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Cancer Research

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Cancer Research

Over the last decade, immunogenetic analysis of B-cell receptor immunoglobulins (BcR IG) has proved instrumental in dissecting chronic lymphocytic leukemia (CLL) pathogenesis. Initially, it was the finding that the level of somatic hypermutations in rearranged IG heavy-chain genes could define two CLL subtypes associated with a different clinical course that drew attention. As the years ensued, this not only continued to hold strong, but also revealed an unprecedented BcR restriction (aptly coined as “stereotypy”), thus cementing the idea that antigenic elements select the leukemic clones. With all this in mind, in the present review, we focus on the CLL BcR IG, a molecule that clearly lies at the heart of disease pathogenesis, and attempt to distil from past and emerging biologic knowledge the most relevant aspects in the context of the immunogenetics of CLL, while at the same time provoking questions that remain unanswered. We juxtapose CLL with mutated BcR IGs against CLL with unmutated BcR IGs due to their striking clinicobiologic differences; however, when considering ontogeny, common derivation of the two mutational subtypes cannot be excluded. The issue of stereotypy is intertwined throughout and we also raise the subject of isotype-switched CLL, which, despite its rarity, contributes intriguing ontogenetic hints. Cancer Res; 74(16); 4211–6. ©2014 AACR.

Distinct cytotoxic agents currently used in the oncological armamentarium mediate their clinical benefit by influencing, directly or indirectly, the immune system in such a way that innate and adaptive immunity contributes to the tumoricidal activity. Now, we bring up evidence that both arms of anticancer immunity can be triggered through the intervention of the intestinal microbiota. Alkylating agents, such as cyclophosphamide, set up the stage for enhanced permeability of the small intestine, facilitating the translocation of selected arrays of Gram-positive bacteria against which the host mounts effector pTh17 cells and memory Th1 responses. In addition, gut commensals, through lipopolysaccharide and other bacterial components, switch the tumor microenvironment, in particular the redox equilibrium and the TNF production of intratumoral myeloid cells during therapies with platinum salts or intratumoral TLR9 agonists combined with systemic anti-IL10R Ab respectively. Consequently, antibiotics can compromise the efficacy of certain chemotherapeutic or immunomodulatory regimens. Cancer Res; 74(16); 4217–21. ©2014 AACR.

The activated RAS/RAF cascade plays a crucial role in lung cancer, but is also known to induce cellular senescence, a major barrier imposed on tumor cells early in tumorigenesis. MYC is a key factor in suppression of RAS/BRAFV600E-induced senescence in vitro. However, it is still unclear whether MYC has the same role during tumor development in vivo. Using a conditional, compound knock-in model of Cre-activated BRAFV600E and tamoxifen-regulatable MycER, we show that tamoxifen-induced activation of MYC accelerated the onset and increased the number and size of BRAFV600E-driven adenomas in a dose-dependent manner, resulting in reduced survival. Furthermore, MYC activation leads to reduced expression of the senescence markers p16INK4A, p21CIP1, and H3K9me3-containing heterochromatin foci, and an increased percentage of Ki67+ tumor cells. This suggests that MYC already early during tumor formation suppresses a BRAFV600E-induced senescence-like state. Initial activation of MYC followed by tamoxifen withdrawal still resulted in an increased number of tumors and reduced survival. However, these tumors were of smaller size, showed increased expression of p16INK4A and p21CIP1, and reduced number of Ki67+ cells, indicating that MYC inactivation restores BRAFV600E-induced senescence. Surprisingly, MYC activation did not promote adenoma to carcinoma progression. This suggests that senescence suppression by MYC is a discrete step in tumor development important for sustained tumor growth but preceding malignant transformation and that additional oncogenic events are required for carcinoma development and metastasis. These findings contribute to our understanding of the neoplastic transformation process, with implications for future treatment strategies. Cancer Res; 74(16); 4222–9. ©2014 AACR.

Pentraxin-3 (PTX3) is a member of the pentraxin family of innate immune regulators, which includes C-reactive protein (CRP). PTX3 has been implicated in angiogenesis, proliferation, and immune escape in cancer. In the present study, we evaluated PTX3 tissue expression and serum concentration as a biomarker to discriminate prostatic inflammation and benign prostatic hyperplasia (BPH) from prostate cancer, and to determine whether PTX3 status may predict progression from BPH to prostate cancer. We analyzed 40 patients with biopsy-proven BPH who underwent a second prostate biopsy 12 to 36 months later when they were diagnosed with prostate cancer or inflammation/BPH (n = 20 patients each group). Furthermore, we evaluated PTX3 serum concentrations in an independent set of patients with biopsy-proven inflammation/BPH (n = 61) and prostate cancer (n = 56). We found reduced PTX3 tissue expression in patients with prostatic inflammation/BPH compared with patients who developed prostate cancer. In the latter group, there was an increase in PTX3 tissue expression between the first and second prostate biopsy. PTX3 serum levels were also higher in patients with prostate cancer than in patients with inflammation/BPH. In contrast, there was no difference in serum PSA or CRP levels in these two groups. ROC curve analysis confirmed the reliability of PTX3 serum levels in predicting prostate cancer development, identifying a cutoff value of 3.25 ng/mL with a sensitivity and a specificity of 89.3% and 88.5%, respectively. In summary, our results encourage further evaluation of PTX3 as a tissue biopsy and blood-borne biomarker to discriminate BPH from prostate cancer. Cancer Res; 74(16); 4230–8. ©2014 AACR.

The capillary wall is the chief barrier to tissue entry of therapeutic nanoparticles, thereby dictating their efficacy. Collagen fibers are an important component of capillary walls, affecting leakiness in healthy or tumor vasculature. Using a computational model along with in vivo systems, we compared how collagen structure affects the diffusion flux of a 1-nm chemotherapeutic molecule [doxorubicin (DOX)] and an 80-nm chemotherapy-loaded pegylated liposome (DOX-PLD) in tumor vasculature. We found a direct correlation between the collagen content around a tumor vessel to the permeability of that vessel permeability to DOX-PLD, indicating that collagen content may offer a biophysical marker of extravasation potential of liposomal drug formulations. Our results also suggested that while pharmacokinetics determined the delivery of DOX and DOX-PLD to the same tumor phenotype, collagen content determined the extravasation of DOX-PLD to different tumor phenotypes. Transport physics may provide a deeper view into how nanotherapeutics cross biological barriers, possibly helping explain the balance between biological and physical aspects of drug delivery. Cancer Res; 74(16); 4239–46. ©2014 AACR.

Mutations of the isocitrate dehydrogenase 1 (IDH1) gene are among the most prevalent in low-grade glioma and secondary glioblastoma, represent an early pathogenic event, and are associated with epigenetically driven modulations of metabolism. Of particular interest is the recently uncovered relationship between the IDH1 mutation and decreased activity of the branched-chain amino acid transaminase 1 (BCAT1) enzyme. Noninvasive imaging methods that can assess BCAT1 activity could therefore improve detection of mutant IDH1 tumors and aid in developing and monitoring new targeted therapies. BCAT1 catalyzes the transamination of branched-chain amino acids while converting α-ketoglutarate (α-KG) to glutamate. Our goal was to use 13C magnetic resonance spectroscopy to probe the conversion of hyperpolarized [1-13C] α-KG to hyperpolarized [1-13C] glutamate as a readout of BCAT1 activity. We investigated two isogenic glioblastoma lines that differed only in their IDH1 status and performed experiments in live cells and in vivo in rat orthotopic tumors. Following injection of hyperpolarized [1-13C] α-KG, hyperpolarized [1-13C] glutamate production was detected both in cells and in vivo, and the level of hyperpolarized [1-13C] glutamate was significantly lower in mutant IDH1 cells and tumors compared with their IDH1-wild-type counterparts. Importantly however, in our cells the observed drop in hyperpolarized [1-13C] glutamate was likely mediated not only by a drop in BCAT1 activity, but also by reductions in aspartate transaminase and glutamate dehydrogenase activities, suggesting additional metabolic reprogramming at least in our model. Hyperpolarized [1-13C] glutamate could thus inform on multiple mutant IDH1-associated metabolic events that mediate reduced glutamate production. Cancer Res; 74(16); 4247–57. ©2014 AACR.

Regulatory T cells (Treg) are supportive to cancer development in most tissues, but their role in colitis-associated colon cancer (CAC) remains unclear. In this study, we investigated the role of CD4+Foxp3+ Treg in a mouse model of CAC and in patients with colon cancer. These Treg were increased strongly in number in a mouse model of CAC and in the peripheral blood of patients with colon cancer, exhibiting an activated phenotype as defined by elevated expression of GARP, CD103, CTLA-4, and IL10, along with an increased suppressive effect on the proliferation and Th1 cytokine expression of CD4+CD25− responder T cells ex vivo. Transient ablation of CD4+Foxp3+ Treg during tumor development in the CAC model suppressed tumor outgrowth and distribution, accompanied by an increased number of CD8+IFNγ/granzyme B-producing effector T cells. Conversely, inactivation of IL10 in Treg did not elevate the antitumor response but instead further boosted tumor development. Our results establish a tumor-promoting function for Treg during CAC formation, but they also suggest that a selective, transient ablation of Treg can evoke antitumor responses, with implications for immunotherapeutic interventions in patients with CAC. Cancer Res; 74(16); 4258–69. ©2014 AACR.

Circulating microRNAs (miRNA) are emerging as important biomarkers of various diseases, including cancer. Intriguingly, circulating levels of several miRNAs are lower in patients with cancer compared with healthy individuals. In this study, we tested the hypothesis that a circulating miRNA might serve as a surrogate of the effects of cancer on miRNA expression or release in distant organs. Here we report that circulating levels of the muscle-enriched miR486 is lower in patients with breast cancer compared with healthy individuals and that this difference is replicated faithfully in MMTV-PyMT and MMTV-Her2 transgenic mouse models of breast cancer. In tumor-bearing mice, levels of miR486 were relatively reduced in muscle, where there was elevated expression of the miR486 target genes PTEN and FOXO1A and dampened signaling through the PI3K/AKT pathway. Skeletal muscle expressed lower levels of the transcription factor MyoD, which controls miR486 expression. Conditioned media (CM) obtained from MMTV-PyMT and MMTV-Her2/Neu tumor cells cultured in vitro were sufficient to elicit reduced levels of miR486 and increased PTEN and FOXO1A expression in C2C12 murine myoblasts. Cytokine analysis implicated tumor necrosis factor α (TNFα) and four additional cytokines as mediators of miR486 expression in CM-treated cells. Because miR486 is a potent modulator of PI3K/AKT signaling and the muscle-enriched transcription factor network in cardiac/skeletal muscle, our findings implicated TNFα-dependent miRNA circuitry in muscle differentiation and survival pathways in cancer. Cancer Res; 74(16); 4270–81. ©2014 AACR.

The BRCA1-associated deubiquitylase BAP1 is mutated in several cancers, most notably mesothelioma and melanoma, where it is thought to promote oncogenesis. In this study, we present evidence that BAP1 functions as part of the DNA damage response (DDR). We found that BAP1 mediates rapid poly(ADP-ribose)-dependent recruitment of the polycomb deubiquitylase complex PR-DUB to sites of DNA damage. Furthermore, we identified BAP1 as a phosphorylation target for the DDR kinase ATM. Functionally, BAP1 promoted repair of DNA double-strand breaks, enhancing cell survival after DNA damage. Our results highlight the importance of ubiquitin turnover at sites of DNA damage, and they provide a mechanism to account for the tumor-suppressive function of BAP1. Cancer Res; 74(16); 4282–94. ©2014 AACR.

Protein phosphatase 2A (PP2A) negatively regulates tumorigenic signaling pathways, in part, by supporting the function of tumor suppressors like p53. The PP2A methylesterase PME-1 limits the activity of PP2A by demethylating its catalytic subunit. Here, we report the finding that PME-1 overexpression correlates with increased cell proliferation and invasive phenotypes in endometrial adenocarcinoma cells, where it helps maintain activated ERK and Akt by inhibiting PP2A. We obtained evidence that PME-1 could bind and regulate protein phosphatase 4 (PP4), a tumor-promoting protein, but not the related protein phosphatase 6 (PP6). When the PP2A, PP4, or PP6 catalytic subunits were overexpressed, inhibiting PME-1 was sufficient to limit cell proliferation. In clinical specimens of endometrial adenocarcinoma, PME-1 levels were increased and we found that PME-1 overexpression was sufficient to drive tumor growth in a xenograft model of the disease. Our findings identify PME-1 as a modifier of malignant development and suggest its candidacy as a diagnostic marker and as a therapeutic target in endometrial cancer. Cancer Res; 74(16); 4295–305. ©2014 AACR.

The recurrence of prostate cancer metastases to bone after androgen deprivation therapy is a major clinical challenge. We identified FN14 (TNFRSF12A), a TNF receptor family member, as a factor that promotes prostate cancer bone metastasis. In experimental models, depletion of FN14 inhibited bone metastasis, and FN14 could be functionally reconstituted with IKKβ-dependent, NFκB signaling activation. In human prostate cancer, upregulated FN14 expression was observed in more than half of metastatic samples. In addition, FN14 expression was correlated inversely with androgen receptor (AR) signaling output in clinical samples. Consistent with this, AR binding to the FN14 enhancer decreased expression. We show here that FN14 may be a survival factor in low AR output prostate cancer cells. Our results define one upstream mechanism, via FN14 signaling, through which the NFκB pathway contributes to prostate cancer metastasis and suggest FN14 as a candidate therapeutic and imaging target for castrate-resistant prostate cancers. Cancer Res; 74(16); 4306–17. ©2014 AACR.

CD40, a member of the TNF receptor family, is expressed on all mature B cells and on most B-cell lymphomas. Recently, we have shown that constitutive activation of CD40 signaling in B cells induced by a fusion protein consisting of the transmembrane part of the Epstein–Barr viral latent membrane protein 1 (LMP1) and the cytoplasmic part of CD40 (LMP1/CD40) drives B-cell lymphoma development in transgenic mice. Because LMP1/CD40-expressing B cells showed an upregulation of CD19, we investigated CD19's function in CD40-driven B-cell expansion and lymphomagenesis. Here, we demonstrate that ablation of CD19 in LMP1/CD40 transgenic mice resulted in a severe loss and reduced lifespan of mature B cells and completely abrogated development of B-cell lymphoma. CD19 is localized to lipid rafts and constitutively activated by the LMP1/CD40 fusion protein in B cells. We provide evidence that the improved survival and malignant transformation of LMP1/CD40-expressing B cells are dependent on activation of the MAPK Erk that is mediated through CD19 in a PI3K-dependent manner. Our data suggest that constitutively active CD40 is dependent on CD19 to transmit survival and proliferation signals. Moreover, we detected a similarly functioning prosurvival pathway involving phosphorylated CD19 and PI3K-dependent Erk phosphorylation in human diffuse large B-cell lymphoma cell lines. Our data provide evidence that CD19 plays an important role in transmitting survival and proliferation signals downstream of CD40 and therefore might be an interesting therapeutic target for the treatment of lymphoma undergoing chronic CD40 signaling. Cancer Res; 74(16); 4318–28. ©2014 AACR.

IL4, a cytokine produced mainly by immune cells, may promote the growth of epithelial tumors by mediating increased proliferation and survival. Here, we show that the type II IL4 receptor (IL4R) is expressed and activated in human breast cancer and mouse models of breast cancer. In metastatic mouse breast cancer cells, RNAi-mediated silencing of IL4Rα, a component of the IL4R, was sufficient to attenuate growth at metastatic sites. Similar results were obtained with control tumor cells in IL4-deficient mice. Decreased metastatic capacity of IL4Rα “knockdown” cells was attributed, in part, to reductions in proliferation and survival of breast cancer cells. In addition, we observed an overall increase in immune infiltrates within IL4Rα knockdown tumors, indicating that enhanced clearance of knockdown tumor cells could also contribute to the reduction in knockdown tumor size. Pharmacologic investigations suggested that IL4-induced cancer cell colonization was mediated, in part, by activation of Erk1/2, Akt, and mTOR. Reduced levels of pAkt and pErk1/2 in IL4Rα knockdown tumor metastases were associated with limited outgrowth, supporting roles for Akt and Erk activation in mediating the tumor-promoting effects of IL4Rα. Collectively, our results offer a preclinical proof-of-concept for targeting IL4/IL4Rα signaling as a therapeutic strategy to limit breast cancer metastasis. Cancer Res; 74(16); 4329–40. ©2014 AACR.

Phyllodes tumors of breast, even histologically diagnosed as benign, can recur locally and have metastatic potential. Histologic markers only have limited value in predicting the clinical behavior of phyllodes tumors. It remains unknown what drives the malignant progression of phyllodes tumors. We found that the expression of myofibroblast markers, α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and stromal cell–derived factor-1 (SDF-1), is progressively increased in the malignant progression of phyllodes tumors. Microarray showed that miR-21 was one of the most significantly upregulated microRNAs in malignant phyllodes tumors compared with benign phyllodes tumors. In addition, increased miR-21 expression was primarily localized to α-SMA–positive myofibroblasts. More importantly, α-SMA and miR-21 are independent predictors of recurrence and metastasis, with their predictive value of recurrence better than histologic grading. Furthermore, miR-21 mimics promoted, whereas miR-21 antisense oligos inhibited, the expression of α-SMA, FAP, and SDF-1, as well as the proliferation and invasion of primary stromal cells of phyllodes tumors. The ability of miR-21 to induce myofibroblast differentiation was mediated by its regulation on Smad7 and PTEN, which regulate the migration and proliferation, respectively. In breast phyllodes tumor xenografts, miR-21 accelerated tumor growth, induced myofibroblast differentiation, and promoted metastasis. This study suggests an important role of myofibroblast differentiation in the malignant progression of phyllodes tumors that is driven by increased miR-21. Cancer Res; 74(16); 4341–52. ©2014 AACR.

Transcriptional repressor Snail is a master regulator of epithelial–mesenchymal transition (EMT), yet the epigenetic mechanism governing Snail to induce EMT is not well understood. Here, we report that in pancreatic ductal adenocarcinoma (PDAC), elevated levels of the ubiquitin E3 ligase Ring1B and Snail, along with elevated monoubiquitination of H2A at K119 (H2AK119Ub1), are highly correlated with poor survival. Mechanistic investigations identified Ring1B as a Snail-interacting protein and showed that the carboxyl zinc fingers of Snail recruit Ring1B and its paralog Ring1A to repress its target promoters. Simultaneous depletion of Ring1A and Ring1B in pancreatic cancer cells decreased Snail binding to the target chromatin, abolished H2AK119Ub1 modification, and thereby compromised Snail-mediated transcriptional repression and cell migration. We found that Ring1B and the SNAG-associated chromatin modifier EZH2 formed distinct protein complexes with Snail and that EZH2 was required for Snail-Ring1A/B recruitment to the target promoter. Collectively, our results unravel an epigenetic mechanism underlying transcriptional repression by Snail, suggest Ring1A/B as a candidate therapeutic target, and identify H2AK119Ub1 as a potential biomarker for PDAC diagnosis and prognosis. Cancer Res; 74(16); 4353–63. ©2014 AACR.

Retinoid X receptor (RXR) regulates key cellular responses such as cell growth and development, and this regulation is frequently perturbed in various malignancies, including hepatocellular carcinoma (HCC). However, the molecule(s) that physically govern this deregulation are mostly unknown. Here, we identified RXR as an interacting partner of astrocyte-elevated gene-1 (AEG-1)/metadherin (MTDH), an oncogene upregulated in all cancers. Upon interaction, AEG-1 profoundly inhibited RXR/retinoic acid receptor (RAR)–mediated transcriptional activation. Consequently, AEG-1 markedly protected HCC and acute myelogenous leukemia (AML) cells from retinoid- and rexinoid-induced cell death. In nontumorigenic cells and primary hepatocytes, AEG-1/RXR colocalizes in the nucleus in which AEG-1 interferes with recruitment of transcriptional coactivators to RXR, preventing transcription of target genes. In tumor cells and AEG-1 transgenic hepatocytes, overexpressed AEG-1 entraps RXR in cytoplasm, precluding its nuclear translocation. In addition, ERK, activated by AEG-1, phosphorylates RXR that leads to its functional inactivation and attenuation of ligand-dependent transactivation. In nude mice models, combination of all-trans retinoic acid (ATRA) and AEG-1 knockdown synergistically inhibited growth of human HCC xenografts. The present study establishes AEG-1 as a novel homeostatic regulator of RXR and RXR/RAR that might contribute to hepatocarcinogenesis. Targeting AEG-1 could sensitize patients with HCC and AML to retinoid- and rexinoid-based therapeutics. Cancer Res; 74(16); 4364–77. ©2014 AACR.

Relatively little progress has been made in determining the in vivo regulation of glutathione S-transferase P (GSTP), particularly the human enzyme hGSTP1, despite being identified as a significant factor in carcinogenesis and development of drug resistance in tumor cell lines. Here, we report the characterization of a transgenic reporter mouse that reveals how hGSTP1 is regulated in vivo by chemopreventive agents. Basal expression was found in crypts and villi of the small and large intestine, bronchiolar epithelial cells, the epidermis and hair follicles, gall bladder epithelium, choroid plexus, and biliary epithelium. Expression was induced in different tissues by the antioxidant chemopreventive agents ethoxyquin and butylated hydroxyanisole. However, genetic deletion of the Nrf2 transcription factor, which directs central genetic programs of detoxification and protection against oxidative stress, increased rather than attenuated GSTP1 expression. In vitro investigations with mouse embryonic fibroblasts revealed factors, in addition to Nrf2, that control the expression of GSTP1, offering further insights into regulation. The new reporter mouse described here provides a useful tool to gain deeper insights into the mechanisms of action of chemopreventive compounds and other environmental agents. Cancer Res; 74(16); 4378–87. ©2014 AACR.

Malignant mesotheliomas are highly aggressive tumors usually caused by exposure to asbestos. Germline-inactivating mutations of BAP1 predispose to mesothelioma and certain other cancers. However, why mesothelioma is the predominate malignancy in some BAP1 families and not others, and whether exposure to asbestos is required for development of mesothelioma in BAP1 mutation carriers are not known. To address these questions experimentally, we generated a Bap1+/− knockout mouse model to assess its susceptibility to mesothelioma upon chronic exposure to asbestos. Bap1+/− mice exhibited a significantly higher incidence of asbestos-induced mesothelioma than wild-type (WT) littermates (73% vs. 32%, respectively). Furthermore, mesotheliomas arose at an accelerated rate in Bap1+/− mice than in WT animals (median survival, 43 weeks vs. 55 weeks after initial exposure, respectively) and showed increased invasiveness and proliferation. No spontaneous mesotheliomas were seen in unexposed Bap1+/− mice followed for up to 87 weeks of age. Mesothelioma cells from Bap1+/− mice showed biallelic inactivation of Bap1, consistent with its proposed role as a recessive cancer susceptibility gene. Unlike in WT mice, mesotheliomas from Bap1+/− mice did not require homozygous loss of Cdkn2a. However, normal mesothelial cells and mesothelioma cells from Bap1+/− mice showed downregulation of Rb through a p16(Ink4a)-independent mechanism, suggesting that predisposition of Bap1+/− mice to mesothelioma may be facilitated, in part, by cooperation between Bap1 and Rb. Drawing parallels to human disease, these unbiased genetic findings indicate that BAP1 mutation carriers are predisposed to the tumorigenic effects of asbestos and suggest that high penetrance of mesothelioma requires such environmental exposure. Cancer Res; 74(16); 4388–97. ©2014 AACR.

Epidemiologic studies associate low serum vitamin D levels with an increased risk of colon cancer and inflammatory diseases such as inflammatory bowel disease (IBD). 129-Smad3tm1Par/J (Smad3−/−) mice are a model of bacteria-driven colitis and colon cancer when infected with Helicobacter bilis (H. bilis). Thus, we used this mouse model to determine whether increased dietary vitamin D would reduce inflammation and colon cancer. Smad3−/− mice were fed purified diet with either maintenance (1 IU vitamin D/g diet; maintenance) or increased concentrations of vitamin D (5 IU vitamin D/g diet; high vitamin D). One week after diet initiation, mice were inoculated with broth or H. bilis and were necropsied at several time points postinoculation to assess inflammation, dysplasia, and neoplasia incidence. At 16 weeks postinfection, 11% of mice fed high vitamin D diet had cancer compared with 41% of mice fed maintenance diet (P = 0.0121). Evaluation at an early time point (1 week postinfection) showed that animals fed high vitamin D had decreased MAPK (p-P38 and p-JNK) activation in lamina propria leukocytes as well as decreased NFκB activation in colonic epithelial cells. Reduction in MAPK and NFκB activation correlated with decreased IBD scores (2.7 vs. 15.5; P < 0.0001) as well as decreased inflammatory cell infiltrates and reduced expression of proinflammatory cytokines in cecal tissue. These findings suggest that increased dietary vitamin D is beneficial in preventing inflammation-associated colon cancer through suppression of inflammatory responses during initiation of neoplasia or early-stage carcinogenesis. Cancer Res; 74(16); 4398–408. ©2014 AACR.

Cancer cell chemoresistance arises in part through the acquisition of apoptotic resistance. Leukemia cells resistant to chemotherapy-induced apoptosis have been found to be sensitive to oridonin, a natural agent with potent anticancer activity. To investigate its mechanisms of action in reversing chemoresistance, we compared the response of human leukemia cells with oridonin and the antileukemia drugs Ara-C and VP-16. Compared with HL60 cells, K562 and K562/ADR cells displayed resistance to apoptosis stimulated by Ara-C and VP-16 but sensitivity to oridonin. Mechanistic investigations revealed that oridonin upregulated BIM-S by diminishing the expression of miR-17 and miR-20a, leading to mitochondria-dependent apoptosis. In contrast, neither Ara-C nor VP-16 could reduce miR-17 and miR-20a expression or could trigger BIM-S–mediated apoptosis. Notably, silencing miR-17 or miR-20a expression by treatment with microRNA (miRNA; miR) inhibitors or oridonin restored sensitivity of K562 cells to VP-16. Synergistic effects of oridonin and VP-16 were documented in cultured cells as well as mouse tumor xenograft assays. Inhibiting miR-17 or miR-20a also augmented the proapoptotic activity of oridonin. Taken together, our results identify a miRNA-dependent mechanism underlying the anticancer effect of oridonin and provide a rationale for its combination with chemotherapy drugs in addressing chemoresistant leukemia cells. Cancer Res; 74(16); 4409–19. ©2014 AACR.

Males have a higher incidence of renal cell carcinoma (RCC) than females, but the reason for this gender difference is unknown. Addressing this question, we report the discovery of an androgen receptor (AR)–induced HIF2α/VEGF signal that drives RCC progression. AR attenuation or augmentation in RCC cells altered their proliferation, migration, and invasion in multiple models in vitro and in vivo. Mechanistic investigations revealed that AR targeting inhibited RCC cell migration and invasion by modulating HIF2α/VEGF signals at the level of mRNA and protein expression. Interrupting HIF2α/VEGF signals with inhibitors of either HIF2α or VEGF was sufficient to suppress RCC progression. Similarly, the specific AR degradation enhancer ASC-J9 was sufficient to suppress AR-induced HIF2α/VEGF signaling and RCC progression in multiple models in vitro and in vivo. Taken together, our results revealed a novel role for AR in RCC initiation and progression with implications for novel therapeutic strategies. Cancer Res; 74(16); 4420–30. ©2014 AACR.

Cancer stem cells (CSC) have garnered significant attention as a therapeutic focus, based on evidence that they may represent an etiologic root of treatment-resistant cells. Indeed, expression of the multidrug resistance protein ATP-binding cassette subfamily G member 2 (ABCG2) confers chemoresistance to CSCs, where it serves as a potential biomarker and therapeutic target. Here, we show that afatinib, a small-molecule inhibitor of the tyrosine kinases EGFR, HER2, and HER4, preferentially eliminated side population cells with CSC character, in both cell lines and patient-derived leukemia cells, by decreasing ABCG2 expression. In these cells, afatinib also acted in parallel to suppress self-renewal capacity and tumorigenicity. Combining afatinib with the DNA-damaging drug topotecan enhanced the antitumor effect of topotecan in vitro and in vivo. Mechanistic investigations suggested that ABCG2 suppression by afatinib did not proceed by proteolysis through the ubiquitin-dependent proteosome, lysosome, or calpain. Instead, we found that afatinib increased DNA methyltransferase activity, thereby leading to methylation of the ABCG2 promoter and to a decrease in ABCG2 message level. Taken together, our results advocate the use of afatinib in combination with conventional chemotherapeutic drugs to improve efficacy by improving CSC eradication. Cancer Res; 74(16); 4431–45. ©2014 AACR.

Obesity is associated with a worse breast cancer prognosis and elevated levels of inflammation, including greater cyclooxygenase-2 (COX-2) expression and activity in adipose-infiltrating macrophages. The product of this enzyme, the proinflammatory eicosanoid prostaglandin E2 (PGE2), stimulates adipose tissue aromatase expression and subsequent estrogen production, which could promote breast cancer progression. This study demonstrates that daily use of a nonsteroidal anti-inflammatory drug (NSAID), which inhibits COX-2 activity, is associated with reduced estrogen receptor α (ERα)–positive breast cancer recurrence in obese and overweight women. Retrospective review of data from ERα-positive patients with an average body mass index of >30 revealed that NSAID users had a 52% lower recurrence rate and a 28-month delay in time to recurrence. To examine the mechanisms that may be mediating this effect, we conducted in vitro studies that utilized sera from obese and normal-weight patients with breast cancer. Exposure to sera from obese patients stimulated greater macrophage COX-2 expression and PGE2 production. This was correlated with enhanced preadipocyte aromatase expression following incubation in conditioned media (CM) collected from the obese-patient, sera-exposed macrophages, an effect neutralized by COX-2 inhibition with celecoxib. In addition, CM from macrophage/preadipocyte cocultures exposed to sera from obese patients stimulated greater breast cancer cell ERα activity, proliferation, and migration compared with sera from normal-weight patients, and these differences were eliminated or reduced by the addition of an aromatase inhibitor during CM generation. Prospective studies designed to examine the clinical benefit of NSAID use in obese patients with breast cancer are warranted. Cancer Res; 74(16); 4446–57. ©2014 AACR.

The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM. Cancer Res; 74(16); 4458–69. ©2014 AACR.

Eph receptor tyrosine kinases are critical for cell–cell communication during normal and oncogenic tissue patterning and tumor growth. Somatic mutation profiles of several cancer genomes suggest EphA3 as a tumor suppressor, but its oncogenic expression pattern and role in tumorigenesis remain largely undefined. Here, we report unexpected EphA3 overexpression within the microenvironment of a range of human cancers and mouse tumor xenografts where its activation inhibits tumor growth. EphA3 is found on mouse bone marrow–derived cells with mesenchymal and myeloid phenotypes, and activation of EphA3+/CD90+/Sca1+ mesenchymal/stromal cells with an EphA3 agonist leads to cell contraction, cell–cell segregation, and apoptosis. Treatment of mice with an agonistic α-EphA3 antibody inhibits tumor growth by severely disrupting the integrity and function of newly formed tumor stroma and microvasculature. Our data define EphA3 as a novel target for selective ablation of the tumor microenvironment and demonstrate the potential of EphA3 agonists for anticancer therapy. Cancer Res; 74(16); 4470–81. ©2014 AACR.

Glioma-initiating cells (GIC), which reside within the perivascular microenvironment to maintain self-renewal capacity, are responsible for glioblastoma initiation, progression, and recurrence. However, the molecular mechanisms controlling crosstalk between GICs and endothelial cells are poorly understood. Here, we report that, in both GICs and endothelial cells, platelet-derived growth factor (PDGF)–driven activation of nitric oxide (NO) synthase increases NO-dependent inhibitor of differentiation 4 (ID4) expression, which in turn promotes JAGGED1–NOTCH activity through suppression of miR129 that specifically represses JAGGED1 suppression. This signaling axis promotes tumor progression along with increased GIC self-renewal and growth of tumor vasculature in the xenograft tumors, which is dramatically suppressed by NOTCH inhibitor. ID4 levels correlate positively with NOS2 (NO synthase-2), HES1, and HEY1 and negatively with miR129 in primary GICs. Thus, targeting the PDGF–NOS–ID4–miR129 axis and NOTCH activity in the perivascular microenvironment might serve as an efficacious therapeutic modality for glioblastoma. Cancer Res; 74(16); 4482–92. ©2014 AACR.

Although antitubulin drugs are used widely to treat human cancer, many patients display intrinsic or acquired drug resistance that imposes major obstacles to successful therapy. Mounting evidence argues that cancer cell apoptosis triggered by antitubulin drugs relies upon activation of the cell-cycle kinase Cdk1; however, mechanistic connections of this event to apoptosis remain obscure. In this study, we identified the antiapoptotic protein YAP, a core component of the Hippo signaling pathway implicated in tumorigenesis, as a critical linker coupling Cdk1 activation to apoptosis in the antitubulin drug response. Antitubulin drugs activated Cdk1, which directly phosphorylated YAP on five sites independent of the Hippo pathway. Mutations in these phosphorylation sites on YAP relieved its ability to block antitubulin drug-induced apoptosis, further suggesting that YAP was inactivated by Cdk1 phosphorylation. Notably, we found that YAP was not phosphorylated and inactivated after antitubulin drug treatment in taxol-resistant cancer cells. Our findings suggest YAP and its phosphorylation status as candidate prognostic markers in predicting antitubulin drug response in patients. Cancer Res; 74(16); 4493–503. ©2014 AACR.

Heparanase has been implicated in cancer but its contribution to the early stages of cancer development is uncertain. In this study, we utilized nontransformed human MCF10A mammary epithelial cells and two genetic mouse models [Hpa-transgenic (Hpa-Tg) and knockout mice] to explore heparanase function at early stages of tumor development. Heparanase overexpression resulted in significantly enlarged asymmetrical acinar structures, indicating increased cell proliferation and decreased organization. This phenotype was enhanced by coexpression of heparanase variants with a mutant H-Ras gene, which was sufficient to enable growth of invasive carcinoma in vivo. These observations were extended in vivo by comparing the response of Hpa-Tg mice to a classical two-stage 12-dimethylbenz(a)anthracene (DMBA)/12-o-tetradecanoylphorbol-13-acetate (TPA) protocol for skin carcinogenesis. Hpa-Tg mice overexpressing heparanase were far more sensitive than control mice to DMBA/TPA treatment, exhibiting a 10-fold increase in the number and size of tumor lesions. Conversely, DMBA/TPA-induced tumor formation was greatly attenuated in Hpa-KO mice lacking heparanase, pointing to a critical role of heparanase in skin tumorigenesis. In support of these observations, the heparanase inhibitor PG545 potently suppressed tumor progression in this model system. Taken together, our findings establish that heparanase exerts protumorigenic properties at early stages of tumor initiation, cooperating with Ras to dramatically promote malignant development. Cancer Res; 74(16); 4504–14. ©2014 AACR.

Hepatocellular carcinoma (HCC) was thought historically to arise from hepatocytes, but gene expression studies have suggested that it can also arise from fetal progenitor cells or their adult progenitor progeny. Here, we report the identification of a unique population of fetal liver progenitor cells in mice that can serve as a cell of origin in HCC development. In the transgenic model used, mice carry the Cited1-CreERTM-GFP BAC transgene in which a tamoxifen-inducible Cre (CreERTM) and GFP are controlled by a 190-kb 5′ genomic region of Cited1, a transcriptional coactivator protein for CBP/p300. Wnt signaling is critical for regulating self-renewal of progenitor/stem cells and has been implicated in the etiology of cancers of rapidly self-renewing tissues, so we hypothesized that Wnt pathway activation in CreERTM-GFP+ progenitors would result in HCC. In livers from the mouse model, transgene-expressing cells represented 4% of liver cells at E11.5 when other markers were expressed, characteristic of the hepatic stem/progenitor cells that give rise to adult hepatocytes, cholangiocytes, and SOX9+ periductal cells. By 26 weeks of age, more than 90% of Cited1-CreERTM-GFP;Ctnnb1ex3(fl) mice with Wnt pathway activation developed HCC and, in some cases, hepatoblastomas and lung metastases. HCC and hepatoblastomas resembled their human counterparts histologically, showing activation of Wnt, Ras/Raf/MAPK, and PI3K/AKT/mTOR pathways and expressing relevant stem/progenitor cell markers. Our results show that Wnt pathway activation is sufficient for malignant transformation of these unique liver progenitor cells, offering functional support for a fetal/adult progenitor origin of some human HCC. We believe this model may offer a valuable new tool to improve understanding of the cellular etiology and biology of HCC and hepatoblastomas and the development of improved therapeutics for these diseases. Cancer Res; 74(16); 4515–25. ©2014 AACR.

The oncogenic fusion gene EWS–WT1 is the defining chromosomal translocation in desmoplastic small round-cell tumors (DSRCT), a rare but aggressive soft tissue sarcoma with a high rate of mortality. EWS–WT1 functions as an aberrant transcription factor that drives tumorigenesis, but the mechanistic basis for its pathogenic activity is not well understood. To address this question, we created a transgenic mouse strain that permits physiologic expression of EWS–WT1 under the native murine Ews promoter. EWS–WT1 expression led to a dramatic induction of many neuronal genes in embryonic fibroblasts and primary DSRCT, most notably the neural reprogramming factor ASCL1. Mechanistic analyses demonstrated that EWS–WT1 directly bound the proximal promoter of ASCL1, activating its transcription through multiple WT1-responsive elements. Conversely, EWS–WT1 silencing in DSRCT cells reduced ASCL1 expression and cell viability. Notably, exposure of DSRCT cells to neuronal induction media increased neural gene expression and induced neurite-like projections, both of which were abrogated by silencing EWS–WT1. Taken together, our findings reveal that EWS–WT1 can activate neural gene expression and direct partial neural differentiation via ASCL1, suggesting agents that promote neural differentiation might offer a novel therapeutic approach to treat DSRCT. Cancer Res; 74(16); 4526–35. ©2014 AACR.

Cancer stem cells, capable of self-renewal and multipotent differentiation, influence tumor behavior through a complex balance of symmetric and asymmetric cell divisions. Mechanisms regulating the dynamics of stem cells and their progeny in human cancer are poorly understood. In Drosophila, mutation of brain tumor (brat) leads to loss of normal asymmetric cell division by developing neural cells and results in a massively enlarged brain composed of neuroblasts with neoplastic properties. Brat promotes asymmetric cell division and directs neural differentiation at least partially through its suppression on Myc. We identified TRIM3 (11p15.5) as a human ortholog of Drosophila brat and demonstrate its regulation of asymmetric cell division and stem cell properties of glioblastoma (GBM), a highly malignant human brain tumor. TRIM3 gene expression is markedly reduced in human GBM samples, neurosphere cultures, and cell lines and its reconstitution impairs growth properties in vitro and in vivo. TRIM3 expression attenuates stem-like qualities of primary GBM cultures, including neurosphere formation and the expression of stem cell markers CD133, Nestin, and Nanog. In GBM stem cells, TRIM3 expression leads to a greater percentage dividing asymmetrically rather than symmetrically. As with Brat in Drosophila, TRIM3 suppresses c-Myc expression and activity in human glioma cell lines. We also demonstrate a strong regulation of Musashi–Notch signaling by TRIM3 in GBM neurospheres and neural stem cells that may better explain its effect on stem cell dynamics. We conclude that TRIM3 acts as a tumor suppressor in GBM by restoring asymmetric cell division. Cancer Res; 74(16); 4536–48. ©2014 AACR.

Tumor hypoxia drives metastatic progression, drug resistance, and posttreatment relapses, but how cancer cells adapt and evolve in response to hypoxic stress is not well understood. In this study, we address this question with the discovery that the receptor tyrosine kinase RON translocates into the nucleus of hypoxic cancer cells. In response to hypoxia, nuclear RON interacts with the hypoxia-inducible factor HIF-1α in a manner that relies on RON tyrosine kinase activity, binding to the c-JUN promoter and activating it. Mechanistic investigations revealed unexpectedly that nuclear RON played a more important role in activation of the c-JUN promoter than HIF-1α, leading to increased cell proliferation, survival adaptation, in vitro migration, and tumorigenicity under hypoxic conditions. Taken together, our results pointed to a novel function for RON as a transcriptional regulator that promotes the survival of cancer cells subjected to hypoxia. These results suggest novel implications for the use of small-molecule inhibitors or monoclonal antibodies targeting the RON kinase in the prevention or treatment of advanced cancer. Cancer Res; 74(16); 4549–62. ©2014 AACR.