E2F1 plays a critical role in cell-cycle progression and the induction of apoptosis in response to DNA damage. The latest evidence has uncovered that this tumor suppressor is most relevant for cancer progression and chemoresistance. Increased abundance of E2F1 triggers invasion and metastasis by activating growth receptor signaling pathways, which in turn promote an antiapoptotic tumor environment. The data shed light on the molecular mechanisms underlying E2F1-induced prometastatic activity and predict its radical switch from a mediator of cell death toward an accelerator of tumor progression. This raises the perspective of new drug targets at late-stage cancer. Cancer Res; 72(3); 571–5. ©2012 AACR.

The origins of tumor-propagating neoplastic stem-like cells [cancer stem cells (CSC)] and their relationship to the bulk population of tumor cells that lack stem-like tumor-propagating features (i.e., transit-amplifying cancer progenitor cells) remain unclear. Recent findings from multiple laboratories show that cancer progenitor cells have the capacity to dedifferentiate and acquire a stem-like phenotype in response to either genetic manipulation or environmental cues. These findings suggest that CSCs and relatively differentiated progenitors coexist in dynamic equilibrium and are subject to bidirectional conversion. In this review, we discuss emerging concepts regarding the stem-like phenotype, its acquisition by cancer progenitor cells, and the molecular mechanisms involved. Understanding the dynamic equilibrium between CSCs and cancer progenitor cells is critical for the development of therapeutic strategies to deplete tumors of their tumor-propagating and treatment-resistant cell subpopulations. Cancer Res; 72(3); 576–80. ©2012 AACR.

The immunosuppressive microenvironment in tumors hampers the induction of antitumor immunity by vaccines or immunotherapies. Toll-like receptor (TLR) ligands have the potential to treat tumors, but they can exert a mixture of positive and negative effects on inflammation in the tumor microenvironment. In this study, we show that specific small molecule inhibitors of phosphoinositide 3-kinase (PI3K) relieve immunosuppression to heighten the proinflammatory effects of TLR ligands that support antitumor immunity. Multiple strategies to inhibit PI3K in dendritic cells (DC) each led to suppression of interleukin (IL)-10 and TGF-β but did affect IL-12 or IL-1β induction by the TLR5 ligand flagellin. In three different mouse models of cancer, combining flagellin with a class I PI3K inhibitor, either with or without a DC vaccine, delayed tumor growth and increased survival, with some animals exhibiting complete rejection and resistance to secondary challenge. Tumor growth suppression was associated with increased accumulation of polyfunctional T cells that secreted multiple effector cytokines, including IFN-γ, IL-17, and IL-2. Therapeutic protection was abolished in mice deficient in IL-17 or deprived of IFN-γ. Together, our results indicate that PI3K inhibition heighten the antitumor properties of TLR ligands, eliciting tumor regression directly but also indirectly by relieving suppressive signals that restrict potent antitumor T-cell responses. These findings suggest important uses for PI3K inhibitors in heightening responses to cancer immunotherapy and immunochemotherapy. Cancer Res; 72(3); 581–91. ©2011 AACR.

Toll-like receptor (TLR) ligands may be a valuable tool to promote antitumor responses by reinforcing antitumor immunity. In addition to their expression in immune cells, functional TLRs are also expressed by many cancer cells, but their significance has been controversial. In this study, we examined the action of TLR ligands on tumor pathophysiology as a result of direct tumor cell effects. B16 murine melanoma cells were stimulated in vitro with a TLR4 ligand (LPS-B16) prior to inoculation into TLR4-deficient mice (Tlr4 lps-del). Under such conditions, B16 cells yielded smaller tumors than nonstimulated B16 cells. The apoptosis/proliferation balance of the cells was not modified by TLR ligand treatment, nor was this effect compromised in immunocompromised nude mice. Mechanistic investigations revealed that IFNβ was the critical factor produced by TLR4-activated tumor cells in mediating their in vivo outgrowth. Transcriptional analysis showed that TLR4 activation on B16 cells induced changes in the expression of type I IFN and type I IFN-related genes. Most importantly, culture supernatants from LPS-B16 cells improved the maturation of bone marrow–derived dendritic cells (BMDC) from TLR4-deficient mice, upregulating the expression of interleukin-12 and costimulatory molecules on those cells. BMDC maturation was blunted by addition of an IFNβ-neutralizing antibody. Moreover, tumor growth inhibition observed in LPS-B16 tumors was abrogated in IFNAR1-deficient mice lacking a functional type I IFN receptor for binding IFN. Together, our findings show that tumor cells can be induced through the TLR4 pathway to produce IFN and positively contribute to the antitumoral immune response. Cancer Res; 72(3); 592–603. ©2011 AACR.

S100A7/psoriasin, a member of the epidermal differentiation complex, is widely overexpressed in invasive estrogen receptor (ER)α-negative breast cancers. However, it has not been established whether S100A7 contributes to breast cancer growth or metastasis. Here, we report the consequences of its expression on inflammatory pathways that impact breast cancer growth. Overexpression of human S100A7 or its murine homologue mS100a7a15 enhanced cell proliferation and upregulated various proinflammatory molecules in ERα-negative breast cancer cells. To examine in vivo effects, we generated mice with an inducible form of mS100a7a15 (MMTV-mS100a7a15 mice). Orthotopic implantation of MVT-1 breast tumor cells into the mammary glands of these mice enhanced tumor growth and metastasis. Compared with uninduced transgenic control mice, the mammary glands of mice where mS100a7a15 was induced exhibited increased ductal hyperplasia and expression of molecules involved in proliferation, signaling, tissue remodeling, and macrophage recruitment. Furthermore, tumors and lung tissues obtained from these mice showed further increases in prometastatic gene expression and recruitment of tumor-associated macrophages (TAM). Notably, in vivo depletion of TAM inhibited the effects of mS100a7a15 induction on tumor growth and angiogenesis. Furthermore, introduction of soluble hS100A7 or mS100a7a15 enhanced chemotaxis of macrophages via activation of RAGE receptors. In summary, our work used a powerful new model system to show that S100A7 enhances breast tumor growth and metastasis by activating proinflammatory and metastatic pathways. Cancer Res; 72(3); 604–15. ©2011 AACR.

Progression of primary prostate cancer to castration-resistant prostate cancer (CRPC) is associated with numerous genetic and epigenetic alterations that are thought to promote survival at metastatic sites. In this study, we investigated gene copy number and CpG methylation status in CRPC to gain insight into specific pathophysiologic pathways that are active in this advanced form of prostate cancer. Our analysis defined and validated 495 genes exhibiting significant differences in CRPC in gene copy number, including gains in androgen receptor (AR) and losses of PTEN and retinoblastoma 1 (RB1). Significant copy number differences existed between tumors with or without AR gene amplification, including a common loss of AR repressors in AR-unamplified tumors. Simultaneous gene methylation and allelic deletion occurred frequently in RB1 and HSD17B2, the latter of which is involved in testosterone metabolism. Lastly, genomic DNA from most CRPC was hypermethylated compared with benign prostate tissue. Our findings establish a comprehensive methylation signature that couples epigenomic and structural analyses, thereby offering insights into the genomic alterations in CRPC that are associated with a circumvention of hormonal therapy. Genes identified in this integrated genomic study point to new drug targets in CRPC, an incurable disease state which remains the chief therapeutic challenge. Cancer Res; 72(3); 616–25. ©2011 AACR.

Subcellular trafficking of key oncogenic signal pathway components is likely to be crucial for neoplastic transformation, but little is known about how such trafficking processes are spatially controlled. In this study, we show how Ras activation causes aberrant nuclear localization of phosphorylated mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK; MEK) MEK1/2 to drive neoplastic transformation. Phosphorylated MEK1/2 was aberrantly located within the nucleus of primary colorectal tumors and human colon cancer cells, and oncogenic activation of Ras was sufficient to induce nuclear accumulation of phosphorylated MEK1/2 and ERK1/2 in intestinal epithelial cells. Enforced nuclear localization of MEK1 in epithelial cells or fibroblasts was sufficient for hyperactivation of ERK1/2, thereby driving cell proliferation, chromosomal polyploidy, and tumorigenesis. Notably, Ras-induced nuclear accumulation of activated MEK1/2 was reliant on downregulation of the spatial regulator Sef, the reexpression of which was sufficient to restore normal MEK1/2 localization and a reversal of Ras-induced proliferation and tumorigenesis. Taken together, our findings indicate that Ras-induced downregulation of Sef is an early oncogenic event that contributes to genetic instability and tumor progression by sustaining nuclear ERK1/2 signaling. Cancer Res; 72(3); 626–35. ©2012 AACR.

Although monoallelic expression (MAE) is a frequent genomic event in normal tissues, its role in tumorigenesis remains unclear. Here we carried out single-nucleotide polymorphism arrays on DNA and RNA from a large cohort of pediatric and adult brain tumor tissues to determine the genome-wide rate of MAE, its role in specific cancer-related genes, and the clinical consequences of MAE in brain tumors. We also used targeted genotyping to examine the role of tumor-related genes in brain tumor development and specifically examined the clinical consequences of MAE at TP53 and IDH1. The genome-wide rate of tumor MAE was higher than in previously described normal tissue and increased with specific tumor grade. Oncogenes, but not tumor suppressors, exhibited significantly higher MAE in high-grade compared with low-grade tumors. This method identified nine novel genes highly associated with MAE. Within cancer-related genes, MAE was gene specific; hTERT was most significantly affected, with a higher frequency of MAE in adult and advanced tumors. Clinically, MAE at TP53 exists only in mutated tumors and increases with tumor aggressiveness. MAE toward the normal allele at IDH1 conferred worse survival even in IDH1 mutated tumors. Taken together, our findings suggest that MAE is tumor and gene specific, frequent in brain tumor subtypes, and may be associated with tumor progression/aggressiveness. Further exploration of MAE at relevant genes may contribute to better understanding of tumor development and determine survival in brain tumor patients. Cancer Res; 72(3); 636–44. ©2011 AACR.

Brain tissue biopsies are required to histologically diagnose brain tumors, but current approaches are limited by tissue characterization at the time of surgery. Emerging technologies such as mass spectrometry imaging can enable a rapid direct analysis of cancerous tissue based on molecular composition. Here, we illustrate how gliomas can be rapidly classified by desorption electrospray ionization-mass spectrometry (DESI-MS) imaging, multivariate statistical analysis, and machine learning. DESI-MS imaging was carried out on 36 human glioma samples, including oligodendroglioma, astrocytoma, and oligoastrocytoma, all of different histologic grades and varied tumor cell concentration. Gray and white matter from glial tumors were readily discriminated and detailed diagnostic information could be provided. Classifiers for subtype, grade, and concentration features generated with lipidomic data showed high recognition capability with more than 97% cross-validation. Specimen classification in an independent validation set agreed with expert histopathology diagnosis for 79% of tested features. Together, our findings offer proof of concept that intraoperative examination and classification of brain tissue by mass spectrometry can provide surgeons, pathologists, and oncologists with critical and previously unavailable information to rapidly guide surgical resections that can improve management of patients with malignant brain tumors. Cancer Res; 72(3); 645–54. ©2011 AACR.

Caveolin-1 (Cav-1), a principal structural component of caveolar membrane domains, contributes to cancer development but its precise functional roles and regulation remain unclear. In this study, we determined the oncogenic function of Cav-1 in preclinical models of pancreatic cancer and in human tissue specimens. Cav-1 expression levels correlated with metastatic potential and epithelial–mesenchymal transition (EMT) in both mouse and human pancreatic cancer cells. Elevated levels in cells promoted EMT, migration, invasion, and metastasis in animal models, whereas RNA interference (RNAi)-mediated knockdown inhibited these processes. We determined that levels of Cav-1 and the Forkhead transcription factor FoxM1 correlated directly in pancreatic cancer cells and tumor tissues. Enforced expression of FoxM1 increased Cav-1 levels, whereas RNAi-mediated knockdown of FoxM1 had the opposite effect. FoxM1 directly bound to the promoter region of Cav-1 gene and positively transactivated its activity. Collectively, our findings defined Cav-1 as an important downstream oncogenic target of FoxM1, suggesting that dysregulated signaling of this novel FoxM1-Cav-1 pathway promotes pancreatic cancer development and progression. Cancer Res; 72(3); 655–65. ©2011 AACR.

XPC protein is a critical DNA damage recognition factor in nucleotide excision repair for which genetic deficiency confers a predisposition to cancer. In this study, we show that XPC has a function that is independent of its canonical function in DNA repair, potentially altering the interpretation of how XPC deficiency leads to heightened cancer susceptibility. XPC enhances apoptosis induced by DNA damage in a p53 nullizygous background, acting downstream of mitochondrial permeabilization and upstream of caspase-9 activation in the DNA damage–induced apoptosis cascade. We found that deficiency in XPC upregulated production of the short isoform of caspase-2 (casp-2S). This upregulation occurred at both protein and mRNA levels through repression of the caspase-2 promoter by XPC protein. Targeted RNAi-mediated downregulation of casp-2S–enhanced UV-induced apoptosis as well as activation of caspase-9 and caspase-6 in XPC-deficient cells, but not in XPC-proficient cells. In addition, XPC overexpression in various p53-deficient cancer cells resistant to cisplatin improved their sensitivity to cisplatin-induced apoptosis. Given that casp-2S functions as an antiapoptotic protein, our findings suggest that XPC enhances DNA damage–induced apoptosis through inhibition of casp-2S transcription. Together, these findings offer a mechanistic foundation to overcome the resistance of highly prevalent p53-deficient tumors to cell death induced by DNA-damaging therapeutic agents, by targeting strategies that inhibit the expression or function of casp-2S. Cancer Res; 72(3); 666–75. ©2011 AACR.

In women, naturally induced anti–human papilloma virus (HPV) serum antibodies are a likely marker of host immune protection against subsequent HPV acquisition and progression to precancerous lesions and cancers. However, it is unclear whether the same is the case in men. In this study, we assessed the risk of incident genital infection and 6-month persistent genital infection with HPV16 in relation to baseline serostatus in a cohort of 2,187 men over a 48-month period. Genital swabs were collected every 6 months and tested for HPV presence. Incidence proportions by serostatus were calculated at each study visit to examine whether potential immune protection attenuated over time. Overall, incidence proportions did not differ statistically between baseline seropositive and seronegative men at any study visit or over the follow-up period. The risk of incident and 6-month persistent infection was not associated with baseline serostatus or baseline serum antibody levels in the cohort. Our findings suggest that baseline HPV seropositivity in men is not associated with reduced risk of subsequent HPV16 acquisition. Thus, prevalent serum antibodies induced by prior infection may not be a suitable marker for subsequent immune protection against genital HPV16 acquisition in men. Cancer Res; 72(3); 676–85. ©2011 AACR.

Although the mitochondrial genome exhibits high mutation rates, common mitochondrial DNA (mtDNA) variation has not been consistently associated with pancreatic cancer. Here, we comprehensively examined mitochondrial genomic variation by sequencing the mtDNA of participants (cases = 286, controls = 283) in a San Francisco Bay Area pancreatic cancer case–control study. Five common variants were associated with pancreatic cancer at nominal statistical significance (P < 0.05) with the strongest finding for mt5460g in the ND2 gene [OR = 3.9; 95% confidence interval (CI), 1.5–10; P = 0.004] which encodes an A331T substitution. Haplogroup K was nominally associated with reduced pancreatic cancer risk (OR = 0.32; 95% CI, 0.13–0.76; P = 0.01) when compared with the most common haplogroup, H. A total of 19 haplogroup-specific rare variants yielded nominal statistically significant associations (P < 0.05) with pancreatic cancer risk, with the majority observed in genes involved in oxidative phosphorylation. Weighted-sum statistics were used to identify an aggregate effect of variants in the 22 mitochondrial tRNAs on pancreatic cancer risk (P = 0.02). While the burden of singleton variants in the HV2 and 12S RNA regions was three times higher among European haplogroup N cases than controls, the prevalence of singleton variants in ND4 and ND5 was two to three times higher among African haplogroup L cases than in controls. Together, the results of this study provide evidence that aggregated common and rare variants and the accumulation of singleton variants are important contributors to pancreatic cancer risk. Cancer Res; 72(3); 686–95. ©2011 AACR.

Endogenous estrogens and estrogen metabolism are hypothesized to be associated with premenopausal breast cancer risk but evidence is limited. We examined 15 urinary estrogens/estrogen metabolites and breast cancer risk among premenopausal women in a case–control study nested within the Nurses' Health Study II (NHSII). From 1996 to 1999, urine was collected from 18,521 women during the mid-luteal menstrual phase. Breast cancer cases (N = 247) diagnosed between collection and June 2005 were matched to two controls each (N = 485). Urinary estrogen metabolites were measured by liquid chromatography-tandem mass spectrometry and adjusted for creatinine level. Relative risks (RR) and 95% confidence intervals (CI) were estimated by multivariate conditional logistic regression. Higher urinary estrone and estradiol levels were strongly significantly associated with lower risk (top vs. bottom quartile RR: estrone = 0.52; 95% CI, 0.30–0.88; estradiol = 0.51; 95% CI, 0.30–0.86). Generally inverse, although nonsignificant, patterns also were observed with 2- and 4-hydroxylation pathway estrogen metabolites. Inverse associations generally were not observed with 16-pathway estrogen metabolites and a significant positive association was observed with 17-epiestriol (top vs. bottom quartile RR = 1.74; 95% CI, 1.08–2.81; Ptrend = 0.01). In addition, there was a significant increased risk with higher 16-pathway/parent estrogen metabolite ratio (comparable RR = 1.61; 95% CI, 0.99–2.62; Ptrend = 0.04). Other pathway ratios were not significantly associated with risk except parent estrogen metabolites/non–parent estrogen metabolites (comparable RR = 0.58; 95% CI, 0.35–0.96; Ptrend = 0.03). These data suggest that most mid-luteal urinary estrogen metabolite concentrations are not positively associated with breast cancer risk among premenopausal women. The inverse associations with parent estrogen metabolites and the parent estrogen metabolite/non–parent estrogen metabolite ratio suggest that women with higher urinary excretion of parent estrogens are at lower risk. Cancer Res; 72(3); 696–706. ©2011 AACR.

The detection of tumor suppressor gene promoter methylation in sputum-derived exfoliated cells predicts early lung cancer. Here, we identified genetic determinants for this epigenetic process and examined their biologic effects on gene regulation. A two-stage approach involving discovery and replication was used to assess the association between promoter hypermethylation of a 12-gene panel and common variation in 40 genes involved in carcinogen metabolism, regulation of methylation, and DNA damage response in members of the Lovelace Smokers Cohort (N = 1,434). Molecular validation of three identified variants was conducted using primary bronchial epithelial cells. Association of study-wide significance (P < 8.2 × 10−5) was identified for rs1641511, rs3730859, and rs1883264 in TP53, LIG1, and BIK, respectively. These single-nucleotide polymorphisms (SNP) were significantly associated with altered expression of the corresponding genes in primary bronchial epithelial cells. In addition, rs3730859 in LIG1 was also moderately associated with increased risk for lung cancer among Caucasian smokers. Together, our findings suggest that genetic variation in DNA replication and apoptosis pathways impacts the propensity for gene promoter hypermethylation in the aerodigestive tract of smokers. The incorporation of genetic biomarkers for gene promoter hypermethylation with clinical and somatic markers may improve risk assessment models for lung cancer. Cancer Res; 72(3); 707–15. ©2011 AACR.

Bcl-2 is an antiapoptotic protein that has also been found to function as a proangiogenic signaling molecule. Improvements in antiangiogenic therapy can be engendered by metronomic dosing. Thus, we hypothesized that BH3-mimetic drugs that antagonize Bcl-2 family proteins may exert a greater efficacy when dosed metronomically. To examine this hypothesis, we employed AT101, an orally available and well-tolerated BH3-mimetic drug that has been established as effective. In a mouse xenograft model of human squamous cell carcinomas (SCC) that includes a humanized vasculature, we explored the effects of docetaxel in combination with either daily (metronomic) or weekly (bolus) doses of AT101. In addition, we explored the effect of single or combination therapy on angiogenesis and survival of endothelial or SCC cells in vitro. Metronomic AT101 therapy increased mouse survival, decreased tumor mitotic index, and decreased tumor microvessel density, compared with bolus therapy. Therapeutic potentiation was achieved by similar overall drug exposure and without altering systemic toxicities. Combinations of AT101 and docetaxel produced additive toxicity in both endothelial and SCC tumor cells. Notably, subapoptotic concentrations of AT101 potently inhibited the angiogenic potential of endothelial cells. Taken together, our findings unveil the efficacious benefits that can be achieved by metronomic delivery of BH3-mimetic drugs, in particular suggesting that SCC patients with might benefit from low-dose continuous administration of these drugs. Cancer Res; 72(3); 716–25. ©2011 AACR.

Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that promotes breast cancer progression by diverse mechanisms that remain somewhat unclear. Here we report pharmacologic evidence of a critical role for sphingosine kinase 1 (SphK1) in producing S1P and mediating tumor-induced hemangiogenesis and lymphangiogenesis in a murine model of breast cancer metastasis. S1P levels increased both in the tumor and the circulation. In agreement, serum S1P levels were significantly elevated in stage IIIA human breast cancer patients, compared with age/ethnicity-matched healthy volunteers. However, treatment with the specific SphK1 inhibitor SK1-I suppressed S1P levels, reduced metastases to lymph nodes and lungs, and decreased overall tumor burden of our murine model. Both S1P and angiopoietin 2 (Ang2) stimulated hemangiogenesis and lymphangiogenesis in vitro, whereas SK1-I inhibited each process. We quantified both processes in vivo from the same specimen by combining directed in vivo angiogenesis assays with fluorescence-activated cell sorting, thereby confirming the results obtained in vitro. Notably, SK1-I decreased both processes not only at the primary tumor but also in lymph nodes, with peritumoral lymphatic vessel density reduced in SK1-I–treated animals. Taken together, our findings show that SphK1-produced S1P is a crucial mediator of breast cancer–induced hemangiogenesis and lymphangiogenesis. Our results implicate SphK1 along with S1P as therapeutic targets in breast cancer. Cancer Res; 72(3); 726–35. ©2012 AACR.

The threonine endopeptidase Taspase1 has a critical role in cancer cell proliferation and apoptosis. In this study, we developed and evaluated small molecule inhibitors of Taspase1 as a new candidate class of therapeutic modalities. Genetic deletion of Taspase1 in the mouse produced no overt deficiencies, suggesting the possibility of a wide therapeutic index for use of Taspase1 inhibitors in cancers. We defined the peptidyl motifs recognized by Taspase1 and conducted a cell-based dual-fluorescent proteolytic screen of the National Cancer Institute diversity library to identify Taspase1 inhibitors (TASPIN). On the basis of secondary and tertiary screens the 4-[(4-arsonophenyl)methyl]phenyl] arsonic acid NSC48300 was determined to be the most specific active compound. Structure–activity relationship studies indicated a crucial role for the arsenic acid moiety in mediating Taspase1 inhibition. Additional fluorescence resonance energy transfer–based kinetic analysis characterized NSC48300 as a reversible, noncompetitive inhibitor of Taspase1 (Ki = 4.22 μmol/L). In the MMTV-neu mouse model of breast cancer and the U251 xenograft model of brain cancer, NSC48300 produced effective tumor growth inhibition. Our results offer an initial preclinical proof-of-concept to develop TASPINs for cancer therapy. Cancer Res; 72(3); 736–46. ©2011 AACR.

Anorexia-cachexia syndrome (ACS) is a major determinant of cancer-related death that causes progressive body weight loss due to depletion of skeletal muscle mass and body fat. Here, we report the development of a novel preclinical murine model of ACS in which lymphomas harbor elevated Myc and activated mTOR signaling. The ACS phenotype in this model correlated with deregulated expression of a number of cytokines, including elevated levels of interleukin-10 which was under the direct translational control of mTOR. Notably, pharmacologic intervention to impair protein synthesis restored cytokine production to near-normal levels, delayed ACS progression, and extended host survival. Together, our findings suggest a new paradigm to treat ACS by strategies which target protein synthesis to block the production of procachexic factors. Cancer Res; 72(3); 747–56. ©2011 AACR.

Despite recent advances in targeted treatments for multiple myeloma, optimal molecular therapeutic targets have yet to be identified. To functionally identify critical molecular targets, we conducted a genome-scale lethality study in multiple myeloma cells using siRNAs. We validated the top 160 lethal hits with four siRNAs per gene in three multiple myeloma cell lines and two non-myeloma cell lines, cataloging a total of 57 potent multiple myeloma survival genes. We identified the Bcl2 family member MCL1 and several 26S proteasome subunits among the most important and selective multiple myeloma survival genes. These results provided biologic validation of our screening strategy. Other essential targets included genes involved in RNA splicing, ubiquitination, transcription, translation, and mitosis. Several of the multiple myeloma survival genes, especially MCL1, TNK2, CDK11, and WBSCR22, exhibited differential expression in primary plasma cells compared with other human primary somatic tissues. Overall, the most striking differential functional vulnerabilities between multiple myeloma and non–multiple myeloma cells were found to occur within the 20S proteasome subunits, MCL1, RRM1, USP8, and CKAP5. We propose that these genes should be investigated further as potential therapeutic targets in multiple myeloma. Cancer Res; 72(3); 757–68. ©2011 AACR.

Resistance to anthracyclines and other chemotherapeutics due to P-glycoprotein (pgp)-mediated export is a frequent problem in cancer treatment. Here, we report that iron oxide–titanium dioxide core-shell nanocomposites can serve as efficient carriers for doxorubicin to overcome this common mechanism of drug resistance in cancer cells. Doxorubicin nanocarriers (DNC) increased effective drug uptake in drug-resistant ovarian cells. Mechanistically, doxorubicin bound to the TiO2 surface by a labile bond that was severed upon acidification within cell endosomes. Upon its release, doxorubicin traversed the intracellular milieu and entered the cell nucleus by a route that evaded pgp-mediated drug export. Confocal and X-ray fluorescence microscopy and flow cytometry were used to show the ability of DNCs to modulate transferrin uptake and distribution in cells. Increased transferrin uptake occurred through clathrin-mediated endocytosis, indicating that nanocomposites and DNCs may both interfere with removal of transferrin from cells. Together, our findings show that DNCs not only provide an alternative route of delivery of doxorubicin to pgp-overexpressing cancer cells but also may boost the uptake of transferrin-tagged therapeutic agents. Cancer Res; 72(3); 769–78. ©2011 AACR.

The protein kinase BRAF is a key component of the RAS–RAF signaling pathway which plays an important role in regulating cell proliferation, differentiation, and survival. Mutations in BRAF at codon 600 promote catalytic activity and are associated with 8% of all human (solid) tumors, including 8% to 10% of colorectal cancers (CRC). Here, we report the preclinical characterization of vemurafenib (RG7204; PLX4032; RO5185426), a first-in-class, specific small molecule inhibitor of BRAFV600E in BRAF-mutated CRC cell lines and tumor xenograft models. As a single agent, vemurafenib shows dose-dependent inhibition of ERK and MEK phosphorylation, thereby arresting cell proliferation in BRAFV600-expressing cell lines and inhibiting tumor growth in BRAFV600E bearing xenograft models. Because vemurafenib has shown limited single-agent clinical activity in BRAFV600E-mutant metastatic CRC, we therefore explored a range of combination therapies, with both standard agents and targeted inhibitors in preclinical xenograft models. In a BRAF-mutant CRC xenograft model with de novo resistance to vemurafenib (RKO), tumor growth inhibition by vemurafenib was enhanced by combining with an AKT inhibitor (MK-2206). The addition of vemurafenib to capecitabine and/or bevacizumab, cetuximab and/or irinotecan, or erlotinib resulted in increased antitumor activity and improved survival in xenograft models. Together, our findings suggest that the administration of vemurafenib in combination with standard-of-care or novel targeted therapies may lead to enhanced and sustained clinical antitumor efficacy in CRCs harboring the BRAFV600E mutation. Cancer Res; 72(3); 779–89. ©2011 AACR.

To overcome drug resistance and reduce the side effects of cisplatin, a widely used antineoplastic agent, major efforts have been made to develop next generation platinum-based anticancer drugs. Because cisplatin–DNA adducts block RNA polymerase II unless removed by transcription-coupled excision repair, compounds that react similarly but elude repair are desirable. The monofunctional platinum agent pyriplatin displays antitumor activity in mice, a cytotoxicity profile in cell cultures distinct from that of cisplatin, and a unique in vitro transcription inhibition mechanism. In this study, we incorporated pyriplatin globally or site specifically into luciferase reporter vectors to examine its transcription inhibition profiles in live mammalian cells. Monofunctional pyriplatin reacted with plasmid DNA as efficiently as bifunctional cisplatin and inhibited transcription as strongly as cisplatin in various mammalian cells. Using repair-defective nucleotide excision repair (NER)-, mismatch repair-, and single-strand break repair–deficient cells, we show that NER is mainly responsible for removal of pyriplatin–DNA adducts. These findings reveal that the mechanism by which pyriplatin generates its antitumor activity is very similar to that of cisplatin, despite the chemically different nature of their DNA adducts, further supporting a role for monofunctional platinum anticancer agents in human cancer therapy. This information also provides support for the validity of the proposed mechanism of action of cisplatin and provides a rational basis for the design of more potent platinum anticancer drug candidates using a monofunctional DNA-damaging strategy. Cancer Res; 72(3); 790–800. ©2011 AACR.

Conventional chemotherapy targets proliferating cancer cells, but most cells in solid tumors are not in a proliferative state. Thus, strategies to enable conventional chemotherapy to target noncycling cells may greatly increase tumor responsiveness. In this study, we used a 3-dimensional tissue culture system to assay diffusible factors that can limit proliferation in the context of the tumor microenvironment, with the goal of identifying targets to heighten proliferative capacity in this setting. We found that supraphysiologic levels of insulin or insulin-like growth factor I (IGF-I) in combination with oxygen supplementation were sufficient to initiate proliferation of quiescence cells in this system. At maximal induction with IGF-I, net tissue proliferation increased 3- to 4-fold in the system such that chemotherapy could trigger a 3- to 6-fold increase in cytotoxicity, compared with control conditions. These effects were confirmed in vivo in colon cancer xenograft models with demonstrations that IGF-I receptor stimulation was sufficient to generate a 45% increase in tumor cell proliferation, along with a 25% to 50% increase in chemotherapy-induced tumor growth delay. Although oxygen was a dominant factor limiting in vitro tumor cell proliferation, we found that oxygen supplementation via pure oxygen breathing at 1 or 2 atmospheres pressure (mimicking hyperbaric therapy) did not decrease hypoxia in the tumor xenograft mouse model and was insufficient to increase tumor proliferation. Thus, our findings pointed to IGF-I receptor stimulation as a rational strategy to successfully increase tumor responsiveness to cytotoxic chemotherapy. Cancer Res; 72(3); 801–9. ©2011 AACR.

Upregulation of the matrix metalloproteinase (MMP)–9 plays a central role in tumor progression and metastasis by stimulating cell migration, tumor invasion, and angiogenesis. To gain insights into MMP-9 expression, we investigated its epigenetic control in a reversible model of cancer that is initiated by infection with intracellular Theileria parasites. Gene induction by parasite infection was associated with trimethylation of histone H3K4 (H3K4me3) at the MMP-9 promoter. Notably, we found that the H3K4 methyltransferase SMYD3 was the only histone methyltransferase upregulated upon infection. SMYD3 is overexpressed in many types of cancer cells, but its contributions to malignant pathophysiology are unclear. We found that overexpression of SMYD3 was sufficient to induce MMP-9 expression in transformed leukocytes and fibrosarcoma cells and that proinflammatory phorbol esters further enhanced this effect. Furthermore, SMYD3 was sufficient to increase cell migration associated with MMP-9 expression. In contrast, RNA interference–mediated knockdown of SMYD3 decreased H3K4me3 modification of the MMP-9 promoter, reduced MMP-9 expression, and reduced tumor cell proliferation. Furthermore, SMYD3 knockdown also reduced cellular invasion in a zebrafish xenograft model of cancer. Together, our results define SMYD3 as an important new regulator of MMP-9 transcription, and they provide a molecular link between SMYD3 overexpression and metastatic cancer progression. Cancer Res; 72(3); 810–20. ©2011 AACR.