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Cancer Research

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Cancer Research

Abnormalities in the TP53 gene and overexpression of MDM2, a transcriptional target and negative regulator of p53, are commonly observed in cancers. The MDM2–p53 feedback loop plays an important role in tumor progression and thus, increased understanding of the pathway has the potential to improve clinical outcomes for cancer patients. Hepatocellular carcinoma (HCC) has emerged as one of the most commonly diagnosed forms of human cancer; yet, the current treatment for HCC is less effective than those used against other cancers. We review the current studies of the MDM2–p53 pathway in cancer with a focus on HCC and specifically discuss the impact of p53 mutations along with other alterations of the MDM2–p53 feedback loop in HCC. We also discuss the potential diagnostic and prognostic applications of p53 and MDM2 in malignant tumors as well as therapeutic avenues that are being developed to target the MDM2–p53 pathway. Cancer Res; 74(24); 7161–7. ©2014 AACR.

T cells are a crucial component of the immune response to infection and cancer. In addition to coordinating immunity in lymphoid tissue, T cells play a vital role at the disease site, which relies on their efficient and specific trafficking capabilities. The process of T-cell trafficking is highly dynamic, involving a series of distinct processes, which include rolling, adhesion, extravasation, and chemotaxis. Trafficking of T cells to the tumor microenvironment is critical for the success of cancer immunotherapies such as adoptive cellular transfer. Although this approach has achieved some remarkable responses in patients with advanced melanoma and hematologic malignancy, the success against solid cancers has been more moderate. One of the major challenges for adoptive immunotherapy is to be able to effectively target a higher frequency of T cells to the tumor microenvironment, overcoming hurdles associated with immunosuppression and aberrant vasculature. This review summarizes recent advances in our understanding of T-cell migration in solid cancer and immunotherapy based on the adoptive transfer of natural or genetically engineered tumor-specific T cells and discusses new strategies that may enhance the trafficking of these cells, leading to effective eradication of solid cancer and metastases. Cancer Res; 74(24); 7168–74. ©2014 AACR.

Cancer cells rapidly evolve a multitude of defense mechanisms to evade the effects of the oncologist's drug arsenal. Unfortunately, clinical strategies to overcome these lag far behind. This mismatch likely underlies our inability to implement new durable treatment strategies. Here, a new form of multidrug resistance, inducible drug glucuronidation, is discussed. This form was discovered while developing means to target a specific oncogene, the eukaryotic translation initiation factor 4E (eIF4E), with its inhibitor ribavirin. In two clinical studies, ribavirin treatment led to substantial clinical responses, but all responding patients eventually relapsed. In most cases, this was due to the overexpression of the sonic hedgehog transcription factor Gli1, which elevated the UDP glucuronsyltransferase UGT1A enzymes. UGT1As add glucuronic acid to many drugs. Indeed, these cells are resistant to not only ribavirin, but also Ara-C, and likely other drugs. Inhibition of Gli1 reduced UGT1As, eliminated drug glucuronides, and renewed sensitivity to ribavirin and Ara-C. These studies highlight that cancer cells and their resistant counterparts metabolize drugs differently from each other as well as from normal cells. Likely, these inducible modifications go beyond glucuronidation. Understanding the extent of inducible drug modifications and the pathways that drive expression of the corresponding enzymatic machinery will better position us to finally make resistance futile. Cancer Res; 74(24); 7175–80. ©2014 AACR.

With increasingly sophisticated technologies in molecular biology and “omic” platforms to analyze patients' tumors, more molecular diversity and complexity in cancer are being observed. Recently, we noted unique genomic profiles in a group of patients with metastatic breast cancer based on an analysis with next-generation sequencing. Among 57 consecutive patients, no two had the same molecular portfolio. Applied genomics therefore appears to represent a disruptive innovation in that it unveils a heterogeneity to metastatic cancer that may be ill-suited to canonical clinical trials and practice paradigms. Upon recognizing that patients have unique tumor landscapes, it is possible that there may be a “mismatch” between our traditional clinical trials system that selects patients based on common characteristics to evaluate a drug (drug-centric approach) and optimal treatment based on curated, individualized drug combinations for each patient (patient-centric approach). Cancer Res; 74(24); 7181–4. ©2014 AACR.

An important aspect of malignant progression is the acquired ability of tumor cells to avoid recognition and destruction by the immune system (immune escape). Clinical cancer progression is also associated with the development of tumor hypoxia, which is mechanistically linked to the acquisition of malignant phenotypes in cancer cells. Despite the well-established role of hypoxia in tumor cell invasion and metastasis, and resistance to therapy, relatively few studies have examined the contribution of hypoxia to cancer immune escape. Accumulating evidence reveals that hypoxia can impair anticancer immunity by altering the function of innate and adaptive immune cells and/or by increasing the intrinsic resistance of tumor cells to the cytolytic activity of immune effectors. Here, we discuss certain aspects of the contribution of hypoxia to tumor immune escape and provide evidence for a novel role of cyclic guanosine monophosphate (cGMP) signaling in the regulation of hypoxia-induced immune escape. Thus, we propose that activation of cGMP signaling in cancer cells may have important immunotherapeutic applications. Cancer Res; 74(24); 7185–90. ©2014 AACR.

DUSP1 is a dual-specificity phosphatase that regulates mitogen-activated protein (MAP) kinase activity. Studies have associated loss of DUSP1 expression with certain cancers, but there has been no report of a mechanism by which this supports tumor progression. In this study, we found DUSP1 mRNA and protein decreased in human head and neck squamous cell carcinoma tissues compared with adjacent nontumor controls. To evaluate the impact of this difference, we compared the susceptibility of Dusp1-deficient mice with oral squamous carcinogenesis induced by 4-nitroquinoline 1-oxide. Dusp1-deficient mice displayed enhanced disease progression, characterized by advanced onset, histologic stage, and tumor burden. In a syngeneic model of tumor progression, subcutaneous injection of EO771 cells formed faster-growing tumors in Dusp1-deficient mice, an effect abrogated by inhibition of p38 MAP kinase with SB203580. Histologic and quantitative assessments demonstrated increased inflammation and deregulated chemokine and cytokine expression in Dusp1-deficient tumor tissues. Specifically, proinflammatory cytokine IL1β was elevated. IL1β production was recapitulated ex vivo in primary bone marrow–derived macrophages from Dusp1-deficient mice. Together, our results clearly establish the role of Dusp1 as a tumor suppressor gene that regulates cancer-associated inflammation. Cancer Res; 74(24); 7191–7. ©2014 AACR.

Cancer cells may overcome growth factor dependence by deregulating oncogenic and/or tumor-suppressor pathways that affect their metabolism, or by activating metabolic pathways de novo with targeted mutations in critical metabolic enzymes. It is unknown whether human prostate tumors develop a similar metabolic response to different oncogenic drivers or a particular oncogenic event results in its own metabolic reprogramming. Akt and Myc are arguably the most prevalent driving oncogenes in prostate cancer. Mass spectrometry–based metabolite profiling was performed on immortalized human prostate epithelial cells transformed by AKT1 or MYC, transgenic mice driven by the same oncogenes under the control of a prostate-specific promoter, and human prostate specimens characterized for the expression and activation of these oncoproteins. Integrative analysis of these metabolomic datasets revealed that AKT1 activation was associated with accumulation of aerobic glycolysis metabolites, whereas MYC overexpression was associated with dysregulated lipid metabolism. Selected metabolites that differentially accumulated in the MYC-high versus AKT1-high tumors, or in normal versus tumor prostate tissue by untargeted metabolomics, were validated using absolute quantitation assays. Importantly, the AKT1/MYC status was independent of Gleason grade and pathologic staging. Our findings show how prostate tumors undergo a metabolic reprogramming that reflects their molecular phenotypes, with implications for the development of metabolic diagnostics and targeted therapeutics. Cancer Res; 74(24); 7198–204. ©2014 AACR.

While viral antigens in human papillomavirus (HPV)-related oropharyngeal cancer (HPVOPC) are attractive targets for immunotherapy, the effects of existing standard-of-care therapies on immune responses to HPV are poorly understood. We serially sampled blood from patients with stage III–IV oropharyngeal cancer undergoing concomitant chemoradiotherapy with or without induction chemotherapy. Circulating immunocytes including CD4+ and CD8+ T cells, regulatory T cells (Treg), and myeloid-derived suppressor cells (MDSC) were profiled by flow cytometry. Antigen-specific T-cell responses were measured in response to HPV16 E6 and E7 peptide pools. The role of PD-1 signaling in treatment-related immunosuppression was functionally defined by performing HPV-specific T-cell assays in the presence of blocking antibody. While HPV-specific T-cell responses were present in 13 of 18 patients before treatment, 10 of 13 patients lost these responses within 3 months after chemoradiotherapy. Chemoradiotherapy decreased circulating T cells and markedly elevated MDSCs. PD-1 expression on CD4+ T cells increased by nearly 2.5-fold after chemoradiotherapy, and ex vivo culture with PD-1–blocking antibody enhanced HPV-specific T-cell responses in 8 of 18 samples tested. Chemoradiotherapy suppresses circulating immune responses in patients with HPVOPC by unfavorably altering effector:suppressor immunocyte ratios and upregulating PD-1 expression on CD4+ T cells. These data strongly support testing of PD-1–blocking agents in combination with standard-of-care chemoradiotherapy for HPVOPC. Cancer Res; 74(24); 7205–16. ©2014 AACR.

DDR2 mutations occur in approximately 4% of lung squamous cell cancer (SCC) where the tyrosine kinase inhibitor dasatinib has emerged as a new therapeutic option. We found that ERK and AKT phosphorylation was weakly inhibited by dasatinib in DDR2-mutant lung SCC cells, suggesting that dasatinib inhibits survival signals distinct from other oncogenic receptor tyrosine kinases (RTK) and/or compensatory signals exist that dampen dasatinib activity. To gain better insight into dasatinib's action in these cells, we assessed altered global tyrosine phosphorylation (pY) after dasatinib exposure using a mass spectrometry-based quantitative phosphoproteomics approach. Overlaying protein–protein interaction relationships upon this dasatinib-regulated pY network revealed decreased phosphorylation of Src family kinases and their targets. Conversely, dasatinib enhanced tyrosine phosphorylation in a panel of RTK and their signaling adaptor complexes, including EGFR, MET/GAB1, and IGF1R/IRS2, implicating a RTK-driven adaptive response associated with dasatinib. To address the significance of this observation, these results were further integrated with results from a small-molecule chemical library screen. We found that dasatinib combined with MET and insulin-like growth factor receptor (IGF1R) inhibitors had a synergistic effect, and ligand stimulation of EGFR and MET rescued DDR2-mutant lung SCC cells from dasatinib-induced loss of cell viability. Importantly, we observed high levels of tyrosine-phosphorylated EGFR and MET in a panel of human lung SCC tissues harboring DDR2 mutations. Our results highlight potential RTK-driven adaptive-resistant mechanisms upon DDR2 targeting, and they suggest new, rationale cotargeting strategies for DDR2-mutant lung SCC. Cancer Res; 74(24); 7217–28. ©2014 AACR.

Modeling the hematogenous spread of cancer cells to distant organs poses one of the greatest challenges in the study of human metastasis. Both tumor cell–intrinsic properties as well as interactions with reactive stromal cells contribute to this process, but identification of relevant stromal signals has been hampered by the lack of models allowing characterization of the metastatic niche. Here, we describe an implantable bioengineered scaffold, amenable to in vivo imaging, ex vivo manipulation, and serial transplantation for the continuous study of human metastasis in mice. Orthotopic or systemic inoculation of tagged human cancer cells into the mouse leads to the release of circulating tumor cells into the vasculature, which seed the scaffold, initiating a metastatic tumor focus. Mouse stromal cells can be readily recovered and profiled, revealing differential expression of cytokines, such as IL1β, from tumor-bearing versus unseeded scaffolds. Finally, this platform can be used to test the effect of drugs on suppressing initiation of metastatic lesions. This generalizable model to study cancer metastasis may thus identify key stromal-derived factors with important implications for basic and translational cancer research. Cancer Res; 74(24); 7229–38. ©2014 AACR.

Adenosine A2A receptor (A2AR) blockade enhances innate and adaptive immune responses. However, mouse genetic studies have shown that A2AR deletion does not inhibit the growth of all tumor types. In the current study, we showed that growth rates for ectopic melanoma and bladder tumors are increased in Adora2a−/− mice within 2 weeks of tumor inoculation. A2AR deletion in the host reduced numbers of CD8+ T cells and effector–memory differentiation of all T cells. To examine intrinsic functions in T cells, we generated mice harboring a T-cell–specific deletion of A2AR. In this host strain, tumor-bearing mice displayed increased growth of ectopic melanomas, decreased numbers of tumor-associated T cells, reduced effector–memory differentiation, and reduced antiapoptotic IL7Rα (CD127) expression on antigen-experienced cells. Intratumoral pharmacologic blockade similarly reduced CD8+ T-cell density within tumors in wild-type hosts. We found that A2AR-proficient CD8+ T cells specific for melanoma cells displayed a relative survival advantage in tumors. Thus, abrogating A2AR signaling appeared to reduce IL7R expression, survival, and differentiation of T cells in the tumor microenvironment. One implication of these results is that the antitumor effects of A2AR blockade that can be mediated by activation of cytotoxic T cells may be overcome in some tumor microenvironments as a result of impaired T-cell maintenance and effector–memory differentiation. Thus, our findings imply that the efficacious application of A2AR inhibitors for cancer immunotherapy may require careful dose optimization to prevent activation-induced T-cell death in tumors. Cancer Res; 74(24); 7239–49. ©2014 AACR.

High concentrations of adenosine in tumor microenvironments inhibit antitumor cytotoxic lymphocyte responses. Although T cells express inhibitory adenosine A2A receptors (A2AR) that suppress their activation and inhibit immune killing of tumors, a role for myeloid cell A2ARs in suppressing the immune response to tumors has yet to be investigated. In this study, we show that the growth of transplanted syngeneic B16F10 melanoma or Lewis lung carcinoma cells is slowed in Adora2af/f–LysMCre+/− mice, which selectively lack myeloid A2ARs. Reduced melanoma growth is associated with significant increases in MHCII and IL12 expression in tumor-associated macrophages and with >90% reductions in IL10 expression in tumor-associated macrophages, dendritic cells (DC), and Ly6C+ or Ly6G+ myeloid-derived suppressor cells (MDSC). Myeloid deletion of A2ARs significantly increases CD44 expression on tumor-associated T cells and natural killer (NK) cells. Depletion of CD8+ T cells or NK cells in tumor-bearing mice indicates that both cell types initially contribute to slowing melanoma growth in mice lacking myeloid A2A receptors, but tumor suppression mediated by CD8+ T cells is more persistent. Myeloid-selective A2AR deletion significantly reduces lung metastasis of melanomas that express luciferase (for in vivo tracking) and ovalbumin (as a model antigen). Reduced metastasis is associated with increased numbers and activation of NK cells and antigen-specific CD8+ T cells in lung infiltrates. Overall, the findings indicate that myeloid cell A2ARs have direct myelosuppressive effects that indirectly contribute to the suppression of T cells and NK cells in primary and metastatic tumor microenvironments. The results indicate that tumor-associated myeloid cells, including macrophages, DCs, and MDSCs all express immunosuppressive A2ARs that are potential targets of adenosine receptor blockers to enhance immune killing of tumors. Cancer Res; 74(24); 7250–9. ©2014 AACR.

Oncolytic virus therapy is being evaluated in clinical trials for human glioma. While it is widely assumed that the immune response of the patient to the virus infection limits the utility of the therapy, investigations into the specific cell type(s) involved in this response have been performed using nonspecific pharmacologic inhibitors or allogeneic models with compromised immunity. To identify the immune cells that participate in clearing an oncolytic infection in glioma, we used flow cytometry and immunohistochemistry to immunophenotype an orthotopic glioma model in immunocompetent mice after Myxoma virus (MYXV) administration. These studies revealed a large resident microglia and macrophage population in untreated tumors, and robust monocyte, T-, and NK cell infiltration 3 days after MYXV infection. To determine the role on the clinical utility of MYXV therapy for glioma, we used a combination of knockout mouse strains and specific immunocyte ablation techniques. Collectively, our experiments identify an important role for tumor-resident myeloid cells and overlapping roles for recruited NK and T cells in the clearance and efficacy of oncolytic MYXV from gliomas. Using a cyclophosphamide regimen to achieve lymphoablation prior and during MYXV treatment, we prevented treatment-induced peripheral immunocyte recruitment and, surprisingly, largely ablated the tumor-resident macrophage population. Virotherapy of cyclophosphamide-treated animals resulted in sustained viral infection within the glioma as well as a substantial survival advantage. This study demonstrates that resistance to MYXV virotherapy in syngeneic glioma models involves a multifaceted cellular immune response that can be overcome with cyclophosphamide-mediated lymphoablation. Cancer Res; 74(24); 7260–73. ©2014 AACR.

Myeloid cells are capable of promoting or eradicating tumor cells and the nodal functions that contribute to their different roles are still obscure. Here, we show that mice with myeloid-specific genetic loss of the NF-κB pathway regulatory kinase IKKβ exhibit more rapid growth of cutaneous and lung melanoma tumors. In a BRAFV600E/PTEN−/− allograft model, IKKβ loss in macrophages reduced recruitment of myeloid cells into the tumor, lowered expression of MHC class II molecules, and enhanced production of the chemokine CCL11, thereby negatively regulating dendritic-cell maturation. Elevated serum and tissue levels of CCL11 mediated suppression of dendritic-cell differentiation/maturation within the tumor microenvironment, skewing it toward a Th2 immune response and impairing CD8+ T cell–mediated tumor cell lysis. Depleting macrophages or CD8+ T cells in mice with wild-type IKKβ myeloid cells enhanced tumor growth, where the myeloid cell response was used to mediate antitumor immunity against melanoma tumors (with less dependency on a CD8+ T-cell response). In contrast, myeloid cells deficient in IKKβ were compromised in tumor cell lysis, based on their reduced ability to phagocytize and digest tumor cells. Thus, mice with continuous IKKβ signaling in myeloid-lineage cells (IKKβCA) exhibited enhanced antitumor immunity and reduced melanoma outgrowth. Collectively, our results illuminate new mechanisms through which NF-κB signaling in myeloid cells promotes innate tumor surveillance. Cancer Res; 74(24); 7274–84. ©2014 AACR.

Interaction of RAGE (the receptor for advanced glycation endproducts) with its ligands can promote tumor progression, invasion, and angiogenesis. Although blocking RAGE signaling has been proposed as a potential anticancer strategy, functional contributions of RAGE expression in the tumor microenvironment (TME) have not been investigated in detail. Here, we evaluated the effect of genetic depletion of RAGE in TME on the growth of gliomas. In both invasive and noninvasive glioma models, animal survival was prolonged in RAGE knockout (Ager−/−) mice. However, the improvement in survival in Ager−/− mice was not due to changes in tumor growth rate but rather to a reduction in tumor-associated inflammation. Furthermore, RAGE ablation in the TME abrogated angiogenesis by downregulating the expression of proangiogenic factors, which prevented normal vessel formation, thereby generating a leaky vasculature. These alterations were most prominent in noninvasive gliomas, in which the expression of VEGF and proinflammatory cytokines were also lower in tumor-associated macrophages (TAM) in Ager−/− mice. Interestingly, reconstitution of Ager−/− TAM with wild-type microglia or macrophages normalized tumor vascularity. Our results establish that RAGE signaling in glioma-associated microglia and TAM drives angiogenesis, underscoring the complex role of RAGE and its ligands in gliomagenesis. Cancer Res; 74(24); 7285–97. ©2014 AACR.

BRAFV600E is a major oncogenic mutation found in approximately 50% of human melanoma that confers constitutive activation of the MAPK pathway and increased melanoma growth. Inhibition of BRAFV600E by oncogene targeting therapy increases overall survival of patients with melanoma, but is unable to produce many durable responses. Adaptive drug resistance remains the main limitation to BRAFV600E inhibitor clinical efficacy and immune-based strategies could be useful to overcome disease relapse. Tumor microenvironment greatly differs between visceral metastasis and primary cutaneous melanoma, and the mechanisms involved in the antimetastatic efficacy of BRAFV600E inhibitors remain to be determined. To address this question, we developed a metastatic BRAFV600E-mutant melanoma cell line and demonstrated that the antimetastatic properties of BRAF inhibitor PLX4720 (a research analogue of vemurafenib) require host natural killer (NK) cells and perforin. Indeed, PLX4720 not only directly limited BRAFV600E-induced tumor cell proliferation, but also affected NK cell functions. We showed that PLX4720 increases the phosphorylation of ERK1/2, CD69 expression, and proliferation of mouse NK cells in vitro. NK cell frequencies were significantly enhanced by PLX4720 specifically in the lungs of mice with BRAFV600E lung metastases. Furthermore, PLX4720 also increased human NK cell pERK1/2, CD69 expression, and IFNγ release in the context of anti-NKp30 and IL2 stimulation. Overall, this study supports the idea that additional NK cell-based immunotherapy (by checkpoint blockade or agonists or cytokines) may combine well with BRAFV600E inhibitor therapy to promote more durable responses in melanoma. Cancer Res; 74(24); 7298–308. ©2014 AACR.

Cancer stem cells (CSC) are thought to play a major role in the development and metastatic progression of pancreatic ductal adenocarcinoma (PDAC), one of the deadliest solid tumors. Likewise, the tumor microenvironment contributes critical support in this setting, including from tumor stromal cells and tumor-associated macrophages (TAM) that contribute structural and paracrine-mediated supports, respectively. Here, we show that TAMs secrete the IFN-stimulated factor ISG15, which enhances CSC phenotypes in PDAC in vitro and in vivo. ISG15 was preferentially and highly expressed by TAM present in primary PDAC tumors resected from patients. ISG15 was secreted by macrophages in response to secretion of IFNβ by CSC, thereby reinforcing CSC self-renewal, invasive capacity, and tumorigenic potential. Overall, our work demonstrates that ISG15 is a previously unrecognized support factor for CSC in the PDAC microenvironment with a key role in pathogenesis and progression. Cancer Res; 74(24); 7309–20. ©2014 AACR.

The oncogene astrocyte elevated gene-1 (AEG-1; MTDH) is highly expressed in glioblastoma multiforme (GBM) and many other types of cancer, where it activates multiple signaling pathways that drive proliferation, invasion, angiogenesis, chemoresistance, radioresistance, and metastasis. AEG-1 activates the Akt signaling pathway and Akt and c-Myc are positive regulators of AEG-1 transcription, generating a positive feedback loop between AEG-1 and Akt in regulating tumorigenesis. Here, we describe in GBM cells a direct interaction between an internal domain of AEG-1 and the PH domain of Akt2, a major driver in GBM. Expression and interaction of AEG-1 and Akt2 are elevated in GBM and contribute to tumor cell survival, proliferation, and invasion. Clinically, in silico gene expression and immunohistochemical analyses of patient specimens showed that AEG-1 and Akt2 expression correlated with GBM progression and reduced patient survival. AEG-1–Akt2 interaction prolonged stabilization of Akt2 phosphorylation at S474, regulating downstream signaling cascades that enable cell proliferation and survival. Disrupting AEG-1–Akt2 interaction by competitive binding of the Akt2-PH domain led to reduced cell viability and invasion. When combined with AEG-1 silencing, conditional expression of Akt2-PH markedly increased survival in an orthotopic mouse model of human GBM. Our study uncovers a novel molecular mechanism by which AEG-1 augments glioma progression and offers a rationale to block AEG-1–Akt2 signaling function as a novel GBM treatment. Cancer Res; 74(24); 7321–32. ©2014 AACR.

Aberrant histone methylation is a frequent event during tumor development and progression. KMT1E (also known as SETDB1) is a histone H3K9 methyltransferase that contributes to epigenetic silencing of both oncogenes and tumor suppressor genes in cancer cells. In this report, we demonstrate that KMT1E acts as a metastasis suppressor that is strongly downregulated in highly metastatic lung cancer cells. Restoring KMT1E expression in this setting suppressed filopodia formation, migration, and invasive behavior. Conversely, loss of KMT1E in lung cancer cells with limited metastatic potential promoted migration in vitro and restored metastatic prowess in vivo. Mechanistic investigations indicated that KMT1E cooperates with the TGFβ-regulated complex SMAD2/3 to repress metastasis through ANXA2. Together, our findings defined an essential role for the KMT1E/SMAD2/3 repressor complex in TGFβ-mediated lung cancer metastasis. Cancer Res; 74(24); 7333–43. ©2014 AACR.

Cachexia is a muscle-wasting syndrome that contributes significantly to morbidity and mortality of many patients with advanced cancers. However, little is understood about how the severe loss of skeletal muscle characterizing this condition occurs. In the current study, we tested the hypothesis that the muscle protein myostatin is involved in mediating the pathogenesis of cachexia-induced muscle wasting in tumor-bearing mice. Myostatin gene inactivation prevented the severe loss of skeletal muscle mass induced in mice engrafted with Lewis lung carcinoma (LLC) cells or in ApcMin/+ mice, an established model of colorectal cancer and cachexia. Mechanistically, myostatin loss attenuated the activation of muscle fiber proteolytic pathways by inhibiting the expression of atrophy-related genes, MuRF1 and MAFbx/Atrogin-1, along with autophagy-related genes. Notably, myostatin loss also impeded the growth of LLC tumors, the number and the size of intestinal polyps in ApcMin/+ mice, thus strongly increasing survival in both models. Gene expression analysis in the LLC model showed this phenotype to be associated with reduced expression of genes involved in tumor metabolism, activin signaling, and apoptosis. Taken together, our results reveal an essential role for myostatin in the pathogenesis of cancer cachexia and link this condition to tumor growth, with implications for furthering understanding of cancer as a systemic disease. Cancer Res; 74(24); 7344–56. ©2014 AACR.

Misexpression of developmental transcription factors occurs often in human cancers, where embryonic programs may be reinstated in a context that promotes or sustains malignant development. In this study, we report the involvement of the kidney development transcription factor Six2 in the metastatic progression of human breast cancer. We found that Six2 promoted breast cancer metastasis by a novel mechanism involving both transcriptional and epigenetic regulation of E-cadherin. Downregulation of E-cadherin by Six2 was necessary for its ability to increase soft agar growth and in vivo metastasis in an immunocompetent mouse model of breast cancer. Mechanistic investigations showed that Six2 represses E-cadherin expression by upregulating Zeb2, in part, through a microRNA-mediated mechanism and by stimulating promoter methylation of the E-cadherin gene (Cdh1). Clinically, SIX2 expression correlated inversely with CDH1 expression in human breast cancer specimens, corroborating the disease relevance of their interaction. Our findings establish Six2 as a regulator of metastasis in human breast cancers and demonstrate an epigenetic function for SIX family transcription factors in metastatic progression through the regulation of E-cadherin. Cancer Res; 74(24); 7357–70. ©2014 AACR.

PTEN mutations are the most common genetic alterations in endometrial cancer. Loss of PTEN and subsequent AKT activation stimulate estrogen receptor α–dependent pathways that play an important role in endometrial tumorigenesis. The major pathologic phenomenon of endometrial cancer is the loss of ovarian steroid hormone control over uterine epithelial cell proliferation and apoptosis. However, the precise mechanism of PTEN/AKT signaling in endometrial cancer remains poorly understood. The progesterone signaling mediator MIG-6 suppresses estrogen signaling and it has been implicated previously as a tumor suppressor in endometrial cancer. In this study, we show that MIG-6 also acts as a tumor suppressor in endometrial cancers associated with PTEN deficiency. Transgenic mice, where Mig-6 was overexpressed in progesterone receptor–expressing cells, exhibited a relative reduction in uterine tumorigenesis caused by Pten deficiency. ERK1/2 was phosphorylated in uterine tumors and administration of an ERK1/2 inhibitor suppressed cancer progression in PRcre/+Ptenf/f mice. In clinical specimens of endometrial cancer, MIG-6 expression correlated inversely with ERK1/2 phosphorylation during progression. Taken together, our findings suggest that Mig-6 regulates ERK1/2 phosphorylation and that it is crucial for progression of PTEN-mutant endometrial cancers, providing a mechanistic rationale for the evaluation of ERK1/2 inhibitors as a therapeutic treatment in human endometrial cancer. Cancer Res; 74(24); 7371–82. ©2014 AACR.

Bone is the most frequent site of metastasis in prostate cancer and patients with bone metastases are deemed incurable. Targeting prostate cancer cells that disseminated to the bone marrow before surgery and before metastatic outgrowth may therefore prevent lethal metastasis. This prompted us to directly analyze the transcriptome of disseminated cancer cells (DCC) isolated from patients with nonmetastatic (UICC stage M0) prostate cancer. We screened 105 bone marrow samples of patients with M0-stage prostate cancer and 18 bone marrow samples of patients without malignancy for the presence of EpCAM+ single cells. In total, we isolated 270 cells from both groups by micromanipulation and globally amplified their mRNA. We used targeted transcriptional profiling to unambiguously identify DCCs for subsequent in-depth analysis. Transcriptomes of all cells were examined for the expression of EPCAM, KRT8, KRT18, KRT19, KRT14, KRT6a, KRT5, KLK3 (PSA), MAGEA2, MAGEA4, PTPRC (CD45), CD33, CD34, CD19, GYPC, SCL4A1 (band 3), and HBA2. Using these transcripts, we found it impossible to reliably identify true DCCs. We then applied combined genome and transcriptome analysis of single cells and found that EpCAM+ cells from controls expressed transcripts thought to be epithelial-specific, whereas true DCCs may express hematopoietic transcripts. These results point to an unexpected transcriptome plasticity of epithelial cancer cells in bone marrow and question common transcriptional criteria to identify DCCs. Cancer Res; 74(24); 7383–94. ©2014 AACR.

Estrogen receptor (ER) coregulator overexpression promotes carcinogenesis and/or progression of endocrine related-cancers in which steroid hormones are powerful mitogenic agents. Recent studies in our laboratory, as well as others, demonstrated that the estrogen receptor coregulator PELP1 is a proto-oncogene. PELP1 interactions with histone demethylase KDM1 play a critical role in its oncogenic functions and PELP1 is a prognostic indicator of decreased survival in patients with breast cancer. However, the in vivo significance of PELP1 deregulation during initiation and progression of breast cancer remains unknown. We generated an inducible, mammary gland-specific PELP1-expressing transgenic (Tg) mouse (MMTVrtTA-TetOPELP1). We found more proliferation, extensive side branching, and precocious differentiation in PELP1-overexpressing mammary glands than in control glands. Aged MMTVrtTA-TetOPELP1 Tg mice had hyperplasia and preneoplastic changes as early as 12 weeks, and ER-positive mammary tumors occurred at a latency of 14 to 16 months. Mechanistic studies revealed that PELP1 deregulation altered expression of a number of known ER target genes involved in cellular proliferation (cyclin D1, CDKs) and morphogenesis (EGFR, MMPs) and such changes facilitated altered mammary gland morphogenesis and tumor progression. Furthermore, PELP1 was hyper-phosphorylated at its CDK phosphorylation site, suggesting an autocrine loop involving the CDK–cyclin D1–PELP1 axis in promoting mammary tumorigenesis. Treatment of PELP1 Tg mice with a KDM1 inhibitor significantly reduced PELP1-driven hyperbranching, reversed alterations in cyclin D1 expression levels, and reduced CDK-driven PELP1 phosphorylation. These results further support the hypothesis that PELP1 deregulation has the potential to promote breast tumorigenesis in vivo and represent a novel model for future investigation into molecular mechanisms of PELP1-mediated tumorigenesis. Cancer Res; 74(24); 7395–405. ©2014 AACR.

Patient-derived human-in-mouse xenograft models of breast cancer (PDX models) that exhibit spontaneous lung metastases offer a potentially powerful model of cancer metastasis. In this study, we evaluated the malignant character of lung micrometastases that emerge in such models after orthotopic implantation of human breast tumor cells into the mouse mammary fat pad. Interestingly, relative to the parental primary breast tumors, the lung metastasis (met)–derived mammary tumors exhibited a slower growth rate and a reduced metastatic potential with a more differentiated epithelial status. Epigenetic correlates were determined by gene array analyses. Lung met–derived tumors displayed differential expression of negative regulators of cell proliferation and metabolism and positive regulators of mammary epithelial differentiation. Clinically, this signature correlated with breast tumor subtypes. We identified hsa-miR-138 (miR-138) as a novel regulator of invasion and epithelial–mesenchymal transition in breast cancer cells, acting by directly targeting the polycomb epigenetic regulator EZH2. Mechanistic investigations showed that GATA3 transcriptionally controlled miR-138 levels in lung metastases. Notably, the miR-138 activity signature served as a novel independent prognostic marker for patient survival beyond traditional pathologic variables, intrinsic subtypes, or a proliferation gene signature. Our results highlight the loss of malignant character in some lung micrometastatic lesions and the epigenetic regulation of this phenotype. Cancer Res; 74(24); 7406–17. ©2014 AACR.

Multiple myeloma is a cytogenetically/molecularly heterogeneous hematologic malignancy that remains mostly incurable, and the identification of a universal and relevant therapeutic target molecule is essential for the further development of therapeutic strategy. Herein, we identified that 3-phosphoinositide–dependent protein kinase 1 (PDPK1), a serine threonine kinase, is expressed and active in all eleven multiple myeloma–derived cell lines examined regardless of the type of cytogenetic abnormality, the mutation state of RAS and FGFR3 genes, or the activation state of ERK and AKT. Our results revealed that PDPK1 is a pivotal regulator of molecules that are essential for myelomagenesis, such as RSK2, AKT, c-MYC, IRF4, or cyclin Ds, and that PDPK1 inhibition caused the growth inhibition and the induction of apoptosis with the activation of BIM and BAD, and augmented the in vitro cytotoxic effects of antimyeloma agents in myeloma cells. In the clinical setting, PDPK1 was active in myeloma cells of approximately 90% of symptomatic patients at diagnosis, and the smaller population of patients with multiple myeloma exhibiting myeloma cells without active PDPK1 showed a significantly less frequent proportion of the disease stage III by the International Staging System and a significantly more favorable prognosis, including the longer overall survival period and the longer progression-free survival period by bortezomib treatment, than patients with active PDPK1, suggesting that PDPK1 activation accelerates the disease progression and the resistance to treatment in multiple myeloma. Our study demonstrates that PDPK1 is a potent and a universally targetable signaling mediator in multiple myeloma regardless of the types of cytogenetic/molecular profiles. Cancer Res; 74(24); 7418–29. ©2014 AACR.

Oncogenic KRAS mutations found in 20% to 30% of all non–small cell lung cancers (NSCLC) are associated with chemoresistance and poor prognosis. Here we demonstrate that activation of the cell protective stress response gene NRF2 by KRAS is responsible for its ability to promote drug resistance. RNAi-mediated silencing of NRF2 was sufficient to reverse resistance to cisplatin elicited by ectopic expression of oncogenic KRAS in NSCLC cells. Mechanistically, KRAS increased NRF2 gene transcription through a TPA response element (TRE) located in a regulatory region in exon 1 of NRF2. In a mouse model of mutant KrasG12D-induced lung cancer, we found that suppressing the NRF2 pathway with the chemical inhibitor brusatol enhanced the antitumor efficacy of cisplatin. Cotreatment reduced tumor burden and improved survival. Our findings illuminate the mechanistic details of KRAS-mediated drug resistance and provide a preclinical rationale to improve the management of lung tumors harboring KRAS mutations with NRF2 pathway inhibitors. Cancer Res; 74(24); 7430–41. ©2014 AACR.

Few studies have examined associations between plasma choline metabolites and risk of colorectal cancer. Therefore, we investigated associations between plasma biomarkers of choline metabolism [choline, betaine, dimethylglycine, and trimethylamine N-oxide (TMAO)] and colorectal cancer risk among postmenopausal women in a case–control study nested within the Women's Health Initiative Observational Study. We selected 835 matched case–control pairs, and cases were further stratified by tumor site (proximal, distal, or rectal) and stage (local/regional or metastatic). Colorectal cancer was assessed by self-report and confirmed by medical records over the mean of 5.2 years of follow-up. Baseline plasma choline metabolites were measured by LC/MS-MS. In multivariable-adjusted conditional logistic regression models, plasma choline tended to be positively associated with rectal cancer risk [OR (95% confidence interval, CI)highest vs. lowest quartile = 2.44 (0.93–6.40); P trend = 0.08], whereas plasma betaine was inversely associated with colorectal cancer overall [0.68 (0.47–0.99); P trend = 0.01] and with local/regional tumors [0.64 (0.42–0.99); P trend = 0.009]. Notably, the plasma betaine:choline ratio was inversely associated with colorectal cancer overall [0.56 (0.39–0.82); P trend = 0.004] as well as with proximal [0.66 (0.41–1.06); P trend = 0.049], rectal [0.27 (0.10–0.78); P trend = 0.02], and local/regional [0.50 (0.33–0.76); P trend = 0.001] tumors. Finally, plasma TMAO, an oxidative derivative of choline produced by intestinal bacteria, was positively associated with rectal cancer [3.38 (1.25–9.16); P trend = 0.02] and with overall colorectal cancer risk among women with lower (vs. higher) plasma vitamin B12 levels (P interaction = 0.003). Collectively, these data suggest that alterations in choline metabolism, which may arise early in disease development, may be associated with higher risk of colorectal cancer. The positive association between plasma TMAO and colorectal cancer risk is consistent with an involvement of the gut microbiome in colorectal cancer pathogenesis. Cancer Res; 74(24); 7442–52. ©2014 AACR.

The high mortality of epithelial ovarian cancer (EOC) is mainly caused by resistance to the available therapies. In EOC, the endothelin-1 (ET-1, EDN1)–endothelin A receptor (ETAR, EDNRA) signaling axis regulates the epithelial–mesenchymal transition (EMT) and a chemoresistant phenotype. However, there is a paucity of knowledge about how ET-1 mediates drug resistance. Here, we define a novel bypass mechanism through which ETAR/β-arrestin-1 (β-arr1, ARRB1) links Wnt signaling to acquire chemoresistant and EMT phenotype. We found that ETAR/β-arr1 activity promoted nuclear complex with β-catenin and p300, resulting in histone acetylation, chromatin reorganization, and enhanced transcription of genes, such as ET-1, enhancing the network that sustains chemoresistance. Silencing of β-arr1 or pharmacologic treatment with the dual ETAR/ETBR antagonist macitentan prevented core complex formation and restored drug sensitivity, impairing the signaling pathways involved in cell survival, EMT, and invasion. In vivo macitentan treatment reduced tumor growth, vascularization, intravasation, and metastatic progression. The combination of macitentan and cisplatinum resulted in the potentiation of the cytotoxic effect, indicating that macitentan can enhance sensitivity to chemotherapy. Investigations in clinical specimens of chemoresistant EOC tissues confirmed increased recruitment of β-arr1 and β-catenin to ET-1 gene promoter. In these tissues, high expression of ETAR significantly associated with poor clinical outcome and chemoresistance. Collectively, our findings reveal the existence of a novel mechanism by which ETAR/β-arr1 signaling is integrated with the Wnt/β-catenin pathway to sustain chemoresistance in EOC, and they offer a solid rationale for clinical evaluation of macitentan in combination with chemotherapy to overcome chemoresistance in this setting. Cancer Res; 74(24); 7453–64. ©2014 AACR.

As receptor-targeted therapeutics become increasingly used in clinical oncology, the ability to quantify protein expression and pharmacokinetics in vivo is imperative to ensure successful individualized treatment plans. Current standards for receptor analysis are performed on extracted tissues. These measurements are static and often physiologically irrelevant; therefore, only a partial picture of available receptors for drug targeting in vivo is provided. Until recently, in vivo measurements were limited by the inability to separate delivery, binding, and retention effects, but this can be circumvented by a dual-tracer approach for referencing the detected signal. We hypothesized that in vivo receptor concentration imaging (RCI) would be superior to ex vivo immunohistochemistry (IHC). Using multiple xenograft tumor models with varying EGFR expression, we determined the EGFR concentration in each model using a novel targeted agent (anti-EGFR affibody-IRDye800CW conjugate) along with a simultaneously delivered reference agent (control affibody-IRDye680RD conjugate). The RCI-calculated in vivo receptor concentration was strongly correlated with ex vivo pathologist-scored IHC and computer-quantified ex vivo immunofluorescence. In contrast, no correlation was observed with ex vivo Western blot analysis or in vitro flow-cytometry assays. Overall, our results argue that in vivo RCI provides a robust measure of receptor expression equivalent to ex vivo immunostaining, with implications for use in noninvasive monitoring of therapy or therapeutic guidance during surgery. Cancer Res; 74(24); 7465–74. ©2014 AACR.

Histone deacetylases (HDAC) that regulate gene expression are being explored as cancer therapeutic targets. In this study, we focused on HDAC6 based on its ability to inhibit cancerous Hsp90 chaperone activities by disrupting Hsp90/p23 interactions. To identify novel HDAC6 inhibitors, we used a dual-luciferase reporter system in cell culture and living mice by bioluminescence imaging (BLI). On the basis of existing knowledge, a library of hydrazone compounds was generated for screening by coupling cinnamic hydroxamates with aldehydes and ketones. Potency and selectivity were determined by in vitro HDAC profiling assays, with further evaluation to inhibit Hsp90(α/β)/p23 interactions by BLI. In this manner, we identified compound 1A12 as a dose-dependent inhibitor of Hsp90(α/β)/p23 interactions, UKE-1 myeloid cell proliferation, p21waf1 upregulation, and acetylated histone H3 levels. 1A12 was efficacious in tumor xenografts expressing Hsp90(α)/p23 reporters relative to carrier control–treated mice as determined by BLI. Small animal 18F-FDG PET/CT imaging on the same cohort showed that 1A12 also inhibited glucose metabolism relative to control subjects. Ex vivo analyses of tumor lysates showed that 1A12 administration upregulated acetylated-H3 by approximately 3.5-fold. Taken together, our results describe the discovery and initial preclinical validation of a novel selective HDAC inhibitor. Cancer Res; 74(24); 7475–86. ©2014 AACR.

Anticancer agent FL118 was recently identified in screening of small-molecule inhibitors of human survivin expression. Although FL118 is a camptothecin analogue, its antitumor potency is much superior to other FDA-approved camptothecin analogues (irinotecan and topotecan). The mechanism of action (MOA) underlying the antitumor effects of FL118 remains to be fully elucidated. Here, we report that FL118 activates tumor suppressor p53 as a novel MOA in p53 wild-type cancer cells. Our studies show that this MOA involves an induction of proteasomal degradation of MdmX, a critical negative regulator of p53, in a manner largely independent of ATM-dependent DNA damage signaling pathway but dependent on E3-competent Mdm2. FL118 inhibits p53 polyubiquitination and monoubiquitination by Mdm2–MdmX E3 complex in cells and in cell-free systems. In contrast, FL118 stimulates Mdm2-mediated MdmX ubiquitination. Coimmunoprecipitation revealed that FL118 slightly decreases Mdm2–p53 interactions and moderately increases Mdm2–MdmX interactions, suggesting a change of targeting specificity of Mdm2–MdmX E3 complex from p53 to MdmX, resulting in accelerated MdmX degradation. As a result, p53 ubiquitination by Mdm2–MdmX E3 complex is reduced, which in turn activates p53 signaling. Activation of the p53 pathway by FL118 induces p53-dependent senescence in colorectal cancer cells. However, in the absence of p53 or in the presence of MdmX overexpression, FL118 promotes p53-independent apoptosis. These two distinct cellular consequences collectively contribute to the potent effects of FL118 to inhibit clonogenic potential of colon cancer cells. This study identifies a potential application of FL118 as an MdmX inhibitor for targeted therapies. Cancer Res; 74(24); 7487–97. ©2014 AACR.

The MAPK phosphatase MKP1 (DUSP1) is overexpressed in many human cancers, including chemoresistant and radioresistant breast cancer cells, but its functional contributions in these settings are unclear. Here, we report that after cell irradiation, MKP1 translocates into mitochondria, where it prevents apoptotic induction by limiting accumulation of phosphorylated active forms of the stress kinase JNK. Increased levels of mitochondrial MKP1 after irradiation occurred in the mitochondrial inner membrane space. Notably, cell survival regulated by mitochondrial MKP1 was responsible for conferring radioresistance in HER2-overexpressing breast cancer cells, due to the fact that MKP1 serves as a major downstream effector in the HER2-activated RAF–MEK–ERK pathway. Clinically, we documented MKP1 expression exclusively in HER2-positive breast tumors, relative to normal adjacent tissue from the same patients. MKP1 overexpression was also detected in irradiated HER2-positive breast cancer stem-like cells (HER2+/CD44+/CD24−/low) isolated from a radioresistant breast cancer cell population after long-term radiation treatment. MKP1 silencing reduced clonogenic survival and enhanced radiosensitivity in these stem-like cells. Combined inhibition of MKP1 and HER2 enhanced cell killing in breast cancer. Together, our findings identify a new mechanism of resistance in breast tumors and reveal MKP1 as a novel therapeutic target for radiosensitization. Cancer Res; 74(24); 7498–509. ©2014 AACR.

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy in need of new, effective, and safe therapies. The recently identified IgM receptor FcμR is overexpressed on malignant B cells in CLL and mediates the rapid internalization and lysosomal shuttling of IgM via its Fc fragment (Fcμ). To exploit this internalization and trafficking pathway for targeted drug delivery, we engineered an IgM-derived protein scaffold (Fcμ) and linked it with the cytotoxic agent monomethylauristatin F. This Fcμ–drug conjugate was selectively toxic for FcμR-expressing cell lines in vitro and for CLL cells but not autologous normal T cells ex vivo. Notably, the cytotoxic activity of the Fcμ–drug conjugate was maintained in CLL cells carrying a 17p deletion, which predicts resistance to standard chemotherapy. Next, we tested the possible therapeutic application of the Fcμ–drug conjugate in immunodeficient NOD/SCID/IL-2Rγnull (NSG) mice engrafted with peripheral blood cells from patients with leukemia. Three intravenous injections of the Fcμ–drug conjugate over a 10-day period were well tolerated and selectively killed the human CLL cells but not the coengrafted autologous human T cells. In summary, we developed a novel strategy for targeted cytotoxic therapy of CLL based on the unique properties of FcμR. FcμR-targeted drug delivery showed potent and specific therapeutic activity in CLL, thus providing proof of concept for FcμR as a valuable therapeutic target in CLL and for IgM-based antibody–drug conjugates as a new targeting platform. Cancer Res; 74(24); 7510–20. ©2014 AACR.

Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits antineoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here, we show that serine withdrawal increases the antineoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation modified the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, a serine-deficient diet reduced serine levels in tumors and significantly enhanced the tumor growth–inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as antineoplastic agents that target cancer cell energy metabolism. Cancer Res; 74(24); 7521–33. ©2014 AACR.

Resistance to DNA-damaging chemotherapy is a barrier to effective treatment that appears to be augmented by p53 functional deficiency in many cancers. In p53-deficient cells in which the G1–S checkpoint is compromised, cell viability after DNA damage relies upon intact intra-S and G2–M checkpoints mediated by the ATR (ataxia telangiectasia and Rad3 related) and Chk1 kinases. Thus, a logical rationale to sensitize p53-deficient cancers to DNA-damaging chemotherapy is through the use of ATP-competitive inhibitors of ATR or Chk1. To discover small molecules that may act on uncharacterized components of the ATR pathway, we performed a phenotype-based screen of 9,195 compounds for their ability to inhibit hydroxyurea-induced phosphorylation of Ser345 on Chk1, known to be a critical ATR substrate. This effort led to the identification of four small-molecule compounds, three of which were derived from known bioactive library (anthothecol, dihydrocelastryl, and erysolin) and one of which was a novel synthetic compound termed MARPIN. These compounds all inhibited ATR-selective phosphorylation and sensitized p53-deficient cancer cells to DNA-damaging agents in vitro and in vivo. Notably, these compounds did not inhibit ATR catalytic activity in vitro, unlike typical ATP-competitive inhibitors, but acted in a mechanistically distinct manner to disable ATR–Chk1 function. Our results highlight a set of novel molecular probes to further elucidate druggable mechanisms to improve cancer therapeutic responses produced by DNA-damaging drugs. Cancer Res; 74(24); 7534–45. ©2014 AACR.

Cancer stem–like cells (CSC) thought to contribute to head and neck squamous carcinomas (HNSCC) may offer attractive therapeutic targets if a tractable approach can be developed. In this study, we report that silencing c-Met is sufficient to suppress sphere formation, tumor initiation, and metastatic properties of HN-CSC. Pharmacologic inhibition of c-Met with the selective inhibitor PF-2341066 preferentially targeted CSC and synergized with conventional chemotherapy to improve efficacy in a mouse xenograft model of HNSCC, impeding tumor growth and reducing metastasis. Mechanistic investigations showed that CSC elimination was due to downregulation of Wnt/β-catenin signaling in HN-CSC and that the Wnt pathway receptor FZD8 was essential for interactions of c-Met and Wnt/β-catenin signaling in HN-CSC. Notably, ectopic expression of FZD8 rescued the impaired phenotype of HN-CSC where c-Met was inhibited. Furthermore, c-Met upregulated FZD8 through the ERK/c-Fos cascade in HN-CSC. Taken together, our results offer a preclinical proof-of-concept for targeting the c-Met/FZD8 signaling axis as a CSC-directed therapy to improve HNSCC treatment. Cancer Res; 74(24); 7546–59. ©2014 AACR.

microRNA (miRNA) dysregulation contributes widely to human cancer but has not been fully assessed in oral cancers. In this study, we conducted a global microarray analysis of miRNA expression in 40 pairs of betel quid–associated oral squamous cell carcinoma (OSCC) specimens and their matched nontumorous epithelial counterparts. Eighty-four miRNAs were differentially expressed in the OSCC specimens compared with the matched tissue. Among these downregulated miRNAs, 19 miRNAs were found and mapped to the chromosome 14q32.2 miRNA cluster region, which resides within a parentally imprinted region designated as Dlk-Dio3 and known to be important in development and growth. Bioinformatic analysis predicted two miRNAs from the cluster region, miR329 and miR410, which could potentially target Wnt-7b, an activator of the Wnt–β-catenin pathway, thereby attenuating the Wnt–β-catenin signaling pathway in OSCC. Stable ectopic expression of Wnt-7b in OSCC cells overexpressing miR329 or miR410 restored proliferation and invasion capabilities abolished by these miRNA. Combining a demethylation agent and a histone deacetylase inhibitor was sufficient to reexpress miR329, miR410, and Meg3, consistent with epigenetic regulation of these miRNA in human OSCC. Specifically, arecoline, a major betel nut alkaloid, reduced miR329, miR410, and Meg3 gene expression. Overall, our results provide novel molecular insights into how betel quid contributes to oral carcinogenesis through epigenetic silencing of tumor-suppressor miRNA that targets Wnt–β-catenin signaling. Cancer Res; 74(24); 7560–72. ©2014 AACR.

Human breast cancers include cancer stem cell populations as well as nontumorigenic cancer cells. Breast cancer stem cells have self-renewal capability and are resistant to conventional chemotherapy. miRNAs regulate the expression of many target genes; therefore, dysregulation of miRNAs has been associated with the pathogenesis of human diseases, including cancer. However, a role for miRNA dysregulation in stemness and drug resistance has yet to be identified. Members of the miR34 family are reportedly tumor-suppressor miRNAs and are associated with various human cancers. Our results confirm that miR34a expression was downregulated in MCF7/ADR cells compared with MCF7 cells. We hypothesized that this reduction was due to the p53 (TP53) mutation in MCF7/ADR cells. In this study, we found that primary and mature miR34a were suppressed by treatment with p53 RNAi or the dominant-negative p53 mutant in MCF7 cells. Ectopic miR34a expression reduced cancer stem cell properties and increased sensitivity to doxorubicin treatment by directly targeting NOTCH1. Furthermore, tumors from nude mice treated with miR34a were significantly smaller compared with those of mice treated with control lentivirus. Our research suggests that the ectopic expression of miR34a represents a novel therapeutic approach in chemoresistant breast cancer treatment. Cancer Res; 74(24); 7573–82. ©2014 AACR.

Increased glucose utilization is a hallmark of human cancer that is used to image tumors clinically. In this widely used application, glucose uptake by tumors is monitored by positron emission tomography of the labeled glucose analogue 2[18F]fluoro-2-deoxy-d-glucose (FDG). Despite its widespread clinical use, the cellular and molecular mechanisms that determine FDG uptake—and that underlie the heterogeneity observed across cancers—remain poorly understood. In this study, we compared FDG uptake in mammary tumors driven by the Akt1, c-MYC, HER2/neu, Wnt1, or H-Ras oncogenes in genetically engineered mice, correlating it to tumor growth, cell proliferation, and expression levels of gene involved in key steps of glycolytic metabolism. We found that FDG uptake by tumors was dictated principally by the driver oncogene and was not independently associated with tumor growth or cellular proliferation. Oncogene downregulation resulted in a rapid decrease in FDG uptake, preceding effects on tumor regression, irrespective of the baseline level of uptake. FDG uptake correlated positively with expression of hexokinase-2 (HK2) and hypoxia-inducible factor-1α (HIF1α) and associated negatively with PFK-2b expression and p-AMPK. The correlation between HK2 and FDG uptake was independent of all variables tested, including the initiating oncogene, suggesting that HK2 is an independent predictor of FDG uptake. In contrast, expression of Glut1 was correlated with FDG uptake only in tumors driven by Akt or HER2/neu. Together, these results demonstrate that the oncogenic pathway activated within a tumor is a primary determinant of its FDG uptake, mediated by key glycolytic enzymes, and provide a framework to interpret effects on this key parameter in clinical imaging. Cancer Res; 74(24); 7583–98. ©2014 AACR.