3L and a membrane-tethered anti-CD28 antibody significantly suppressed the growth of subcutaneous tumors.

Conclusions: Tumor-located ligation of CD3 and CD28 can activate both innate (NKT cells) and adaptive (CD4+ and CD8+ T cells) responses to create a tumor-destructive environment to control tumor growth, but modulation of regulatory T cells is necessary to unmask local adaptive antitumor responses.
Data from Clinical and Biological Efficacy of Recombinant Human Interleukin-21 in Patients with Stage IV Malignant Melanoma without Prior Treatment: A Phase IIa Trial
Authors: Davis Ian D. , Brady Ben , Kefford Richard F. , Millward Michael , Cebon Jonathan , Skrumsager Birte K. , Mouritzen Ulrik , Hansen Lasse Tengbjerg , Skak Kresten , Lundsgaard Dorthe , Frederiksen Klaus Stensgaard , Kristjansen Paul E.G. , McArthur Grant
Published: April 2023

Abstract
Purpose: Human interleukin-21 (IL-21) is a class I cytokine that mediates activation of CD8+ T cells, natural killer (NK) cells, and other cell types. We report final clinical and biological results of a phase II study of recombinant human IL-21 (rIL-21) in patients with metastatic melanoma.

Experimental Design: Open-label, single-arm, two-stage trial. Eligibility criteria: unresectable metastatic melanoma, measurable disease by Response Evaluation Criteria in Solid Tumors, no prior systemic therapy (adjuvant IFN permitted), adequate major organ function, good performance status, no significant autoimmune disease, and life expectancy at least 4 months. Primary objective: antitumor efficacy (response rate). Secondary objectives: safety, blood biomarkers, and generation of anti-rIL-21 antibodies. rIL-21 (30 μg/kg/dose) was administered by intravenous bolus injection in 8-week cycles (5 dosing days followed by 9 days of rest for 6 weeks and then 2 weeks off treatment).

Results: Stage I of the study comprised 14 patients. One confirmed complete response (CR) was observed, and as per protocol, 10 more patients were accrued to stage II (total n = 24: 10 female and 14 male). Best tumor response included one confirmed CR and one confirmed partial response, both with lung metastases. Treatment was overall well tolerated. Biomarker analyses showed increases in serum soluble CD25, frequencies of CD25+ NK and CD8+ T cells, and mRNA for IFN-γ, perforin, and granzyme B in CD8+ T and NK cells.

Conclusions: rIL-21 administered at 30 μg/kg/d in 5-day cycles every second week is biologically active and well tolerated in patients with metastatic melanoma. Confirmed responses, including one CR, were observed.
Data from Stereotyped B-Cell Receptor Is an Independent Risk Factor of Chronic Lymphocytic Leukemia Transformation to Richter Syndrome
Authors: Rossi Davide , Spina Valeria , Cerri Michaela , Rasi Silvia , Deambrogi Clara , De Paoli Lorenzo , Laurenti Luca , Maffei Rossana , Forconi Francesco , Bertoni Francesco , Zucca Emanuele , Agostinelli Claudio , Cabras Antonello , Lucioni Marco , Martini Maurizio , Magni Michele , Deaglio Silvia , Ladetto Marco , Nomdedeu Josep F. , Besson Caroline , Ramponi Antonio , Canzonieri Vincenzo , Paulli Marco , Marasca Roberto , Larocca Luigi M. , Carbone Antonino , Pileri Stefano A. , Gattei Valter , Gaidano Gianluca
Published: April 2023

Abstract
Purpose: Few biological prognosticators are useful for prediction of Richter syndrome (RS), representing the transformation of chronic lymphocytic leukemia (CLL) to aggressive lymphoma. Stereotyped B-cell receptors (BCR) may have prognostic effect in CLL progression. We tested the prognostic effect of stereotyped BCR for predicting RS transformation.

Experimental Design: The prevalence of stereotyped BCR was compared in RS (n = 69) versus nontransformed CLL (n = 714) by a case-control analysis. Subsequently, the effect of stereotyped BCR at CLL diagnosis on risk of RS transformation was actuarially assessed in a consecutive CLL series (n = 753).

Results: RS (n = 69) displayed a higher prevalence of stereotyped BCR (P < 0.001) compared with nontransformed CLL. The actuarial risk of RS transformation was significantly higher in CLL carrying stereotyped BCR (P < 0.001). Among BCR subsets most represented in CLL, subset 8 using IGHV4-39/IGHD6-13/IGHJ5 carried the highest risk of RS transformation [hazard ratio (HR), 24.50; P < 0.001]. Multivariate analysis selected stereotyped BCR (HR, 3.33; P = 0.001) and IGHV4-39 usage (HR, 4.03; P = 0.004) as independent predictors of RS transformation. The combination of IGHV4-39 usage and stereotyped BCR in the same patient identified CLL with a very high risk of RS transformation (5-year risk, 68.7%). The risk carried by stereotyped BCR and IGHV4-39 usage was specific for RS transformation and had no effect on CLL progression without transformation.

Conclusions: Analysis of BCR features may help identify CLL patients at risk of RS. A close monitoring and a careful biopsy policy may help early recognition of RS in CLL patients using stereotyped BCR, particularly if combined with IGHV4-39.
Data from The Expression of Three Genes in Primary Non–Small Cell Lung Cancer Is Associated with Metastatic Spread to the Brain
Authors: Grinberg-Rashi Helena , Ofek Efrat , Perelman Marina , Skarda Jozef , Yaron Pnina , Hajdúch Marián , Jacob-Hirsch Jasmin , Amariglio Ninette , Krupsky Meir , Simansky David A. , Ram Zvi , Pfeffer Raphael , Galernter Ilana , Steinberg David M. , Ben-Dov Issachar , Rechavi Gideon , Izraeli Shai
Published: April 2023

Abstract
Purpose: Brain metastases affect 25% of patients with non–small cell lung cancer (NSCLC). We hypothesized that the expression of genes in primary NSCLC tumors could predict brain metastasis and be used for identification of high-risk patients, who may benefit from prophylactic therapy.

Experimental Design: The expression of 12 genes was measured by real-time quantitative reverse transcriptase PCR in 142 frozen NSCLC tissue samples. Univariate and multivariate Cox regression analysis was used to analyze the correlation between gene expression and the occurrence of brain metastasis. Immunohistochemistry on independent samples was used to verify the findings.

Results: A score based on the expression levels of three genes, CDH2 (N-cadherin), KIFC1, and FALZ, was highly predictive of brain metastasis in early and advanced lung cancer. The probability of remaining brain metastasis–free at 2 years after diagnosis was 90.0 ± 9.5% for patients with stage I/stage II tumors and low score compared with 62.7 ± 12% for patients with high score (P < 0.01). In patients with more advanced lung cancer, the brain metastasis–free survival at 24 months was 89% for patients with low score compared with only 37% in patients with high score (P < 0.02). These results were confirmed by immunohistochemical detection of N-cadherin in independent cohort of primary NSCLC.

Conclusions: The expression levels of three genes in primary NSCLC tumors may be used to identify patients at high risk for brain metastasis who may benefit from prophylactic therapy to the central nervous system.
Data from Microarray-Based Identification of Tenascin C and Tenascin XB, Genes Possibly Involved in Tumorigenesis Associated with Neurofibromatosis Type 1
Authors: Lévy Pascale , Ripoche Hugues , Laurendeau Ingrid , Lazar Vladimir , Ortonne Nicolas , Parfait Béatrice , Leroy Karen , Wechsler Janine , Salmon Isabelle , Wolkenstein Pierre , Dessen Philippe , Vidaud Michel , Vidaud Dominique , Bièche Ivan
Published: April 2023

Abstract
Purpose: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a complex variety of clinical manifestations. The hallmark of NF1 is the onset of heterogeneous (dermal or plexiform) benign neurofibromas. Plexiform neurofibromas can give rise to malignant peripheral nerve sheath tumors, which are resistant to conventional therapies.

Experimental Design: To identify new signaling pathways involved in the malignant transformation of plexiform neurofibromas, we applied a 22,000-oligonucleotide microarray approach to a series of plexiform neurofibromas and malignant peripheral nerve sheath tumors. Changes in the expression of selected genes were then confirmed by real-time quantitative reverse transcription-PCR.

Results: We identified two tenascin gene family members that were significantly deregulated in both human NF1-associated tumors and NF1-deficient primary cells: Tenascin C (TNC) was up-regulated whereas tenascin XB (TNXB) was down-regulated during tumor progression. TNC activation is mainly due to the up-regulation of large TNC splice variants. Immunohistochemical studies showed that TNC transcripts are translated into TNC protein in TNC-overexpressing tumors. Aberrant transcriptional activation of TNC seems to be principally mediated by activator protein transcription factor complexes.

Conclusion:TNXB and TNC may be involved in the malignant transformation of plexiform neurofibromas. Anti-TNC antibodies, already used successfully in clinical trials to treat malignant human gliomas, may be an appropriate new therapeutic strategy for NF1.
Data from The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Selective Cytotoxicity Against CD138-Positive Multiple Myeloma Cells In vitro and In vivo
Authors: Ikeda Hiroshi , Hideshima Teru , Fulciniti Mariateresa , Lutz Robert J. , Yasui Hiroshi , Okawa Yutaka , Kiziltepe Tanyel , Vallet Sonia , Pozzi Samantha , Santo Loredana , Perrone Giulia , Tai Yu-Tzu , Cirstea Diana , Raje Noopur S. , Uherek Christoph , Dälken Benjamin , Aigner Silke , Osterroth Frank , Munshi Nikhil , Richardson Paul , Anderson Kenneth C.
Published: April 2023

Abstract
Purpose: We investigated the antitumor effect of murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives against multiple myeloma (MM) cells in vitro and in vivo.

Experimental Design: We examined the growth inhibitory effect of BT062-SPDB-DM4, BT062-SMCC-DM1, and BT062-SPP-DM1 against MM cell lines and primary tumor cells from MM patients. We also examined in vivo activity of these agents in murine MM cell xenograft model of human and severe combined immunodeficient (SCID) mice bearing implant bone chips injected with human MM cells (SCID-hu model).

Results: Anti-CD138 immunoconjugates significantly inhibited growth of MM cell lines and primary tumor cells from MM patients without cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. In MM cells, they induced G2-M cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. Nonconjugated nBT062 completely blocked cytotoxicity induced by nBT062-maytansinoid conjugate, confirming that specific binding is required for inducing cytotoxicity. Moreover, nBT062-maytansinoid conjugates blocked adhesion of MM cells to bone marrow stromal cells. The coculture of MM cells with bone marrow stromal cells protects against dexamethasone-induced death but had no effect on the cytotoxicity of immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human MM and SCID-hu mouse model.

Conclusion: These results provide the preclinical framework supporting evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in MM.
Data from Isolation and Molecular Profiling of Bone Marrow Micrometastases Identifies TWIST1 as a Marker of Early Tumor Relapse in Breast Cancer Patients
Authors: Watson Mark A. , Ylagan Lourdes R. , Trinkaus Kathryn M. , Gillanders William E. , Naughton Michael J. , Weilbaecher Katherine N. , Fleming Timothy P. , Aft Rebecca L.
Published: April 2023

Abstract
Purpose: Micrometastatic cells detected in the bone marrow have prognostic significance in breast cancer. These cells are heterogeneous and likely do not exhibit uniform biological behavior. To understand the molecular diversity of disseminated cancer cells that reside in bone marrow, we enriched this cell population and did global gene expression profiling in the context of a prospective clinical trial involving women with clinical stage II/III breast cancer undergoing neoadjuvant chemotherapy.

Experimental Design: Enrichment of TACSTD1 (EpCAM)–expressing cells from bone marrow of breast cancer patients was achieved using immunomagnetic beads. Gene expression profiles were compared between enriched cell populations and whole bone marrow from 5 normal volunteers and 23 breast cancer patients after neoadjuvant chemotherapy treatment. Enriched cells from bone marrow samples of breast cancer patients before treatment or at 1 year follow-up were also analyzed (total of 87 data sets). The expression of transcripts specifically detected in enriched cell populations from breast cancer patients was correlated with 1-year clinical outcome using quantitative reverse transcription-PCR in an independent cohort of bone marrow samples.

Results: Analysis of EpCAM-enriched bone marrow cells revealed specific expression of a subgroup of transcripts, including the metastasis regulator, TWIST1. Most transcripts identified, including TWIST1, were not expressed in enriched populations of bone marrow from normal volunteers, suggesting that this expression profile reflects a signature of breast cancer bone marrow micrometastases that persist after chemotherapy. In an independent set of bone marrow samples obtained before any treatment, TWIST1 expression correlated with early disease relapse.

Conclusions: Disseminated breast cancer cells present in bone marrow after chemotherapy possess unique transcriptional signatures. Genes whose expression is overrepresented in these cell populations, such as TWIST1, may prove to be excellent markers of early distant relapse in breast cancer patients.
Data from Inhibition of Poly(ADP-Ribose) Polymerase-1 Enhances Temozolomide and Topotecan Activity against Childhood Neuroblastoma
Authors: Daniel Rachel A. , Rozanska Agata L. , Thomas Huw D. , Mulligan Evan A. , Drew Yvette , Castelbuono Deborah J. , Hostomsky Zdenek , Plummer E. Ruth , Boddy Alan V. , Tweddle Deborah A. , Curtin Nicola J. , Clifford Steven C.
Published: April 2023

Abstract
Purpose: High-risk neuroblastoma is characterized by poor survival rates, and the development of improved therapeutic approaches is a priority. Temozolomide and topotecan show promising clinical activity against neuroblastoma. Poly(ADP-ribose) polymerase-1 (PARP-1) promotes DNA repair and cell survival following genotoxic insult; we postulated that its inhibition may enhance the efficacy of these DNA-damaging drugs in pediatric cancers.

Experimental Design: We evaluated the chemosensitizing properties of the PARP inhibitor AG014699 (Pfizer, Inc.) in combination with temozolomide and topotecan, against human neuroblastoma cells and xenografts, alongside associated pharmacologic and toxicologic indices.

Results: Addition of PARP-inhibitory concentrations of AG014699 significantly potentiated growth inhibition by both topotecan (1.5- to 2.3-fold) and temozolomide (3- to 10-fold) in vitro, with equivalent effects confirmed in clonogenic assays. In two independent in vivo models (NB1691 and SHSY5Y xenografts), temozolomide caused a xenograft growth delay, which was enhanced by co-administration of AG014699, and resulted in complete and sustained tumor regression in the majority (6 of 10; 60%) of cases. Evidence of enhanced growth delay by topotecan/AG014699 co-administration was observed in NB1691 xenografts. AG014699 metabolites distributed rapidly into the plasma (Cmax, 1.2-1.9 nmol/L at 30 min) and accumulated in xenograft tissues (Cmax, 1-2 μmol/L at 120 min), associated with a sustained suppression of PARP-1 enzyme activity. Doses of AG014699 required for potentiation were not toxic per se.

Conclusions: These data show enhancement of temozolomide and topotecan efficacy by PARP inhibition in neuroblastoma. Coupled with the acceptable pharmacokinetic, pharmacodynamic, and toxicity profiles of AG014699, our findings provide strong rationale for investigation of PARP inhibitors in pediatric early clinical studies.
Data from Methylation Induced Gene Silencing of HtrA3 in Smoking-Related Lung Cancer
Authors: Beleford Daniah , Liu Zhixue , Rattan Ramandeep , Quagliuolo Lucio , Boccellino Mariarosaria , Baldi Alfonso , Maguire Jacie , Staub Julie , Molina Julian , Shridhar Viji
Published: April 2023

Abstract
Purpose: Some 85% of lung cancers are smoking related. Here, we investigate the role of serine protease HtrA3 in smoking-related lung cancer.

Experimental Design: We assess HtrA3 methylation and its corresponding expression in the human bronchial cell line BEAS-2B following cigarette smoke carcinogen treatment, in lung cancer cell lines and in primary lung tumors from light, moderate, and heavy smokers. We also show the effects of HtrA3 downregulation on MTT reduction and clonogenic survival with etoposide and cisplatin treatment and the corresponding effects of HtrA3 re-expression during treatment.

Results: We show for the first time that HtrA3 expression is reduced or completely lost in over 50% of lung cancer cell lines and primary lung tumors from heavy smokers. Treatment of HtrA3-deficient cell lines with 5-aza-2′-deoxycytidine resulted in a dose-dependent increase in HtrA3 transcription. Further, sequence analysis of bisulfite-modified DNA from lung cancer cell lines and from primary lung tumors showed an increased frequency of methylation within the first exon of HtrA3 with a corresponding loss of HtrA3 expression, particularly in tumors from smokers. In BEAS-2B, treatment with the cigarette smoke carcinogen 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone resulted in HtrA3 downregulation with a corresponding increase in methylation. Additional studies indicate resistance to etoposide and cisplatin cytotoxicity as a functional consequence of HtrA3 loss. Finally, immunohistochemical analysis of primary lung tumors revealed a strong correlation between low HtrA3 expression and heavy smoking history.

Conclusions: Collectively, these results suggest that cigarette smoke–induced methylation of HtrA3 could contribute to the etiology of chemoresistant disease in smoking-related lung cancer. Clin Cancer Res; 16(2); 398–409