Coregulators (coactivators and corepressors) occupy the driving seat for actions of all nuclear receptors, and consequently, selective receptor modulator drugs. The potency and selectivity for subreactions of transcription reside in the coactivators, and thus, they are critically important for tissue-selective gene function. Each tissue has a "quantitative finger print" of coactivators based on its relative inherited concentrations of these molecules. When the cellular concentration of a coactivator is altered, genetic dysfunction usually leads to a pathologic outcome. For example, many cancers overexpress "growth coactivators." In this way, the cancer cell can hijack these coactivator molecules to drive proliferation and metastasis. The present review contains summaries of selective coactivators and corepressors that have been demonstrated to play important roles in the malignant process and emphasizes their importance for future therapeutic interventions. [Cancer Res 2009;69(21):8217–22]

There is increasing evidence that alterations in chromatin remodeling play a significant role in human disease. The SWI/SNF chromatin remodeling complex family mobilizes nucleosomes and functions as a master regulator of gene expression and chromatin dynamics whose functional specificity is driven by combinatorial assembly of a central ATPase and association with 10 to 12 unique subunits. Although the biochemical consequence of SWI/SNF in model systems has been extensively reviewed, the present article focuses on the evidence linking SWI/SNF perturbations to cancer initiation and tumor progression in human disease. [Cancer Res 2009;69(21):8223–30]

Uterine leiomyosarcoma (ULMS) is a rare gynecologic malignancy with a low survival rate. Currently, there is no effective treatment for ULMS. Infrequent occurrences of human ULMS hamper the understanding of the initiation and progression of the disease, thereby limiting the ability to develop efficient therapies. To elucidate the roles of the p53 and BRCA1 tumor suppressor genes in gynecologic malignancies, we generated mice in which p53 and/or BRCA1 can be conditionally deleted using anti-Müllerian hormone type II receptor (Amhr2)–driven Cre recombinase. We showed that conditional deletion of p53 in mice results in the development of uterine tumors that resemble human ULMS and that concurrent deletion of p53 and BRCA1 significantly accelerates the progression of these tumors. This finding led to our hypothesis that BRCA1 may play a role in human ULMS development. Consistent with this hypothesis, we showed that the BRCA1 protein is absent in 29% of human ULMS and that BRCA1 promoter methylation is the likely mechanism of BRCA1 downregulation. These data indicate that the loss of BRCA1 function may be an important step in the progression of ULMS. Our findings provide a rationale for investigating therapies that target BRCA1 deficiency in ULMS. [Cancer Res 2009;69(21):8231–5]

N'-nitrosonornicotine (NNN) is a strong carcinogen present in unburned tobacco and cigarette smoke. We here analyze data obtained in two studies, in which a biomarker of exposure to NNN—the sum of NNN and its pyridine-N-glucuronide, called total NNN—was quantified in the urine of people who had stopped smoking and used various nicotine replacement therapy (NRT) products. In 13 of 34 nicotine gum or lozenge users from both studies, total NNN at one or more time points after biochemically confirmed smoking cessation was comparable with, or considerably higher than, the baseline levels. For most of the subjects who used the nicotine patch as a smoking cessation aid, urinary total NNN at all post–quit time points was <37% of their mean baseline levels. These results indicate that endogenous formation of significant amounts of NNN may occur sporadically in some users of oral NRT. Given the carcinogenicity of NNN and the frequent use of nicotine gum as a smoking cessation aid, further studies are needed so that preventive measures can be developed. [Cancer Res 2009;69(21):8236–40]

Emerging evidence indicates that the highly regenerative human endometrium harbors rare populations of epithelial progenitor cells. In tumors of other regenerative epithelial tissues, rare cancer stem cells (CSC) have been identified that may have originated from normal epithelial stem/progenitor cells. We hypothesized that CSC are responsible for epithelial neoplasia associated with endometrial carcinoma, the most common gynecologic malignancy in women. Stem cell characteristics of single cells isolated from endometrial carcinoma tissues from women ages 62 ± 11.8 years (n = 34) were assessed. Twenty-five of 28 endometrial carcinoma samples contained a small population of clonogenic cells [0.24% (0-1.84%)], with no significant difference in cloning efficiency between the three grades of endometrial carcinoma or between endometrial carcinoma and normal endometrial epithelial samples. Isolated endometrial carcinoma cells transplanted under the kidney capsule of immunocompromised mice in serial dilution (2 x 10⁶-1 x 10⁴ cells) generated tumors in 8 of 9 samples with morphologies similar to the parent tumors. These tumors recapitulated cytokeratin, vimentin, estrogen receptor-, and progesterone receptor expression of the parent tumor, indicating that tumor-initiating cells likely differentiated into cells comprising the endometrial carcinoma tissue. Individual clones underwent serial clonal subculture 2.5 to 4 times, with a trend of increasing number of subclonings with increasing tumor grade, indicating increasing self-renewal with greater malignancy. Clonally derived endometrial carcinoma cells also expressed the self-renewal genes BMI-1, NANOG, and SOX-2. Isolated cells from primary tumors were serially transplanted 3 to 5 times in nonobese diabetic/severe combined immunodeficient mice, showing self-renewal in vivo. This evidence of cells with clonogenic, self-renewing, differentiating, and tumorigenic properties suggests that a CSC population may be responsible for production of endometrial carcinoma tumor cells. [Cancer Res 2009;69(21):8241–8]

Disruption of Runx1/AML1 subnuclear localization, either by a single amino acid substitution or by a chromosomal translocation [e.g., t(8;21)], is linked to the etiology of acute myeloid leukemia (AML). Here, we show that this defect induces a select set of micro-RNAs (miR) in myeloid progenitor cells and AML patients with t(8;21). Both Runx1 and the t(8;21)-encoded AML1-ETO occupy the miR-24-23-27 locus and reciprocally control miR-24 transcription. miR-24 directly downregulates mitogen-activated protein kinase (MAPK) phosphatase-7 and enhances phosphorylation of both c-jun-NH₂-kinase and p38 kinases. Expression of miR-24 stimulates myeloid cell growth, renders proliferation independent of interleukin-3, and blocks granulocytic differentiation. Thus, compromised Runx1 function induces a miR-dependent mechanism that, through MAPK signaling, enhances myeloid proliferation but blocks differentiation—key steps that contribute to leukemia. [Cancer Res 2009;69(21):8249–55]

Urothelial carcinoma of the renal pelvis is a deadly disease with an unclear tumorigenic mechanism. We conducted gene expression profiling on a set of human tumors of this type and identified a phosphatidylinositol 3-kinase (PI3K)/AKT activation expression signature in 76.9% (n = 13) of our samples. Sequence analysis found both activating mutations of PIK3CA (13.6%, n = 22) and loss of heterozygosity at the PTEN locus (25%, n = 8). In contrast, none of the other subtypes of kidney neoplasms (e.g., clear-cell renal cell carcinoma) harbored PIK3CA mutations (n = 87; P < 0.001). Immunohistochemical analysis of urothelial carcinoma samples found loss of PTEN protein expression (36.4%, n = 11) and elevation of phosphorylated mammalian target of rapamycin (mTOR; 63.6%, n = 11). To confirm the role of the PI3K/AKT pathway in urothelial carcinoma, we generated mice containing biallelic inactivation of Pten in the urogenital epithelia. These mice developed typical renal pelvic urothelial carcinomas, with an incidence of 57.1% in mice older than 1 year. Laser capture microdissection followed by PCR confirmed the deletion of Pten exons 4 and 5 in the animal tumor cells. Immunohistochemical analyses showed increased phospho-mTOR and phospho-S6K levels in the animal tumors. Renal lymph node metastases were found in 15.8% of the animals with urothelial carcinoma. In conclusion, we identified and confirmed an important role for the PI3K/AKT pathway in the development of urothelial carcinoma and suggested that inhibitors of this pathway (e.g., mTOR inhibitor) may serve as effective therapeutic agents. [Cancer Res 2009;69(21):8256–64]

The CXC receptor-1 (CXCR1) is a coreceptor for interleukin-8 (IL-8) and is expressed on both normal and tumor cells. The function of CXCR1 in prostate cancer was investigated by silencing its expression, using RNA interference. We established stable cell colonies of PC-3 cells, depleted of CXCR1, using lentiviral plasmids (pLK0.1puro) generating small hairpin RNA (shRNA) against CXCR1 mRNA. Stable shRNA transfectants (PLK1–PLK5) that express significantly reduced CXCR1 mRNA (≥90% down) and protein (≥43% down) or vector-only transfectants (PC-3V) were characterized. PLK cells showed reduced cell proliferation (down, ≥66%), due to cell cycle arrest at G₁-S phase, decreases in Cyclin D1, CDK4, phosphorylated Rb, and extracellular signal-regulated kinase 1/2 levels compared with those in PC-3V cells. CXCR1 depletion lead to increases in spontaneous apoptosis by mitochondria-mediated intrinsic mechanism and increases in proapoptotic proteins (BAD, 40%; BAX, 12%), but decreases in antiapoptotic proteins (BCL2, down 38%; BCL_xL, 20%). PLK2 cells grew as slow-growing tumors (decrease of 54%), compared with that of PC3V tumors in athymic mice. Ex vivo analyses of PLK2 tumor tissues showed reduced expression of Cyclin D1 and vascular endothelial growth factor, and increased apoptosis activity. Other IL-8–expressing prostate cancer cell lines also exhibited similar phenotypes when CXCR1 was depleted by CXCR1 shRNA transfection. In contrast to these cells, CXCR1 depletion had little effect on IL-8 ligand–deficient LNCaP cells. RNA interference rescue using mutated CXCR1 plasmids reversed the silencing effect of PLK2, thus demonstrating the specificity of phenotypic alteration by CXCR1 shRNA. These studies establish that CXCR1 promotes IL-8–mediated tumor growth. [Cancer Res 2009;69(21):8265–74]

Many cancers, including breast cancer, harbor loss-of-function mutations in the catalytic domain of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or have reduced PTEN expression through loss of heterozygosity and/or epigenetic silencing mechanisms. However, specific phenotypic effects of PTEN inactivation in human cancer cells remain poorly defined without a direct causal connection between the loss of PTEN function and the development or progression of cancer. To evaluate the biological and clinical relevance of reduced or deleted PTEN expression, a novel in vitro model system was generated using human somatic cell knockout technologies. Targeted homologous recombination allowed for a single and double allelic deletion, which resulted in reduced and deleted PTEN expression, respectively. We determined that heterozygous loss of PTEN in the nontumorigenic human mammary epithelial cell line MCF-10A was sufficient for activation of the phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase pathways, whereas the homozygous absence of PTEN expression led to a further increased activation of both pathways. The deletion of PTEN was able to confer growth factor–independent proliferation, which was confirmed by the resistance of the PTEN^–/– MCF-10A cells to small-molecule inhibitors of the epidermal growth factor receptor. However, neither heterozygous nor homozygous loss of PTEN expression was sufficient to promote anchorage-independent growth, but the loss of PTEN did confer apoptotic resistance to cell rounding and matrix detachment. Finally, MCF-10A cells with the reduction or loss of PTEN showed increased susceptibility to the chemotherapeutic drug doxorubicin but not paclitaxel. [Cancer Res 2009;69(21):8275–83]

Cells are incessantly exposed to many sources of genotoxic stress. A critical unresolved issue is how the resulting activation of the p53 tumor suppressor can lead to either cell cycle arrest or apoptosis depending on the extent of DNA damage. The present study shows that the level of Krüppel-like factor 4 (KLF4) expression is inversely correlated with the extent of DNA damage. KLF4 is activated by p53 following cytostatic, mild DNA damage, whereas it is strongly repressed via enhanced turnover of mRNA on severe DNA damage that irreversibly drives cells to apoptosis. Blocking the repression of KLF4 on severe DNA damage suppresses p53-mediated apoptosis, whereas ablation of the KLF4 induction on mild DNA damage shifts the p53 response from cell cycle arrest to cell death. Our results suggest that coordinate regulation of KLF4 expression depending on the extent of DNA damage may be an important mechanism that dictates the life and death decisions of p53. [Cancer Res 2009;69(21):8284–92]

There is a great need for the development of novel chemotherapeutic agents that overcome the emergence of multidrug resistance (MDR) in cancer. We catalogued the National Cancer Institute's DTP drug repository in search of compounds showing increased toxicity in MDR cells. By comparing the sensitivity of parental cell lines with MDR derivatives, we identified 22 compounds possessing MDR-selective activity. Analysis of structural congeners led to the identification of 15 additional drugs showing increased toxicity in Pgp-expressing cells. Analysis of MDR-selective compounds led to the formulation of structure activity relationships and pharmacophore models. This data mining coupled with experimental data points to a possible mechanism of action linked to metal chelation. Taken together, the discovery of the MDR-selective compound set shows the robustness of the developing field of MDR-targeting therapy as a new strategy for resolving Pgp-mediated MDR. [Cancer Res 2009;69(21):8293–301]

Conventional development of multivariate gene expression models (GEM) predicting therapeutic response of cancer patients is based on analysis of patients treated with specific regimens, which limits generalization to different or novel drug combinations. We overcome this limitation by developing GEMs based on in vitro drug sensitivities and microarray analyses of the NCI-60 cancer cell line panel. These GEMs were evaluated in blind fashion as predictors of tumor response and/or patient survival in seven independent cohorts of patients with breast (n = 275), bladder (n = 59), and ovarian (n= 143) cancer treated with multiagent chemotherapy, of which 233 patients were from prospectively enrolled clinical trials. In all studies, GEMs effectively stratified tumor response and patient survival independent of established clinical and pathologic tumor variables. In bladder cancer patients treated with neoadjuvant methotrexate, vinblastine, Adriamycin (doxorubicin), and cisplatin, the 3-year overall survival for those with favorable GEM scores was 81% versus 33% for those with less favorable scores (P = 0.002). GEMs for breast cancer patients treated with 5-fluorouracil, Adriamycin (doxorubicin), and cyclophosphamide and ovarian cancer patients treated with platinum-containing regimens also stratified patient survival [5-year overall survival 100% versus 74% (P = 0.05) and 3-year overall survival 68% versus 43% (P = 0.008), respectively]. Importantly, clinical prediction using our in vitro GEM was superior to that of conventionally derived GEMs. We show a facile yet effective approach to GEM derivation that identifies patients most likely to benefit from selected multiagent therapy. Use of such in vitro–based GEMs may provide a robust and generalizable approach to personalized cancer therapy. [Cancer Res 2009;69(21):8302–9]

Acute leukemia is a hematopoietic malignancy for which the accurate measurement of minimal residual disease is critical to determining prognosis and treatment. Although bone marrow aspiration and light microscopy remain the current standard of care for detecting residual disease, these approaches cannot reliably discriminate less than 5% lymphoblast cells. To improve the detection of leukemia cells in the marrow, we developed a novel apparatus that utilizes antibodies conjugated to superparamagnetic iron oxide nanoparticles (SPION) and directed against the acute leukemia antigen CD34, coupled with a "magnetic needle" biopsy. Leukemia cell lines expressing high or minimal CD34 were incubated with anti-CD34–conjugated SPIONs. Three separate approaches including microscopy, superconducting quantum interference device magnetometry, and in vitro magnetic needle extraction were then used to assess cell sampling. We found that CD34-conjugated nanoparticles preferentially bind high CD34-expressing cell lines. Furthermore, the magnetic needle enabled identification of both cell line and patient leukemia cells diluted into normal blood at concentrations below those normally found in remission marrow samples. Finally, the magnetic needle enhanced the percentage of lymphoblasts detectable by light microscopy by 10-fold in samples of fresh bone marrow aspirate approximating minimal residual disease. These data suggest that bone marrow biopsy using antigen-targeted magnetic nanoparticles and a magnetic needle for the evaluation of minimal residual disease in CD34-positive acute leukemias can significantly enhance sensitivity compared with the current standard of care. [Cancer Res 2009;69(21):8310–6]

The activating mutation BRAF^V600E is a frequent genetic event in papillary thyroid carcinomas (PTC) that predicts a poor prognosis, leading to loss of sodium/iodide symporter (NIS) expression and subsequent radioiodide-refractory metastatic disease. The molecular basis of such an aggressive behavior induced by BRAF remains unclear. Here, we show a mechanism through which BRAF induces NIS repression and promotes epithelial to mesenchimal transition and invasion based on the operation of an autocrine transforming growth factor (TGF)β loop. BRAF induces secretion of functional TGFβ and blocking TGFβ/Smad signaling at multiple levels rescues BRAF-induced NIS repression. Although this mechanism is MAP/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK independent, secreted TGFβ cooperates with MEK-ERK signaling in BRAF-induced cell migration, Matrigel invasion, and EMT. Consistent with this process, TGFβ and other key components of TGFβ signaling, such as TβRII and pSmad2, are overexpressed in human PTC, suggesting a widespread activation of this pathway by locally released TGFβ. Moreover, this high TGFβ/Smad activity is associated with PTC invasion, nodal metastasis, and BRAF status. Interestingly, TGFβ is overexpressed in the invasive front, whereas NIS is preferentially expressed in the central regions of the tumors, suggesting that this negative correlation between TGFβ and NIS occurs locally inside the tumor. Our study describes a novel mechanism of NIS repression in thyroid cancer and provides evidence that TGFβ may play a key role in promoting radioiodide resistance and tumor invasion during PTC progression. [Cancer Res 2009;69(21):8317–25]

The metastatic cell population, ranging from solitary cells to actively growing metastases, is heterogeneous and unlikely to respond uniformly to treatment. However, quantification of the entire experimental metastatic cell population in whole organs is complicated by requirements of an imaging modality with the large field of view and high spatial resolution necessary to detect both single cells and metastases in the same organ. Thus, it is difficult to assess differential responses of these distinct metastatic populations to therapy. Here, we develop a magnetic resonance imaging (MRI) technique capable of quantifying the full population of metastatic cells in a secondary organ. B16F1 mouse melanoma cells were labeled with micron-sized iron oxide particles (MPIO) and injected into mouse liver via the mesenteric vein. Livers were removed immediately or at day 9 or 11, following doxorubicin or vehicle control treatment, and imaged using a 3T clinical magnetic resonance scanner and custom-built gradient coil. Both metastases (>200 µm) and MPIO-labeled single cells were detected and quantified from MR images as areas of hyperintensity or hypointensity (signal voids), respectively. We found that 1mg/kg doxorubicin treatment inhibited metastasis growth (n = 11 per group; P = 0.02, t test) but did not decrease the solitary metastatic cell population in the same livers (P > 0.05). Thus, the technique presented here is capable of quickly quantifying the majority of the metastatic cell population, including both growing metastases and solitary cells, in whole liver by MRI and can identify differential responses of growing metastases and solitary cells to therapy. [Cancer Res 2009;69(21):8326–31]

Altered expression of microRNAs (miRNA), an abundant class of small nonprotein-coding RNAs that mostly function as negative regulators of protein-coding gene expression, is common in cancer. Here, we analyze the regulation of miRNA expression in response to estrogen, a steroid hormone that is involved in the development and progression of breast carcinomas and that is acting via the estrogen receptors (ER) transcription factors. We set out to thoroughly describe miRNA expression, by using miRNA microarrays and real-time reverse transcription-PCR (RT-PCR) experiments, in various breast tumor cell lines in which estrogen signaling has been induced by 17β-estradiol (E₂). We show that the expression of a broad set of miRNAs decreases following E₂ treatment in an ER-dependent manner. We further show that enforced expression of several of the repressed miRNAs reduces E₂-dependent cell growth, thus linking expression of specific miRNAs with estrogen-dependent cellular response. In addition, a transcriptome analysis revealed that the E₂-repressed miR-26a and miR-181a regulate many genes associated with cell growth and proliferation, including the progesterone receptor gene, a key actor in estrogen signaling. Strikingly, miRNA expression is also regulated in breast cancers of women who had received antiestrogen neoadjuvant therapy. Overall, our data indicate that the extensive alterations in miRNA regulation upon estrogen signaling pathway play a key role in estrogen-dependent functions and highlight the utility of considering miRNA expression in the understanding of antiestrogen resistance of breast cancer. [Cancer Res 2009;69(21):8332–40]

In a subset of lung adenocarcinomas, the epidermal growth factor receptor (EGFR) is activated by kinase domain mutations and/or gene amplification, but the interaction between the two types of abnormalities is complex and unclear. For this study, we selected 99 consecutive never-smoking women of East Asian origin with lung adenocarcinomas that were characterized by histologic subtype. We analyzed EGFR mutations by PCR-capillary sequencing, EGFR copy number abnormalities by fluorescence and chromogenic in situ hybridization and quantitative PCR, and EGFR expression by immunohistochemistry with both specific antibodies against exon 19 deletion–mutated EGFR and total EGFR. We compared molecular and clinicopathologic features with disease-free survival. Lung adenocarcinomas with EGFR amplification had significantly more EGFR exon 19 deletion mutations than adenocarcinomas with disomy, and low and high polysomy (100% versus 54%, P = 0.009). EGFR amplification occurred invariably on the mutated and not the wild-type allele (median mutated/wild-type ratios 14.0 versus 0.33, P = 0.003), was associated with solid histology (P = 0.008), and advanced clinical stage (P = 0.009). EGFR amplification was focally distributed in lung cancer specimens, mostly in regions with solid histology. Patients with EGFR amplification had a significantly worse outcome in univariate analysis (median disease-free survival, 16 versus 31 months, P = 0.01) and when adjusted for stage (P = 0.027). Lung adenocarcinomas with EGFR amplification have a unique association with exon 19 deletion mutations and show distinct clinicopathologic features associated with a significantly worsened prognosis. In these cases, EGFR amplification is heterogeneously distributed, mostly in areas with a solid histology. [Cancer Res 2009;69(21):8341–8]

Whether energy balance during early life and/or adulthood is related to glioma risk is unknown. We therefore investigated height, body mass index (BMI), and physical activity in relation to glioma risk in the prospective NIH-AARP Diet and Health Study. Participants completed a baseline questionnaire (sent in 1995-1996) inquiring about height, weight, and potential confounders. A second questionnaire (sent in 1996) inquired about physical activity during ages 15 to 18, 19 to 29, and 35 to 39 years and the past 10 years and body weight at ages 18, 35, and 50 years. During follow-up from 1995/1996 to 2003, we documented 480 cases of glioma among 499,437 respondents to the baseline questionnaire and 257 cases among 305,681 respondents to the second questionnaire. Glioma risk among tall persons (≥1.90 m) was twice that of short persons [<1.60 m; multivariate relative risk (RR), 2.12; 95% confidence interval (95% CI), 1.18-3.81; P_trend = 0.006]. Risk among participants who were obese (BMI 30.0-34.9 kg/m²) at age 18 years was nearly four times that of persons of normal weight (BMI 18.5-24.9 kg/m²) at age 18 years (RR, 3.74; 95% CI, 2.03-6.90; P_trend = 0.003); 11 cases were obese at age 18 years. Risk among participants who were active during ages 15 to 18 years was 36% lower than that of persons who were inactive during ages 15 to 18 years (RR, 0.64; 95% CI, 0.44-0.93; P_trend = 0.02). BMI and physical activity after age 18 years were unrelated to glioma risk. Adult height, BMI during adolescence, and physical activity during adolescence were each associated with glioma risk, supporting a role for early-life energy balance in glioma carcinogenesis. [Cancer Res 2009;69(21):8349–55]

DNA damage stabilizes the p53 tumor suppressor protein that determines the cell fate by either cell cycle arrest or cell death induction. Noxa, the BH3-only Bcl-2 family protein, was shown to be a key player in p53-induced cell death through the mitochondrial dysfunction; however, the molecular mechanism by which Noxa induces the mitochondrial dysfunction to cause cell death in response to genotoxic agents is largely unknown. Here, we show that the mitochondrial-targeting domain (MTD) of Noxa is a prodeath domain. Peptide containing MTD causes massive necrosis in vitro through cytosolic calcium increase; it is released from the mitochondria by opening the mitochondrial permeability transition pore. MTD peptide–induced cell death can be inhibited by calcium chelator BAPTA-AM. Moreover, MTD peptide shows the potent tumor-killing activities in mice by joining with tumor-homing motifs. [Cancer Res 2009;69(21):8356–65]

Pyrrolo-1,5-benzoxazepine-15 (PBOX-15) is a novel microtubule depolymerization agent that induces cell cycle arrest and subsequent apoptosis in a number of cancer cell lines. Chronic lymphocytic leukemia (CLL) is characterized by clonal expansion of predominately nonproliferating mature B cells. Here, we present data suggesting PBOX-15 is a potential therapeutic agent for CLL. We show activity of PBOX-15 in samples taken from a cohort of CLL patients (n = 55) representing both high-risk and low-risk disease. PBOX-15 exhibited cytotoxicity in CLL cells (n = 19) in a dose-dependent manner, with mean IC₅₀ of 0.55 µmol/L. PBOX-15 significantly induced apoptosis in CLL cells (n = 46) including cells with poor prognostic markers: unmutated IgV_H genes, CD38 and zeta-associated protein 70 (ZAP-70) expression, and fludarabine-resistant cells with chromosomal deletions in 17p. In addition, PBOX-15 was more potent than fludarabine in inducing apoptosis in fludarabine-sensitive cells. Pharmacologic inhibition and small interfering RNA knockdown of caspase-8 significantly inhibited PBOX-15–induced apoptosis. Pharmacologic inhibition of c-jun NH₂-terminal kinase inhibited PBOX-15–induced apoptosis in mutated IgV_H and ZAP-70^– CLL cells but not in unmutated IgV_H and ZAP-70⁺ cells. PBOX-15 exhibited selective cytotoxicity in CLL cells compared with normal hematopoietic cells. Our data suggest that PBOX-15 represents a novel class of agents that are toxic toward both high-risk and low-risk CLL cells. The need for novel treatments is acute in CLL, especially for the subgroup of patients with poor clinical outcome and drug-resistant disease. This study identifies a novel agent with significant clinical potential. [Cancer Res 2009;69(21):8366–75]

The novel proteasome inhibitor NPI-0052 has been shown to sensitize tumor cells to apoptosis by various chemotherapeutic drugs and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), although the mechanisms involved are not clear. We hypothesized that NPI-0052–mediated sensitization may result from NF-B inhibition and downstream modulation of the metastasis inducer Snail and the metastasis suppressor/immunosurveillance cancer gene product Raf-1 kinase inhibitory protein (RKIP). Human prostate cancer cell lines were used as models, as they express different levels of these proteins. We show that NPI-0052 inhibits both NF-B and Snail and induces RKIP expression, thus resulting in cell sensitization to CDDP and TRAIL. The direct role of NF-B inhibition in sensitization was corroborated with the NF-B inhibitor DHMEQ, which mimicked NPI-0052 in sensitization and inhibition of Snail and induction of RKIP. The direct role of Snail inhibition by NPI-0052 in sensitization was shown with Snail small interfering RNA, which reversed resistance and induced RKIP. Likewise, the direct role of RKIP induction in sensitization was revealed by both overexpression of RKIP (mimicking NPI-0052) and RKIP small interfering RNA that inhibited NPI-0052–mediated sensitization. These findings show that NPI-0052 modifies the NF-B-Snail-RKIP circuitry in tumor cells and results in downstream inhibition of antiapoptotic gene products and chemoimmunosensitization. The findings also identified Snail and RKIP as targets for reversal of resistance. [Cancer Res 2009;69(21):8376–85]

Taxol chemotherapy is one of the few therapeutic options for men with castration-resistant prostate cancer (CRPC). However, the working mechanisms for Taxol are not fully understood. Here, we showed that treatment of 22Rv1, a PTEN-positive CRPC cell line, with paclitaxel and its semisynthetic analogue docetaxel decreases expression of the androgen receptor (AR)–activated genes prostate-specific antigen (PSA) and Nkx3.1 but increases expression of the AR repression gene maspin, suggesting that Taxol treatment inhibits AR activity. This was further supported by the observation that the activity of AR luciferase reporter genes was inhibited by paclitaxel. In contrast, paclitaxel treatment failed to inhibit AR activity in the PTEN-null CRPC cell line C4-2. However, pretreatment of C4-2 cells with the phosphoinositide 3-kinase inhibitor LY294002 restored paclitaxel inhibition of the AR. Treatment of 22Rv1 xenografts in mice with docetaxel induced mitotic arrest and a decrease in PSA expression in tumor cells adjacent to vascular vessels. We further showed that paclitaxel induces nuclear accumulation of FOXO1, a known AR suppressive nuclear factor, and increases the association of FOXO1 with AR proteins in the nucleus. FOXO1 knockdown with small interfering RNA attenuated the inhibitory effect of paclitaxel on AR transcriptional activity, expression of PSA and Nkx3.1, and cell survival. These data reveal a previously uncharacterized, FOXO1-mediated, AR-inhibitory effect of Taxol in CRPC cells that may play an important role in Taxol-mediated inhibition of CRPC growth. [Cancer Res 2009;69(21):8386–94]

The transmembrane serine protease hepsin is one of the most highly upregulated genes in prostate cancer. Here, we investigated its tumor-promoting activity by use of a mouse orthotopic prostate cancer model. First, we compared the tumor growth of low hepsin-expressing LnCaP-17 cells with hepsin-overexpressing LnCaP-34 cells. After implantation of cells into the left anterior prostate lobe, LnCaP-34 tumors not only grew faster based on increased serum prostate-specific antigen levels but also metastasized to local lymph nodes and, most remarkably, invaded the contralateral side of the prostate at a rate of 100% compared with only 18% for LnCaP-17 tumors. The increased tumor growth was not due to nonspecific gene expression changes and was not predicted from the unaltered in vitro growth and invasion of LnCaP-34 cells. A likely explanation is that the in vivo effects of hepsin were mediated by specific hepsin substrates present in the tumor stroma. In a second study, mice bearing LnCaP-34 tumors were treated with a PEGylated form of Kunitz domain-1, a potent hepsin active site inhibitor derived from hepatocyte growth factor activator inhibitor-1 (K_i^app 0.30 ± 0.02 nmol/L). Treatment of established tumors with PEGylated Kunitz domain-1 decreased contralateral prostate invasion (46% weight reduction) and lymph node metastasis (50% inhibition). Moreover, serum prostate-specific antigen level remained reduced during the entire treatment period, reaching a maximal reduction of 76% after 5 weeks of dosing. The findings show that hepsin promotes invasive prostate tumor growth and metastasis and suggest that active site-directed hepsin inhibition could be effective in prostate cancer therapy. [Cancer Res 2009;69(21):8395–402]

Breast cancer is a highly heterogeneous disease with distinct histologic subtypes. Targeted therapies such as endocrine therapy and growth factor receptor inhibitors have had a significant impact on the treatment of metastatic breast cancer patients. Unfortunately, resistance to these agents eventually occurs, and currently represents a significant clinical problem in the management of breast cancers. Inhibitors of histone deacetylases (HDACi) exhibit anticancer activity in a variety of tumor cell models and have been shown to target mechanisms of resistance to a number of targeted agents. It is unclear, however, if there are specific breast cancer subtypes for which an HDACi may be more or less effective. Here, we report that the class I isoform–selective HDACi entinostat (SNDX-275) preferentially inhibits cell proliferation/survival and inactivates downstream signaling in erbB2-overexpressing compared with basal breast cancer cells. SNDX-275 reduces the levels of both erbB2 and erbB3, as well as significantly decreases P-erbB2, P-erbB3, P-Akt, and P-MAPK in erbB2-overexpressing cells. Additionally, SNDX-275 promotes apoptosis and induces cell cycle arrest predominantly at G₁ phase in erbB2-overexpressing cells, whereas SNDX-275 mainly induces G₂-M arrest in basal breast cancer cells. The cellular bias of SNDX-275 is shown to be related partly to the levels of erbB3 expression that directly impact the ability of SNDX-275 to inhibit proliferation/survival of the erbB2-overexpressing breast cancer cells. These findings show that SNDX-275 may be developed as a novel therapeutic agent to treat breast cancers with coexpression of both erbB2 and erbB3. [Cancer Res 2009;69(21):8403–11]

Renal cell carcinoma is frequently infiltrated by cells of the immune system. This makes it important to understand interactions between cancer cells and immune cells so they can be manipulated to bring clinical benefit. Here, we analyze subsets and functions of T lymphocytes infiltrating renal cell tumors directly ex vivo following mechanical disaggregation and without any culture step. Subpopulations of memory and effector CD4⁺ Th1, Th2, and Th17 and CD8⁺ Tc1 cells were identified based on surface phenotype, activation potential, and multicytokine production. Compared with the same patient's peripheral blood, T lymphocytes present inside tumors were found to be enriched in functional CD4⁺ cells of the Th1 lineage and in effector memory CD8⁺ cells. Additionally, several populations of CD4⁺ and CD8⁺ regulatory T cells were identified that may synergize to locally dampen antitumor T-cell responses. [Cancer Res 2009;69(21):8412–9]

Spontaneous immune responses in cancer patients have been described. Yet their clinical relevance and the conditions for their generation remain unclear. We characterized conditions that determine immune responses in primary breast cancer patients. We used tetramer analysis, ex vivo IFN- ELISPOT, cytotoxicity assays, and ELISA in 207 untreated patients and 12 Her-2/neu–specific CD8 T-cell lines to evaluate tumor-specific T cells (TC) in the bone marrow or MUC1-specific antibodies in the blood. Multiplex analysis was performed to quantify 27 intratumoral cytokines, chemokines, and growth factors. Results were compared with multiple pathologic and clinical parameters of the patients and tumors. Forty percent of the patients showed tumor-specific TC responses. These correlated with tumors of high differentiation, estrogen receptor expression, and low proliferative activity, and with a reduced cancer mortality risk. High tumor cell differentiation correlated with increased intratumoral, but not plasma, concentrations of IFN- and reduced transforming growth factor (TGF)β1. In an in vitro priming experiment these two cytokines increased or inhibited, respectively, the capacity of dendritic cells to induce tumor-reactive TC. Tumor-specific B-cell responses, mainly of IgM isotype, were detectable in 50% of the patients and correlated with advanced tumor stage, increased TGFβ1, reduced IFN-, and absence of TC responses. We show here that different types of immune responses are linked to distinct cytokine microenvironments and correlate with prognosis-relevant differences in tumor pathobiology. These findings shed light on the relation between immune response and cancer prognosis. [Cancer Res 2009;69(21):8420–8]

The Sleeping Beauty (SB) transposon system has been used as a somatic mutagen to identify candidate cancer genes. In previous studies, efficient leukemia/lymphoma formation on an otherwise wild-type genetic background occurred in mice undergoing whole-body mobilization of transposons, but was accompanied by high levels of embryonic lethality. To explore the utility of SB for large-scale cancer gene discovery projects, we have generated mice that carry combinations of different transposon and transposase transgenes. We have identified a transposon/transposase combination that promotes highly penetrant leukemia/lymphoma formation on an otherwise wild-type genetic background, yet does not cause embryonic lethality. Infiltrating gliomas also occurred at lower penetrance in these mice. SB-induced or accelerated tumors do not harbor large numbers of chromosomal amplifications or deletions, indicating that transposon mobilization likely promotes tumor formation by insertional mutagenesis of cancer genes, and not by promoting wide-scale genomic instability. Cloning of transposon insertions from lymphomas/leukemias identified common insertion sites at known and candidate novel cancer genes. These data indicate that a high mutagenesis rate can be achieved using SB without high levels of embryonic lethality or genomic instability. Furthermore, the SB system could be used to identify new genes involved in lymphomagenesis/leukemogenesis. [Cancer Res 2009;69(21):8429–37]

In this study, we used single nucleotide polymorphism and comparative genomic hybridization array to study DNA copy number changes and loss of heterozygosity for 28 patients affected by Sézary syndrome (SS), a rare form of cutaneous T-cell lymphoma (CTCL). Our data identified, further confirming previous studies, recurrent losses of 17p13.2-p11.2 and 10p12.1-q26.3 occurring in 71% and 68% of cases, respectively; common gains were detected for 17p11.2-q25.3 (64%) and chromosome 8/8q (50%). Moreover, we identified novel genomic lesions recurring in >30% of tumors: loss of 9q13-q21.33 and gain of 10p15.3-10p12.2. Individual chromosomal aberrations did not show a significant correlation with prognosis; however, when more than three recurrent chromosomal alterations (gain or loss) were considered, a statistical association was observed using Kaplan-Meier survival analysis. Integrating mapping and transcriptional data, we were able to identify a total of 113 deregulated transcripts in aberrant chromosomal regions that included cancer-related genes such as members of the NF-B pathway (BAG4, BTRC, NKIRAS2, PSMD3, and TRAF2) that might explain its constitutive activation in CTCL. Matching this list of genes with those discriminating patients with different survival times, we identify several common candidates that might exert critical roles in SS, such as BUB3 and PIP5K1B. Altogether, our study confirms and maps more precisely the regions of gain and loss and, combined to transcriptional profiles, suggests a novel set of genes of potential interest in SS. [Cancer Res 2009;69(21):8438–46]

Disruption of circadian rhythms, daily oscillations in biological processes that are regulated by an endogenous clock, has been linked to tumorigenesis. Normal and malignant tissues often show asynchronies in cell proliferation and metabolic rhythms. Cancer chronotherapy takes biological time into account to improve the therapy. However, alterations of the circadian clock machinery genes have rarely been reported in human cancer. Herein, we show that the BMAL1 gene, a core component of the circadian clock, is transcriptionally silenced by promoter CpG island hypermethylation in hematologic malignancies, such as diffuse large B-cell lymphoma and acute lymphocytic and myeloid leukemias. We also describe how BMAL1 reintroduction in hypermethylated leukemia/lymphoma cells causes growth inhibition in colony assays and nude mice, whereas BMAL1 depletion by RNA interference in unmethylated cells enhances tumor growth. We also show that BMAL1 epigenetic inactivation impairs the characteristic circadian clock expression pattern of genes such as C-MYC, catalase, and p300 in association with a loss of BMAL1 occupancy in their respective promoters. Furthermore, the DNA hypermethylation–associated loss of BMAL1 also prevents the recruitment of its natural partner, the CLOCK protein, to their common targets, further enhancing the perturbed circadian rhythm of the malignant cells. These findings suggest that BMAL1 epigenetic inactivation contributes to the development of hematologic malignancies by disrupting the cellular circadian clock. [Cancer Res 2009;69(21):8447–54]

The usurping of translational control by sustained activation of translation initiation factors is oncogenic. Here, we show that the primary negative regulators of these oncogenic initiation factors—the 4E-BP protein family—operate as guardians of a translational control checkpoint in lung tumor defense. When challenged with the tobacco carcinogen 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone (NNK), 4ebp1^–/–/4ebp2^–/– mice showed increased sensitivity to tumorigenesis compared with their wild-type counterparts. The 4E-BP–deficient state per se creates pro-oncogenic, genome-wide skewing of the molecular landscape, with translational activation of genes governing angiogenesis, growth, and proliferation, and translational activation of the precise cytochrome p450 enzyme isoform (CYP2A5) that bioactivates NNK into mutagenic metabolites. Our study provides in vivo proof for a translational control checkpoint in lung tumor defense. [Cancer Res 2009;69(21):8455–62]

p53 is a master transcription factor that prevents neoplasia and genomic instability. It is an important target for anticancer drug design. Understanding the molecular mechanisms behind its transcriptional activities in normal cells is a prerequisite to further understand the deregulation effected by mutant p53 in cancerous cells. Currently, how p53 coordinates transcription programs in response to stress remains unclear. One theory proposes that stresses induce pre-binding events that direct p53 to bind to specific response elements, whereas a second posits that, in response to stress, p53 binds most response elements and post-binding events then regulate transcription initiation. It is critical to establish the relevance of both theories and investigate whether stresses induce specific p53-binding patterns correlated with effector gene induction. Using unique in cellulo genomic footprinting experiments, we studied p53 binding to the five response elements of p21 in response to stresses and monitored p21 mRNA variant transcription. We show clear footprints of p53 bound to response elements in living cells and reveal that the binding of p53 to response elements is transient, subject to dynamic changes during stress responses, and influenced by response element pentamer orientations. We show further that stresses lead to specific p53-binding patterns correlated with particular p21 mRNA variant transcription profiles and that p53 binding is necessary but not sufficient to induce p21 transcription. Our results indicate that pre- and post-binding events act together to regulate adapted stress responses; this paves the way to the unification of pre- and post-binding event theories. [Cancer Res 2009;69(21):8463–71]

Urothelial carcinoma of the bladder (UCC) is a common disease that arises by at least two different molecular pathways. The biology of UCC is incompletely understood, making the management of this disease difficult. Recent evidence implicates a regulatory role for microRNA in cancer. We hypothesized that altered microRNA expression contributes to UCC carcinogenesis. To test this hypothesis, we examined the expression of 322 microRNAs and their processing machinery in 78 normal and malignant urothelial samples using real-time rtPCR. Genes targeted by differentially expressed microRNA were investigated using real-time quantification and microRNA knockdown. We also examined the role of aberrant DNA hypermethylation in microRNA downregulation. We found that altered microRNA expression is common in UCC and occurs early in tumorogenesis. In normal urothelium from patients with UCC, 11% of microRNAs had altered expression when compared with disease-free controls. This was associated with upregulation of Dicer, Drosha, and Exportin 5. In UCC, microRNA alterations occur in a tumor phenotype–specific manner and can predict disease progression. High-grade UCC were characterized by microRNA upregulation, including microRNA-21 that suppresses p53 function. In low-grade UCC, there was downregulation of many microRNA molecules. In particular, loss of microRNAs-99a/100 leads to upregulation of FGFR3 before its mutation. Promoter hypermethylation is partly responsible for microRNA downregulation. In conclusion, distinct microRNA alterations characterize UCC and target genes in a pathway-specific manner. These data reveal new insights into the disease biology and have implications regarding tumor diagnosis, prognosis and therapy. [Cancer Res 2009;69(21):8472–81]

Complete loss or deletion of the long arm of chromosome 5 is frequent in myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). The putative gene(s) deleted and responsible for the pathogenesis of these poor prognosis hematologic disorders remain controversial. This study is a comprehensive analysis of previously implicated and novel genes for epigenetic inactivation in AML and MDS. In 146 AML cases, methylation of CTNNA1 was frequent, and more common in AML patients with 5q deletion (31%) than those without 5q deletion (14%), whereas no methylation of other 5q genes was observed. In 31 MDS cases, CTNNA1 methylation was only found in high-risk MDS (≥RAEB2), but not in low-risk MDS (<RAEB2), indicating that CTNNA1 methylation might be important in the transformation of MDS to AML. CTNNA1 expression was lowest in AML/MDS patients with CTNNA1 methylation, although reduced expression was found in some patients without promoter methylation. Repressive chromatin marks (H3K27me3) at the promoter were identified in CTNNA1-repressed AML cell lines and primary leukemias, with the most repressive state correlating with DNA methylation. These results suggest progressive, acquired epigenetic inactivation at CTNNA1, including histone modifications and promoter CpG methylation, as a component of leukemia progression in patients with both 5q– and non-5q– myeloid malignancies. [Cancer Res 2009;69(21):8482–90]

The E2F and MYC transcription factors are critical regulators of cell proliferation and contribute to the development of human cancers. Here, we report on the identification of a novel E2F target gene, ATAD2, the predicted protein product of which contains both a bromodomain and an ATPase domain. The pRB-E2F pathway regulates ATAD2 expression, which is limiting for the entry into the S phase of the cell cycle. We show that ATAD2 binds the MYC oncogene and stimulates its transcriptional activity. ATAD2 maps to chromosome 8q24, 4.3 Mb distal to MYC, in a region that is frequently found amplified in cancer. Consistent with this, we show that ATAD2 expression is high in several human tumors and that the expression levels correlate with clinical outcome of breast cancer patients. We suggest that ATAD2 links the E2F and MYC pathways and contributes to the development of aggressive cancer through the enhancement of MYC-dependent transcription. [Cancer Res 2009;69(21):8491–8]

KRAS and BRAF mutations are frequently observed in human colon cancers. These mutations occur in a mutually exclusive manner, and each is associated with distinctive biological features. We showed previously that K-ras can interact with hypoxia to activate multiple signaling pathways. Many hypoxic responses are mediated by hypoxia-inducible factor (HIF)-1 and HIF-2, and we sought to define the roles of mutant KRAS and BRAF in the induction of HIF-1 and HIF-2 in colon cancer cells. Ectopic expression of mutant K-ras in Caco2 cells enhanced the hypoxic induction of only HIF-1, whereas mutant BRAF enhanced both HIF-1 and HIF-2. Knockout or knockdown of mutant KRAS in DLD-1 and HCT116 cells impaired the hypoxic induction of only HIF-1. HIF-1 mRNA levels were comparable in cells with and without a KRAS mutation. However, the rate of HIF-1 protein synthesis was higher in cells with a KRAS mutation, and this was suppressed by the phosphoinositide 3-kinase inhibitor LY294002. In contrast, knockdown of mutant BRAF in HT29 cells suppressed both HIF-1 and HIF-2. Although BRAF regulated mRNA levels of both HIF-1 and HIF-2, knockdown of BRAF or treatment with the MEK inhibitor PD98059 impaired the translation of only HIF-2. Our data reveal that oncogenic KRAS and BRAF mutations differentially regulate the hypoxic induction of HIF-1 and HIF-2 in colon cancer, and this may potentially contribute to the phenotypic differences of KRAS and BRAF mutations in colon tumors. [Cancer Res 2009;69(21):8499–506]

Optimal delivery of chemotherapy intensity is dependent on host- and tumor-specific characteristics. In this article, the chemotherapy late intensity schedule is revised to account for tumor growth instability, where a small tumor cell fraction emerges that exhibits a higher proliferation rate than the parent strain. Modeling this instability as simplified two-population dynamics, we find that: (a) if this instability precedes the onset of treatment, the slope of the linear increase of the drug concentration for the standard "Norton-Simon late intensity schedule" changes and the initial value of the dose strongly depends on the ratio of the two tumor cell populations and on their distinct growth rates; and (b) if the instability trails the initial treatment, the effective chemotherapeutic drug concentration changes as well. Both cases point toward testable potential refinements of the Norton-Simon late intensity schedule. [Cancer Res 2009;69(21):8507–15]

Oncolytic viral vectors have shown promise as antitumor therapeutic agents but their effectiveness is complicated by induction of antiviral antibody responses and rapid host clearance of recombinant vectors. We developed a recombinant oncolytic vaccinia virus expressing the 4-1BBL T-cell costimulatory molecule (rV-4-1BBL) and showed modest tumor regression in the poorly immunogenic B16 murine melanoma model. To improve the therapeutic potential of this vector, we tested the antitumor activity of local intratumoral injection in the setting of host lymphodepletion, which has been shown to augment vaccination and adoptive T-cell therapy. In this model, rV-4-1BBL injection in the setting of lymphodepletion promoted MHC class I expression, reduced antiviral antibody titers, promoted viral persistence, and rescued effector-memory CD8⁺ T cells, significantly improving the therapeutic effectiveness of the oncolytic vector. These data suggest that vaccination with rV-4-1BBL in the setting of host nonmyeloablative lymphodepletion represents a logical strategy for improving oncolytic vaccination in melanoma, and perhaps other cancers as well. [Cancer Res 2009;69(21):8516–25]