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Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta superfamily, induces regression of the Müllerian duct in male embryos. In this report, we demonstrate MIS type II receptor expression in normal breast tissue and in human breast cancer cell lines, breast fibroadenoma, and ductal adenocarcinomas. MIS inhibited the growth of both estrogen receptor (ER)-positive T47D and ER-negative MDA-MB-231 breast cancer cell lines, suggesting a broader range of target tissues for MIS action. Inhibition of growth was manifested by an increase in the fraction of cells in the G(1) phase of the cell cycle and induction of apoptosis. Treatment of breast cancer cells with MIS activated the NFkappaB pathway and selectively up-regulated the immediate early gene IEX-1S, which, when overexpressed, inhibited breast cancer cell growth. Dominant negative IkappaBalpha expression ablated both MIS-mediated induction of IEX-1S and inhibition of growth, indicating that activation of the NFkappaB signaling pathway was required for these processes. These results identify the NFkappaB-mediated signaling pathway and a target gene for MIS action and suggest a putative role for the MIS ligand and its downstream interactors in the treatment of ER-positive as well as negative breast cancers.

The breast cancer susceptibility gene BRCA1 encodes a protein implicated in the cellular response to DNA damage, with postulated roles in homologous recombination as well as transcriptional regulation. To identify downstream target genes, we established cell lines with tightly regulated inducible expression of BRCA1. High-density oligonucleotide arrays were used to analyze gene expression profiles at various times following BRCA1 induction. A major BRCA1 target is the DNA damage–responsive gene GADD45. Induction of BRCA1 triggers apoptosis through activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), a signaling pathway potentially linked to GADD45 gene family members. The p53-independent induction of GADD45 by BRCA1 and its activation of JNK/SAPK suggest a pathway for BRCA1-induced apoptosis.

Two studies provide evidence that epithelial tumour cells do not need to transition to a mesenchymal-cell state to form metastases, but that this process does contribute to drug resistance. See Article p.472 & Letter p.525 It has been suggested that epithelial-to-mesenchymal transition (EMT), in which epithelial cells depolarize and adopt a fibroblast-like morphology, is a requirement for metastasis to occur. Other studies imply that the importance of EMT relies on cell-culture-based manipulation of EMT regulators. In this issue of Nature, two groups present results that suggest that EMT is not a prerequisite for metasasis. Dingcheng Gao and colleagues trace the fate of cells that have undergone EMT in mouse model for breast-to-lung metastasis. They find that although some cells undergo EMT in a primary epithelial tumour, the lung metastases mainly contain cells that have not undergone EMT. However, cells that have undergone EMT appear more resistant to chemotherapy. A microRNA that targets key EMT regulators is shown not to affect metastasis, but to reduce survival of EMT cells following chemotherapy. Raghu Kalluri and colleagues delete Twist or Snail — transcription factors that induce EMT — in a mouse model for pancreatic ductal adenocarcinoma. This leads to an increase in cell proliferation, and a greater sensitivity to chemotherapeutic agent gemcitabine, with no effect on invasion and metastasis.

WT1 encodes a tumor suppressor that is expressed in cells of the developing kidney and is inactivated in Wilms tumor, a pediatric kidney cancer. The adenovirus E1B 55K gene product contributes to the transformation of primary baby rat kidney (BRK) cells by binding and inactivating the product of the p53 tumor suppressor. We have previously demonstrated that WT1 and p53 are present within a protein complex in vivo. We now show that WT1 is physically associated with E1B 55K in adenovirus-transformed cells, an interaction that is mediated by the first two zinc fingers of WT1. Immunodepletion of p53 abrogates the coimmunoprecipitation of E1B 55K and WT1, consistent with the presence of a trimeric protein complex containing these three proteins. In the presence of E1B 55K, WT1 which is normally localized in the nucleus, is retained within a very high molecular weight complex and sequestered in the characteristic perinuclear cytoplasmic body that contains E1B 55K and p53. Expression of E1B 55K in osteosarcoma cells that undergo apoptosis following expression of WT1 inhibits WT1-mediated cell death. We conclude that E1B 55K may target WT1 along with p53, resulting in the functional inactivation of both tumor suppressor gene products by this viral oncoprotein.

The Wilms tumor suppressor WT1 encodes a zinc finger transcription factor that is expressed in glomerular podocytes during a narrow window in kidney development. By immunoprecipitation and protein microsequencing analysis, we have identified a major cellular protein associated with endogenous WT1 to be the inducible chaperone Hsp70. WT1 and Hsp70 are physically associated in embryonic rat kidney cells, in primary Wilms tumor specimens and in cultured cells with inducible expression of WT1. Colocalization of WT1 and Hsp70 is evident within podocytes of the developing kidney, and Hsp70 is recruited to the characteristic subnuclear clusters that contain WT1. The amino-terminal transactivation domain of WT1 is required for binding to Hsp70, and expression of that domain itself is sufficient to induce expression of Hsp70 through the heat shock element (HSE). Substitution of a heterologous Hsp70-binding domain derived from human DNAJ is sufficient to restore the functional properties of a WT1 protein with an amino-terminal deletion, an effect that is abrogated by a point mutation in DNAJ that reduces binding to Hsp70. These observations indicate that Hsp70 is an important cofactor for the function of WT1, and suggest a potential role for this chaperone during kidney differentiation.

Chromosomal translocations resulting in chimaeric transcription factors underlie specific malignancies, but few authentic target genes regulated by these fusion proteins have been identified. Desmoplastic small round-cell tumour (DSRT) is a multiphenotypic primitive tumour characterized by massive reactive fibrosis surrounding nests of tumour cells. The t(11;22)(p13;q12) chromosomal translocation that defines DSRT produces a chimaeric protein containing the potential transactivation domain of the Ewing-sarcoma protein (EWS) fused to zinc fingers 2-4 of the Wilms tumour suppressor and transcriptional repressor WT1 (refs 2,3). By analogy with other EWS fusion products, the EWS-WT1 chimaera may encode a transcriptional activator whose target genes overlap with those repressed by WT1 (ref. 4). To characterize its functional properties, we generated osteosarcoma cell lines with tightly regulated inducible expression of EWS-WT1. Expression of EWS-WT1 induced the expression of endogenous platelet-derived growth factor-A (PDGFA), a potent secreted mitogen and chemoattractant whose promoter contains the many potential WT1-binding sites. Native PDGFA was not regulated by wild-type WT1, indicating a difference in target gene specificity between this tumour suppressor and its oncogenic derivative. PDGFA was expressed within tumour cells in primary DSRT specimens, but it was absent in Wilms tumours expressing WT1 and Ewing sarcomas with an EWS-Fli translocation. We conclude that the oncogenic fusion of EWS to WT1 in DSRT results in the induction of PDGFA, a potent fibroblast growth factor that contributes to the characteristic reactive fibrosis associated with this unique tumour.

Insulin-like growth factor 2 (IGF2), a developmentally regulated and maternally imprinted gene, is frequently overexpressed in pediatric cancers. Although loss of imprinting (LOI) at fetal promoters contributes to increased IGF2 in tumors, the magnitude of IGF2 expression suggests the involvement of additional regulatory mechanisms. A microRNA (miRNA) screen of primary Wilms' tumors identified specific overexpression of miR-483-5p, which is embedded within the IGF2 gene. Unexpectedly, the IGF2 mRNA itself is transcriptionally up-regulated by miR-483-5p. A nuclear pool of miR-483-5p binds directly to the 5′ untranslated region (UTR) of fetal IGF2 mRNA, enhancing the association of the RNA helicase DHX9 to the IGF2 transcript and promoting IGF2 transcription. Ectopic expression of miR-483-5p in IGF2-dependent sarcoma cells is correlated with increased tumorigenesis in vivo. Together, these observations suggest a functional positive feedback loop of an intronic miRNA on transcription of its host gene.

RAF and MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase) inhibitors are effective in treating patients with BRAF-mutant melanoma. However, most responses are partial and short-lived, and many patients fail to respond at all. We found that suppression of TORC1 activity in response to RAF or MEK inhibitors, as measured by decreased phosphorylation of ribosomal protein S6 (P-S6), effectively predicted induction of cell death by the inhibitor in BRAF-mutant melanoma cell lines. In resistant melanomas, TORC1 activity was maintained after treatment with RAF or MEK inhibitors, in some cases despite robust suppression of mitogen-activated protein kinase (MAPK) signaling. In in vivo mouse models, suppression of TORC1 after MAPK inhibition was necessary for induction of apoptosis and tumor response. Finally, in paired biopsies obtained from patients with BRAF-mutant melanoma before treatment and after initiation of RAF inhibitor therapy, P-S6 suppression predicted significantly improved progression-free survival. Such a change in P-S6 could be readily monitored in real time by serial fine-needle aspiration biopsies, making quantitation of P-S6 a valuable biomarker to guide treatment in BRAF-mutant melanoma.

Circulating tumor cells (CTCs) are shed into the bloodstream from primary and metastatic tumor deposits. Their isolation and analysis hold great promise for the early detection of invasive cancer and the management of advanced disease, but technological hurdles have limited their broad clinical utility. We describe an inertial focusing-enhanced microfluidic CTC capture platform, termed "CTC-iChip," that is capable of sorting rare CTCs from whole blood at 10(7) cells/s. Most importantly, the iChip is capable of isolating CTCs using strategies that are either dependent or independent of tumor membrane epitopes, and thus applicable to virtually all cancers. We specifically demonstrate the use of the iChip in an expanded set of both epithelial and nonepithelial cancers including lung, prostate, pancreas, breast, and melanoma. The sorting of CTCs as unfixed cells in solution allows for the application of high-quality clinically standardized morphological and immunohistochemical analyses, as well as RNA-based single-cell molecular characterization. The combination of an unbiased, broadly applicable, high-throughput, and automatable rare cell sorting technology with generally accepted molecular assays and cytology standards will enable the integration of CTC-based diagnostics into the clinical management of cancer.

Cells in Transit(ion) Epithelial-mesenchymal transition (EMT) is a developmental program that converts adherent epithelial cells to a migratory mesenchymal state. This cell-fate change has been linked to tumor metastasis in preclinical models. To investigate whether EMT occurs in human cancer, Yu et al. (p. 580 ) isolated circulating tumor cells (CTCs) from breast cancer patients and analyzed their expression of epithelial and mesenchymal markers by RNA–in situ hybridization and RNA sequencing. Biphenotypic cells expressing both types of markers were rare in primary breast tumors but were enriched among CTCs, as were cells expressing only mesenchymal markers. Serial blood samples from one patient revealed that CTCs in the mesenchymal state declined in number when the patient responded to therapy but rebounded when the disease began to progress—a pattern repeated when a different therapy was administered. Thus, EMT may facilitate tumor cell dissemination in humans.

WT1 encodes a zinc-finger protein, expressed as distinct isoforms, that is inactivated in a subset of Wilms tumors. Both constitutional and somatic mutations disrupting the DNA-binding domain of WT1 result in a potentially dominant-negative phenotype. In generating inducible cell lines expressing wild-type isoforms of WT1 and WT1 mutants, we observed dramatic differences in the subnuclear localization of the induced proteins. The WT1 isoform that binds with high affinity to a defined DNA target, WT1(-KTS), was diffusely localized throughout the nucleus. In contrast, expression of an alternative splicing variant with reduced DNA binding affinity, WT1 (+KTS), or WT1 mutants with a disrupted zinc-finger domain resulted in a speckled pattern of expression within the nucleus. Although similar in appearance, the localization of WT1 variants to subnuclear clusters was clearly distinct from that of the essential splicing factor SC35, suggesting that WT1 is not directly involved in pre-mRNA splicing. Localization to subnuclear clusters required the N terminus of WT1, and coexpression of a truncated WT1 mutant and wild-type WT1(-KTS) resulted in their physical association, the redistribution of WT1(-KTS) from a diffuse to a speckled pattern, and the inhibition of its transactivational activity. These observations suggest that different WT1 isoforms and WT1 mutants have distinct subnuclear compartments. Dominant-negative WT1 proteins physically associate with wild-type WT1 in vivo and may result in its sequestration within subnuclear structures.

The Wilms' tumor-suppressor gene product WT1 coimmunoprecipitates with p53 from baby rat kidney (BRK) cells and Wilms' tumor specimens, and expression of WT1 in BRK cells is associated with increased levels of endogenous wild-type p53 protein. To study the effect of WT1 on p53 function, we cotransfected expression constructs into Saos-2 cells, an osteosarcoma cell line without endogenous expression of either gene. Expression of WT1 resulted in increased steady-state levels of p53, attributable to a prolongation in protein half-life, and associated with protection against papillomavirus E6-mediated degradation of p53. This effect mapped to zinc fingers 1 and 2 of WT1 and was not observed with the closely related EGR1 protein. The stabilized p53 demonstrated enhanced binding to its target DNA sequence and increased trans-activation of a promoter containing this RGC site, but reduced transcriptional repression of a TATA-containing promoter lacking this site. Expression of WT1 inhibited p53-mediated apoptosis triggered by UV irradiation or by expression of temperature-sensitive p53 in the wild-type conformation, but did not affect p53-mediated cell cycle arrest. We conclude that WT1 protein can stabilize p53, modulate its trans-activational properties, and inhibit its ability to induce apoptosis. This effect may contribute to the elevated levels of wild-type p53 protein that are observed in Wilms' tumors.

Identification of candidate metastasis genes has traditionally resulted from comparison of primary and metastatic tumor specimens. However, Circulating Tumor Cells (CTCs) contain metastatic precursors that are present transiently in the bloodstream and their analysis may reveal additional pathways that are induced for a limited time, as they invade and survive within the vasculature. By comparing transcriptome profiles of CTCs from breast, prostate and lung cancers with their primary tumor of origin, we observed consistent and significant induction of the β-globin gene (HBB) within CTCs. In contrast, expression of α-globin, its binding partner within hematopoietic cells, is not coordinately upregulated. The tumor-specific origin of HBB was further confirmed by analysis of human xenografts-derived CTCs in mice, where human-specific HBB polymorphisms are readily distinguishable in the murine background. In cultured cancer cells, we further demonstrate that induction of HBB is triggered by exposure to reactive oxygen species (ROS), and we identify KLF-family transcriptional regulators that mediate this effect. To investigate the function of aberrant β-globin expression within CTCs, we performed shRNA-mediated knockdown of HBB in breast CTC-derived cultures. Cells with depleted HBB expression displayed elevated intracellular ROS levels, increased sensitivity to hydrogen peroxide, and impaired metastatic potential in mouse models. Taken together, these observations suggest that β-globin, a component of functional hemoglobin in red blood cells, is deregulated in disseminated tumor cells, where it may function as a ROS scavenger, reducing oxidative stress and facilitating cancer metastasis. Citation Format: Yu Zheng, David T. Miyamoto, Ben S. Wittner, James P. Sullivan, Nicola Aceto, Nicole Vincent Jordan, Min Yu, Nezihi Murat Karabacak, Valentine Comaills, Robert Morris, Rushil Desai, Niyati Desai, Erin Emmons, Richard J. Lee, Chin-Lee Wu, Lecia V. Sequist, Wilhelm Haas, David T. Ting, Mehmet Toner, Sridhar Ramaswamy, Shyamala Maheswaran, Daniel A. Haber. Induction of β-globin protects circulating tumor cells from oxidative stress during dissemination. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2679.

Today, circulating tumor cells (CTCs) can provide important prognostic information about a patient. However, to one day help guide decisions for precision medicine, more and increasingly sophisticated information will have to be extracted from each patient blood sample. We describe a new microfluidic CTC isolation technology, the Harpoon CTC Isolator and Chip (Hrp Isolator). From a single sample the Hrp Isolator can enable the analysis of plasma components such as ctDNA, as well as the ability to enrich all circulating tumor cell types, regardless of their antigenic expression. To enrich the CTCs from 7.5 mL of whole blood, the Hrp Isolator removes the red blood cells and platelets based on their small size and the white blood cells by negative depletion using magnetic particles. The time required to process 7.5 mLs of blood is approximately 90 minutes. In contrast, the CELLSEARCH® CTC Test (CS), the only US FDA-cleared test, uses antigens expressed by CTCs to target the CTCs and positively select them out of blood. To enumerate CTCs from patient samples, the output from the Hrp Isolator was then loaded into and run on the CELLSEARCH® System using the CS test (Hybrid method). CTCs were enumerated from 37 breast and prostate cancer patients by CS and by the Hybrid method. At least 1 CTC was found in 23 (62%) patient samples by the Hybrid method versus 17 (46%) by CS. The product of the Hrp Isolator enrichment contained on average the same numbers of CTCs as were found by CS (mean = 13.3 ± 18 by CS versus 12.0 ± 16 by Hybrid) in patient samples having 75), the Hybrid method may have underestimated the total Hrp Isolator CTC count. An important feature of the Hrp Isolator is its ability to enrich CTCs that are negative or low expressers of epithelial cell adhesion molecule (EpCAM) or cytokeratin (CK). These cells could have been missed by positive-selection technologies such as CS and may not have been counted using the Hybrid method. CTC spiked samples were run on the Hrp Isolator and the CTC enriched product was spun onto microscope slides, stained, and examined using a Vectra Multispectral Imaging System (Hrp+Vec method). Using the Hrp+Vec method, 79% to 100% of spiked CTCs were recovered depending on the slide fixation method used. Also, using Hrp+Vec we confirmed the ability of the Harpoon Isolator to enrich EpCAM-/CK+ and EpCAM+/CK- CTCs from patient samples. This establishes the existence of “non-canonical” tumor cell types in circulation and adds to the biological diversity of the CTC population in circulation. These results emphasize the need to further characterize all tumor cell populations and to determine what, if any, clinical significance may be assigned to these diverse CTC types. Citation Format: David Chianese, Mark Connelly, Carrie Morano, Thai Bui, Steven Gross, Renouard Sanders, Tom Barber, Ravi Kapur, Shyamala Maheswaran, Mehmet Toner, Daniel Haber. Isolation of canonical and non-canonical circulating tumor cells (CTCs) by negative depletion using the Harpoon CTC Isolator and Chip. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2422.

An ssa1-homologous genomic fragment cloned from Pasteurella haemolytica serotype 2 (ST2) enabled transformation of Escherichia coli DH5 alpha to a serotype 1 (ST1) phenotype through expression of the ST1-specific antigen (Ssa1). The Ssa1 protein expressed by ssa1-transformed E. coli was susceptible to heat and protease treatment and was distinct from P. haemolytica ST1-specific capsular polysaccharide. Electrophoretic analysis of in vitro-translated proteins, as well as the predicted amino acid sequence, demonstrated that Ssa1 proteins encoded from either ST1- or ST2-derived ssa1 genes were essentially identical. A comparison of the nucleotide sequences of ssa1 genes derived from P. haemolytica ST1 and ST2 revealed greater than 99% homology. Amino acid sequence homology of the predicted products of ST1- and ST2-derived ssa1 genes was greater than 98%. Northern (RNA) blot studies revealed that the presence of an increased level of ssa1 transcript in P. haemolytica ST1 grown as surface-adherent cultures on solid medium was correlated with a serologically detectable Ssa1 protein. Expression of the ssa1 transcript in ST1 was similarly upregulated by a high iron concentration in the growth medium.

Bovine tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) cDNAs were generated by reverse transcription and then by PCR amplification from lipopolysaccharide (LPS)-stimulated alveolar macrophage RNA. The amplified cDNAs were cloned into pPow and expressed in Escherichia coli DH5 alpha. The expressed proteins were confirmed as TNF-alpha and IL-1 beta by Western blot (immunoblot) analysis and bioassays. We then used the cloned genes as probes in Northern (RNA) blots and investigated the kinetics of TNF-alpha and IL-1 beta mRNA expression in bovine alveolar macrophages stimulated with purified LPS from Pasteurella haemolytica 12296. The effect of LPS on TNF-alpha and IL-1 beta gene expression was dose dependent, and induction was observed at a concentration of 0.01 microgram/ml. Both TNF-alpha and IL-1 beta mRNA expression were detectable within 0.5 h after stimulation with 1 microgram of LPS per ml, peaked at 1 to 2 h, steadily declined up to 16 h, and were undetectable by 24 h. Secreted TNF-alpha measured by bioassay peaked at 4 h and accumulated at a lesser concentration in conditioned medium throughout the 24 h. By contrast, secreted IL-1 beta was induced at 8 h and reached a maximal concentration at 24 h after stimulation. The ability of LPS to induce TNF-alpha and IL-1 beta gene expression and secretion of bioactive proteins were suppressed by polymyxin B. Our findings support a role for LPS from P. haemolytica in the induction of inflammatory cytokines in bovine pneumonic pasteurellosis.

In this study, we used an in vitro coculture system to determine which virulence factor from Pasteurella haemolytica A1 was responsible for augmenting bovine polymorphonuclear neutrophil (PMN)-mediated killing of bovine pulmonary artery endothelial cells (BPAEC). A 51Cr release cytotoxicity assay was used as a measure of BPAEC killing. The mechanisms associated with this BPAEC killing were also studied. Our results demonstrated that the leukotoxin and not the lipopolysaccharide from P. haemolytica was responsible for augmenting the PMN-mediated killing of BPAEC. Furthermore, this augmented killing was related to the stimulation of PMNs by the leukotoxin. Killing of BPAEC by leukotoxin-stimulated PMNs was diminished in the presence of the H2O2 inactivator, catalase. The membrane-permeant H2O2, hydroxyl radical (HO.) scavenger 1,3-dimethyl-2 thiourea, and the HO. scavenger dimethyl sulfoxide but not the myeloperoxidase inhibitor sodium azide attenuated this BPAEC killing. Pretreatment of BPAEC with a 21-aminosteroid (U74500A), a potent iron chelator-antioxidant, provided the most effective protection against BPAEC killing induced by leukotoxin-stimulated PMNs. These data were compatible with the concept that the H2O2 generated by leukotoxin-stimulated PMNs interacts with intracellular iron in the endothelial cell to form highly reactive HO.. We suggest that HO. may be a key factor in BPAEC killing. Furthermore, since the elastase-specific inhibitor N-methoxy-succinyl-Ala-Ala-Pro-Val-chloromethyl ketone (CMK) also attenuated BPAEC killing and both CMK and 1,3-dimethyl-2 thiourea functioned additively in protecting against BPAEC killing, we conclude that both HO. and elastase may jointly contribute to BPAEC killing induced by leukotoxin-stimulated PMNs. This study broadens our understanding of how leukotoxin-stimulated PMNs injure lung endothelial cells and provides new insight into the pathogenesis of bovine pneumonic pasteurellosis.

Rare circulating tumor cells (CTCs) are present in the blood of patients with metastatic epithelial cancers but have been difficult to measure routinely. We report a quantitative automated imaging system for analysis of prostate CTCs, taking advantage of prostate-specific antigen (PSA), a unique prostate tumor–associated marker. The specificity of PSA staining enabled optimization of criteria for baseline image intensity, morphometric measurements, and integration of multiple signals in a three-dimensional microfluidic device. In a pilot analysis, we detected CTCs in prostate cancer patients with localized disease, before surgical tumor removal in 8 of 19 (42%) patients (range, 38 to 222 CTCs per milliliter). For 6 of the 8 patients with preoperative CTCs, a precipitous postoperative decline (

Selective kinase inhibitors have had a substantial impact on the field of medical oncology. Whereas these agents can elicit dramatic clinical responses in some settings, their activity is generally limited to a subset of treated patients whose tumor cells harbor a specific genetic lesion. We have established an automated platform for examining the sensitivity to various molecularly targeted inhibitors across a large panel of human tumor-derived cell lines to identify additional genotype-correlated responses that may be clinically relevant. Among the inhibitors tested in a panel of 602 cell lines derived from a variety of human cancers, we found that a selective inhibitor of the anaplastic lymphoma kinase (ALK) potently suppressed growth of a small subset of tumor cells. This subset included lines derived from anaplastic large cell lymphomas, non–small-cell lung cancers, and neuroblastomas. ALK is a receptor tyrosine kinase that was first identified as part of a protein fusion derived from a chromosomal translocation detected in the majority of anaplastic large cell lymphoma patients, and has recently been implicated as an oncogene in a small fraction of non–small-cell lung cancers and neuroblastomas. Significantly, sensitivity in these cell lines was well correlated with specific ALK genomic rearrangements, including chromosomal translocations and gene amplification. Moreover, in such cell lines, ALK kinase inhibition can lead to potent suppression of downstream survival signaling and an apoptotic response. These findings suggest that a subset of lung cancers, lymphomas, and neuroblastomas that harbor genomic ALK alterations may be clinically responsive to pharmacologic ALK inhibition. [Cancer Res 2008;68(9):3389–95]

The transforming growth factor-β (TGF-β) superfamily consists of over 35 structurally related proteins, which regulate a myriad of cellular processes. Signaling by these growth regulatory molecules is initiated by ligand-induced hetero-oligomerization of distinct type II and type I serine/threonine kinase receptors that activate receptor-activated Smad proteins as well as Smad-independent pathways. TGF-β-related ligands and receptors are expressed in the prostate gland and aberrant expression and function of this class of signaling molecules are likely to regulate development and tumorigenesis of the prostate. The functional significance of TGF-β in prostate cancer has been reviewed in several articles. The objective of this review is to evaluate the expression and functional significance of activins, inhibins, and BMP signaling in prostate development and tumorigenesis, and their role as modulators and biomarkers of prostate cancer progression.

Isolation of circulating tumor cells (CTCs) in the blood allows noninvasive tumor sampling from patients with cancer. Molecular analysis of single CTCs may uncover heterogeneous cellular pathways that underlie disease progression and resistance to therapy. Using a microfluidic device, we isolated individual CTCs from patients with metastatic prostate cancer. Single candidate CTCs were micromanipulated after cell surface staining for epithelial (EpCAM) and mesenchymal (CDH11) markers, and then subjected to single cell RNA-sequencing. Digital gene expression profiles of lineage-confirmed CTCs were compared with each other, with primary prostate tumors, and with annotated markers of cellular signaling pathways. Single prostate CTCs displayed considerable heterogeneity in transcriptional profiles, but clustered according to patient of origin, indicating higher diversity in CTCs across different individuals (mean correlation 0.10 for CTCs within patients vs. 0.0014 for CTCs across patients, P=2.0x10E-11). Compared to primary tumors, CTCs were significantly enriched in 37 molecular pathways (FDR Citation Format: David T. Miyamoto, Yu Zheng, Ben S. Wittner, Richard J. Lee, Huili Zhu, Katherine T. Broderick, Rushil Desai, Brian W. Brannigan, Kshitij S. Arora, Douglas M. Dahl, Lecia V. Sequist, Matthew R. Smith, Ravi Kapur, Chin-Lee Wu, Toshi Shioda, Sridhar Ramaswamy, David T. Ting, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Single cell RNA-sequencing of circulating tumor cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr IA09.

Mullerian-inhibiting substance (MIS), a transforming growth factor-β family member, activates the nuclear factor-κB (NF-κB) pathway and induces the expression of B-cell translocation gene 2 (BTG2), IFN regulatory factor-1 (IRF-1), and the chemokine Gro-β. Inhibiting NF-κB activation with a phosphorylation-deficient IκBα mutant abrogated MIS-mediated induction of all three genes. Expression of dominant-negative Smad1, in which serines at the COOH-terminal SSVS motif are converted to alanines, suppressed MIS-induced Smad1 phosphorylation and impaired MIS-stimulated Gro-β promoter-driven reporter expression and Gro-β mRNA. Suppressing Smad1 expression using small interfering RNA also mitigated MIS-induced Gro-β mRNA, suggesting that regulation of Gro-β expression by MIS was dependent on activation of NF-κB as well as Smad1. However, induction of IRF-1 and BTG2 mRNAs by MIS was independent of Smad1 activation. Characterization of κB-binding sequences within Gro-β, BTG2, and IRF-1 promoters showed that MIS stimulated binding of p50 and p65 subunits to all three sites, whereas phosphorylated Smad1 (phospho-Smad1) protein was detectable only in the NF-κB complex bound to the κB site of the Gro-β promoter. Consistent with these observations, chromatin immunoprecipitation assays showed recruitment of both phospho-Smad1 and p65 to the Gro-β promoter in vivo, whereas p65, but not phospho-Smad1, was recruited to the BTG2 promoter. These results show a novel interaction between MIS-stimulated Smad1 and NF-κB signaling in which enhancement of NF-κB DNA binding and gene expression by phospho-Smad1 is dependent on the sequence of the κB consensus site within the promoter. [Cancer Res 2007;67(6):2747–56]

The B-cell translocation gene-2 (BTG2) is present in the nuclei of epithelial cells in many tissues, including the mammary gland where its expression is regulated during glandular proliferation and differentiation in pregnancy. In immortalized mammary epithelial cells and breast cancer cells, BTG2 protein localized predominantly to the nucleus and cytoplasm, respectively. The highly conserved domains (BTG boxes A, B, and C) were required for regulating localization, suppression of cyclin D1 and growth inhibitory function of BTG2. Expression analysis of BTG2 protein in human breast carcinoma (n = 148) revealed the loss of nuclear expression in 46% of tumors, whereas it was readily detectable in the nuclei of adjacent normal glands. Loss of nuclear BTG2 expression in estrogen receptor-α (ERα)–positive breast tumors correlated significantly with increased histologic grade and tumor size. Consistent with its ability to suppress cyclin D1 transcription, loss of nuclear BTG2 expression in ER-positive breast carcinomas showed a significant correlation with cyclin D1 protein overexpression, suggesting that loss of BTG2 may be a factor involved in deregulating cyclin D1 expression in human breast cancer. (Cancer Res 2006; 66(14): 7075-82)

Mullerian inhibiting substance (MIS) inhibits breast cancer cell growth in vitro . To extend the use of MIS to treat breast cancer, it is essential to test the responsiveness of mammary tumor growth to MIS in vivo . Mammary tumors arising in the C3(1) T antigen mouse model expressed the MIS type II receptor, and MIS in vitro inhibited the growth of cells derived from tumors. Administration of MIS to mice was associated with a lower number of palpable mammary tumors compared with vehicle-treated mice ( P = 0.048), and the mean mammary tumor weight in the MIS-treated group was significantly lower compared with the control group ( P = 0.029). Analysis of proliferating cell nuclear antigen (PCNA) expression and caspase-3 cleavage in tumors revealed that exposure to MIS was associated with decreased proliferation and increased apoptosis, respectively, and was not caused by a decline in T antigen expression. The effect of MIS on tumor growth was also evaluated on xenografted human breast cancer cell line MDA-MB-468, which is estrogen receptor- and retinoblastoma-negative and expresses mutant p53, and thus complements the C3(1)Tag mouse mammary tumors that do not express estrogen receptor and have functional inactivation of retinoblastoma and p53. In agreement with results observed in the transgenic mice, MIS decreased the rate of MDA-MB-468 tumor growth and the gain in mean tumor volume in severe combined immunodeficient mice compared with vehicle-treated controls ( P = 0.004). These results suggest that MIS can suppress the growth of mammary tumors in vivo .

Circulating Tumor Cells (CTCs) are present at extraordinarily low levels in the blood of patients with advanced epithelial cancers. Our group has developed and tested three generations of microfluidic devices for the isolation of CTCs, each with further improvements in automation, processing throughput and downstream analytical capabilities. Isolation and molecular analyses of these cells has a number of significant potential clinical applications: rising or declining numbers of CTCs are well correlated with tumor progression or therapeutic response. Furthermore, CTCs bear genetic hallmarks of the primary cancer, offering a noninvasive “liquid biopsy” to enable initial and serial monitoring of tumor genotypes during cancer therapy. Most significantly, CTCs hold the key to understanding the process of blood-borne metastasis. Using next generation RNA sequencing strategies, we have initiated studies of expression profiles within CTCs. These have identified cellular pathways implicated in resistance of epithelial cells to anoikis in the circulation and in epithelial-mesenchymal transition - a phenomenon implicated in cancer invasion. The heterogeneity of CTC populations is evident in single cell imaging analyses and sequencing studies, with implications for understanding the complexity of therapeutic responses. Taken together, the application of novel bioengineering technologies to the isolation and analysis of CTCs across multiple different cancer types offers a unique window into the properties of cancer cells that invade into the bloodstream and give rise to metastases. Citation Format: Daniel A. Haber, Min Yu, Emre Ozkumar, David Ting, David Miyamoto, Richard Lee, Ajay Shah, Aditya Bardia, Shannon Stott, Mehmet Toner, Shyamala Maheswaran. Molecular characterization of single circulating tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY30-01. doi:10.1158/1538-7445.AM2013-SY30-01

WT1 encodes a zinc finger transcription factor that is inactivated in a subset of Wilms' tumors. We have recently shown that introduction of wild-type WT1 into a Wilms' tumor-derived cell line, RM1, results in growth suppression, consistent with its function as a tumor suppressor gene. WT1-mediated growth suppression was also observed in other cells derived from embryonal tumors, including two osteosarcoma cell lines, U2OS and Saos-2, notable for the respective presence or absence of wild-type p53. To further characterize the functional properties of WT1, multiple U2OS and Saos-2 cell lines were established, expressing either wild-type WT1 splicing variants or naturally occurring mutants under control of a tightly regulated tetracycline repressable promoter. Induction of WT1 in these cells resulted in programmed cell death. This effect was preferentially mediated by WT1 isoform B (encoding alternative splice I, lacking alternative splice II "KTS"), and it was independent of p53, occurring in both U2OS and Saos-2 cells. WT1-mediated apoptosis was associated with transcriptional repression of the epidermal growth factor receptor (EGFR) and reduced synthesis of endogenous EGFR protein synthesis. Constitutive expression of EGFR abrogated WT1-mediated cell death. We conclude that wild-type WT1 can induce apoptosis in embryonal cancer cells, presumably through the withdrawal of required growth factor survival signals, and that EGFR is a physiological target gene for WT1.

This report demonstrates that in addition to interferons and cytokines, members of the TGF beta superfamily such as Mullerian inhibiting substance (MIS) and activin A also regulate IRF-1 expression. MIS induced IRF-1 expression in the mammary glands of mice in vivo and in breast cancer cells in vitro and stimulation of IRF-1 by MIS was dependent on activation of the NF kappa B pathway. In the rat mammary gland, IRF-1 expression gradually decreased during pregnancy and lactation but increased at involution. In breast cancer, the IRF-1 protein was absent in 13% of tumors tested compared with matched normal glands. Consistent with its growth suppressive activity, expression of IRF-1 in breast cancer cells induced apoptosis. Treatment of breast cancer cells with MIS and interferon gamma (IFN-gamma) co-stimulated IRF-1 and CEACAM1 expression and synergistic induction of CEACAM1 by a combination of MIS and IFN-gamma was impaired by antisense IRF-1 expression. Furthermore, a combination of IFN-gamma and MIS inhibited the growth of breast cancer cells to a greater extent than either one alone. Both reagents alone significantly decreased the fraction of cells in the S-phase of the cell cycle, an effect not enhanced when they were used in combination. However, MIS promoted IFN-gamma-induced apoptosis demonstrating a functional interaction between these two classes of signaling molecules in regulation of breast cancer cell growth.

Mullerian-inhibiting substance (MIS) is a member of the transforming growth factor β superfamily, a class of molecules that regulates growth, differentiation, and apoptosis in many cells. MIS type II receptor in the Mullerian duct is temporally and spatially regulated during development and becomes restricted to the most caudal ends that fuse to form the prostatic utricle. In this article, we have demonstrated MIS type II receptor expression in the normal prostate, human prostate cancer cell lines, and tissue derived from patients with prostate adenocarcinomas. MIS induced NF–κB DNA binding activity and selectively up-regulated the immediate early gene IEX-1S in both androgen-dependent and independent human prostate cancer cells in vitro . Dominant negative IκBα expression ablated both MIS-induced increase of IEX-1S mRNA and inhibition of growth, indicating that activation of NF–κB signaling was required for these processes. Androgen also induced NF–κB DNA binding activity in prostate cancer cells but without induction of IEX-1S mRNA, suggesting that MIS-mediated increase in IEX-1S was independent of androgen-mediated signaling. Administration of MIS to male mice induced IEX-1S mRNA in the prostate in vivo , suggesting that MIS may function as an endogenous hormonal regulator of NF–κB signaling and growth in the prostate gland.

A human Wilms tumor cell line (RM1) was developed to test the tumor suppressor activity of WT1, a zinc finger transcription factor that is expressed in the developing human kidney and is mutationally inactivated in a subset of Wilms tumors. Transfection of each of four wild-type WT1 isoforms suppressed the growth of RM1 cells. The endogenous WT1 transcript in these cells was devoid of exon 2 sequences, a splicing alteration that was also detected in varying amounts in all Wilms tumors tested but not in normal kidney. Production of this abnormal transcript, which encodes a functionally altered protein, may represent a distinct mechanism for inactivating WT1 in Wilms tumors.

The metastatic spread of breast cancer, typically to bone, lung, liver and brain, accounts for the vast majority of cancer-related deaths. Breast cancer metastases are thought to be derived primarily from individual migratory cancer cells, reaching distant sites through the bloodstream and initiating proliferation within distant organs. In addition to these single circulating tumor cells (CTCs), CTC-clusters have been detected in the blood of patients with cancer. In patients with breast cancer, we find that presence of such CTC-clusters is correlated with decreased progression-free survival. To study their functional role, we used mouse models, demonstrating that breast cancer cells injected intravascularly as clusters are more prone to survive and colonize the lungs than similarly injected single cancer cells. Primary orthotopic mammary tumors comprised of differentially tagged cells give rise to oligoclonal CTC-clusters, with 40-fold increased metastatic potential to the lung, compared with single CTCs. Using in vivo flow cytometry, we show that CTC-clusters are rapidly cleared from peripheral vessels, consistent with their trapping in small capillaries. Together, our observations suggest that primary tumor cells break off into the vasculature as CTC-clusters, and exhibit greatly enhanced metastatic propensity. Citation Format: Nicola Aceto, Aditya Bardia, Joel A. Spencer, Ben S. Wittner, Min Yu, Maria C. Donaldson, Adam Pely, Amanda Engstrom, Huili Zhu, Brian W. Brannigan, Ravi Kapur, Shannon L. Stott, Toshi Shioda, Sridhar Ramaswamy, David T. Ting, Charles P. Lin, Mehmet Toner, Daniel A. Haber, Shyamala Maheswaran. Circulating tumor cell clusters are precursors of breast cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-192. doi:10.1158/1538-7445.AM2014-LB-192

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal adult malignancy due to the propensity of this disease to metastasize. Circulating tumor cells (CTCs) are thought to be enriched for cells with metastatic potential and their characterization offers a means to understand the biological underpinnings of the distal spread of cancer. However, CTCs are rare cells in the blood and their isolation has posed a signifcant technological challenge. Multiple platforms have emerged in capturing these cells with most relying on a positive antibody based selection process (e.g. EpCAM). Our initial molecular characterization of pancreatic CTCs in the genetically engineered LSL-KrasG12D, Trp53flox/flox or +, Pdx1-Cre (KPC) mouse model utilized a microfluidic anti-EpCAM device followed by RNA sequencing to identify aberrant non-canonical WNT signaling in CTC populations (Yu M*, Ting DT*, et al. Nature 2012). Wnt2 was found to increase metastatic potential by enhancing anoikis resistance, a requirement of CTC survival, through activation of Tak1 kinase. However, this study was limited in only providing a partial CTC signature given the variable purity of these purified cell populations and analysis of bulk CTC populations could not provide the resolution to truly assess heterogeneity of these cells. Furthermore, a recent study has shown that cells from the mouse model may disseminate into circulation at an early point in PDAC development through epithelial-to-mesenchymal transition (EMT), which would generate CTCs with very low EpCAM expression and therefore would be missed in an EpCAM based capture device (Rhim AD et al. Cell 2012). We have overcome these barriers by employing a novel microfluidic isolation device to capture high numbers of CTCs that can be isolated as single cells (Ozkumur E*, Shah AM*, et al. Science Translational Medicine 2013). This device achieves high efficiency negative depletion of normal blood cells providing an enriched population of CTCs in solution that are not biased by a particular extracellular epitope and without any antibody interactions that could affect expression profiles. This has provided a method to truly understand the key transcriptional programs that differentiate CTCs from their primary tumors on an isogenic mouse background. Five tumor-bearing KPC mice generated a total of 168 single CTCs that were morphologically intact and subjected to a modified single cell RNA sequencing protocol (Tang F et al., Nature Protocols 2010). A total of 75 (45%) of these single CTCs were of sufficient quality for RNA sequencing indicating that the majority (55%) of intact CTCs selected likely had lost viability in the process of hematogenous transit. Single cell RNA-sequencing was also performed on 12 normal leukocytes (WBCs) from a control mouse, 12 mouse embryonic fibroblasts (MEFs), 16 single cells from the mouse NB508 pancreatic cancer cell line, and 34 (min 8 replicates) samples from primary tumors matched to the CTCs. Unsupervised hierarchical clustering of single cell samples demonstrated clear separation of MEFs, the NB508 pancreatic cancer cell line, and normal WBCs supporting the technical validity of the sequencing approach. Analysis of candidate CTCs identified three major CTC clusters, which were all distinct form matched primary tumors as well as from the NB508 cancer cell line. The must abundant CTC cluster comprised 41 of 75 cells (55%) and was defined by presence of epithelial markers (Krt7, Krt8, Krt18, Krt19) consistent with a “classical” CTC phenotype (CTC-c). The second CTC cluster was defined by enrichment of platelet markers CD41 (Itga2b) and CD61 (Itgb3) (CTC-plt) and a third having enrichment of cellular proliferation genes including Mki67 (CTC-pro). Single cell heterogeneity was assessed by intra-cluster correlation coeffficients, where lower values reflect higher heterogeneity. Not surprisingly, single cell heterogeneity was much higher in CTCs (mean correlation coefficient 0.42, 95% CI 0.36-0.47) compared to cancer cell lines (mean 0.86, 95% CI 0.80-0.91, p-value 1.2 x 10-15), but was notably similar comparing CTCs to single primary tumor cells (mean 0.38, 95% CI 0.28-0.47). Focusing on the dominant CTC-c cells, we used a non-parametric differential gene expression analysis including a rank product (RP) methodology suitable for large variations in absolute transcript levels found in single cell expression data (Breitling R et al. FEBS Letters 2004). Using a stringent FDR of ≤ 0.01, CTC-c cells had 878 genes with increased expression and 774 genes with reduced expression when compared with matched primary tumors. CTC-c cells were enriched for MAPK, as well as WNT, TGF-β, Neurotrophin, Toll-like receptor, and B-cell receptor signaling pathways. Analysis of a panel of EMT genes with significant differential expression revealed that CTC-c cells were in a biphenotypic state with universal loss of the epithelial markers E-cadherin (Cdh1) and Muc1, while the mesenchymal genes Cdh11 and Vim were found to be expressed much more heterogeneously amongst individual CTCs. Proposed pancreatic cancer stem cell genes were also evaluated and the Aldh1a1 and Aldh1a2 genes were found to be significantly enriched in CTC-c. Expression of both Aldh1a1 and Aldh1a2 in matched primary tumors was done through RNA in situ hybridization (RNA-ISH) revealing a heterogeneous distribution of these stem cell genes in both the stromal and epithelial compartments of the tumor. This highlighted the potential relevance of these stem cell markers in tumor cells dynamically shifting between epithelial and non-epithelial states. To provide further insight into the potential region from which CTCs emanate from the primary tumor, we selected the most highly enriched CTC transcripts found in ≥ 90% of all classical CTCs. Three genes met these criteria, which were decorin (Dcn), insulin-like growth factor binding protein 5 (Igfbp5), and Kruppel-like factor 4 (Klf4). Each of these genes has been previously implicated in pancreatic cancer development and were evaluated by RNA-ISH in primary tumor specimens to determine if they colocalized in particular tumor cells. Dcn is an extracellular matrix proteoglycan known to be expressed in a wide range of tumor stroma and by RNA-ISH was found primarily in the stromal elements of the tumor. However, both Igfbp5 and Klf4 were found to be focally expressed in cells at the epithelial-stromal interface. Although these genes are co-expressed in a minority of primary tumor cells, they are co-expressed at high levels in 85% of all classical CTCs. Together with the mixed epithelial/mesenchymal marks and enrichment of Aldh1a2 cells in stromal elements, these data point to the majority of viable CTCs emanating from the epithelial/stromal interface. In summary, we have successfully purified individual pancreatic CTCs using a novel microfluidic device and provided the first comprehensive single cell transcriptome analysis of these rare but exceptional cells. Three major classes of CTCs have been identified that would not have been possible without a single cell approach and we have characterized the major pathways that define these different subsets. The most abundant CTCs were found to have robust expression of keratin genes and are defined by a mixed E/M state with enrichment of Aldh1a1 and Aldh1a2 stem cell genes. These classical CTCs are marked by the co-expression of Igfbp5 and Klf4, which appear to localize to the epithelial-stromal interface in primary tumors. Ultimately, CTC cultures and functional testing will determine the contribution of these genes to CTC metastatic potential. This deep analysis of CTCs at single cell resolution has provided new biological insight into the metastatic cascade that will inform the development of novel therapies to treat this deadly disease. Citation Format: David T. Ting, Ben S. Wittner, Ajay M. Shah, David T. Miyamoto, Brian W. Brannigan, Kristina Xega, Jordan Ciciliano, Olivia C. MacKenzie, Julie Trautwein, Mohammad Shahid, Haley L. Ellis, Na Qu, Nabeel Bardeesy, Miguel N. Rivera, Ravi Kapur, Sridhar Ramaswamy, Toshi Shioda, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Diversity of circulating tumor cells in a mouse pancreatic cancer model identified by single cell RNA sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr NG04. doi:10.1158/1538-7445.AM2014-NG04

Treatment of EGFR-mutant lung cancer with erlotinib results in dramatic tumor regression but it is invariably followed by drug resistance. In characterizing early transcriptional changes following drug treatment of mutant EGFR-addicted cells, we identified the stem cell transcriptional regulator SOX2 as being rapidly and specifically induced, both in vitro and in vivo. Suppression of SOX2 sensitizes cells to erlotinib-mediated apoptosis, whereas its ectopic expression reduces drug-induced cell death. We show that erlotinib relieves EGFR-dependent suppression of FOXO6, leading to SOX2 induction and repression of the pro-apoptotic BH3-only genes BIM and BMF. Together, these observations point to a physiological feedback mechanism that attenuates oncogene addiction-mediated cell death and may contribute to the acquisition of drug resistance. Citation Format: Stephen Michael Rothenberg, Kyle Concannon, Sarah Cullen, Alexa B. Turke, Anthony C. Faber, Jeffrey A. Engelman, Shyamala Maheswaran, Daniel A. Haber. Inhibition of mutant EGFR in lung cancer cells triggers SOX2-FOXO6 dependent survival pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4773. doi:10.1158/1538-7445.AM2014-4773

Glioblastoma (GBM), the most common and aggressive form of glioma, is often heterogeneous, diffusely invasive and hypervascularized. Despite being aggressive, extracranial metastases are rare, suggesting constraints on hematologic dissemination or distant survival of primary GBM cells. We recently developed a novel microfluidic device, termed the CTC-iChip, for efficient, antigen agnostic capture of rare circulating tumor cells (CTCs). Using the CTC-iChip, we set out to determine whether GBM cells can be detected in the blood of mice using two orthotopic xenograft models representing diffuse (GBM8) and focal (GBM24) GBM. CTCs were identified in 5/11 and 2/5 mice bearing GBM8 and GBM24 xenografts, respectively. CTC numbers did not correlate with tumor size or latency. We then expanded our analysis to patients and identified CTCs in 12/38 GBM patients with a median 5.3 cells per ml (range: 0 to 48.2 cells per ml). To understand the molecular characteristics of GBM CTCs, we analyzed the expression of 49 GBM-specific and stem cell transcripts in single GBM CTCs from both mouse xenografts models as well as GBM patients. The expression of mesenchymal gene transcripts including VIM, TGFBR2, TGFB, and SERPINE1 was elevated in CTCs compared with cultured cells and matched primary tumor cells. Moreover, CTCs exhibited lower proliferative indices and decreased neural lineage transcripts. To determine the pathophysiologic significance of this expression profile and the prospective origin of GBM CTCs, RNA-ISH was performed on xenografts and patient tumor samples. In the highly migratory GBM8 xenograft model, broad tumor cell expression of CTC/mesenchymal transcripts was observed. However, in the GBM24 focal tumor growth model, CTC/mesenchymal gene transcripts were predominantly expressed in regions of tumor invasion, migration along fiber tracts, and around regions of necrosis. Similarly, RNA-ISH analysis of 6 GBM patient tumors revealed prominent expression of CTC/mesenchymal genes in peri-necrotic and migratory pseudopalisading cells. Finally, RNA-ISH analysis of a rare case of metastatic GBM revealed a significant enrichment of GBM/mesenchymal gene expressing tumor cells within lung and lymph node metastases. Taken together, these data provide the first evidence for the hematologic circulation of GBM cells and reveal the involvement of mesenchymal features in the pathophysiology of malignant brain tumors. Citation Format: James P. Sullivan, Brian V. Nahed, Andrew S. Chi, Marissa N. Madden, Samantha M. Oliveira, Simeon Springer, Hiroaki Wakimoto, Deepak Bhere, Khalid Shah, Phil Spuhler, Ajay M. Shah, David N. Louis, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Molecular characterization of circulating glioblastoma cells identifies a mesenchymal-like tumor cell subpopulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4004. doi:10.1158/1538-7445.AM2014-4004

Müllerian inhibiting substance (MIS) inhibits breast cancer cell growth in vitro through interference with cell cycle progression and induction of apoptosis, a process associated with NFkappaB activation and up-regulation of one of its important target genes, IEX-1S (Segev, D. L., Ha, T., Tran, T. T., Kenneally, M., Harkin, P., Jung, M., MacLaughlin, D. T., Donahoe, P. K., and Maheswaran, S. (2000) J. Biol. Chem. 275, 28371-28379). Here we demonstrate that MIS activates the NFkappaB signaling cascade, induces IEX-1S mRNA, and inhibits the growth of MCF10A, an immortalized human breast epithelial cell line with characteristics of normal cells. In vivo, an inverse correlation was found to exist between various stages of mammary growth and MIS type II receptor expression. Receptor mRNA significantly diminished during puberty, when the ductal system branches and invades the adipose stroma and during the expansive growth at lactation, but it was up-regulated during involution, a time of regression and apoptosis. Peripartum variations in MIS type II receptor expression correlated with NFkappaB activation and IEX-1S mRNA expression. Administration of MIS to female mice induced NFkappaB DNA binding and IEX-1S mRNA expression in the breast. Furthermore, exposure to MIS in vivo increased apoptosis in the mouse mammary ductal epithelium. Thus, MIS may function as an endogenous hormonal regulator of NFkappaB signaling and growth in the breast.

Inflammatory cytokines are suspected to contribute to the pathogenesis of bovine pneumonic pasteurellosis (BPP) through neutrophil recruitment, leukocyte activation, and the induction of a broad array of soluble inflammatory mediators. An in vivo experimental model of BPP was used to characterize the pulmonary expression kinetics of tumor necrosis factor alpha (TNFalpha), interleukin-1 beta (IL-1beta), and interleukin-8 (IL-8) genes and proteins during the acute phase of disease development. Cytokine expression in bronchoalveolar lavage (BAL) fluid, BAL cells, and pneumonic lung parenchyma was quantitated by northern blot analysis, enzyme-linked immunosorbent assay (ELISA), and in situ hybridization at 2, 4, 8, 16, and 24 hours after endobronchial inoculation of Pasteurella (Mannheimia) haemolytica. Expression of TNFalpha, IL-1beta, and IL-8 was significantly increased in the airways and lung lesions of infected calves as compared with mock-infected controls. Although kinetic patterns varied, peak levels of cytokine mRNA occured within 8 hours postinfection (PI), and peak cytokine concentrations occurred within 16 hours PI. In all samples, IL-8 was expressed to the greatest extent and TNFalpha was least expressed. Expression of TNFalpha was restricted to alveolar macrophages. Alveolar and interstitial macrophages produced IL-1beta and IL-8 in the first 4 hours; bronchial and bronchiolar epithelial cells were also significant sources of IL-8 during this period. By 8 hours PI, neutrophils were the dominant source of both IL-1beta and IL-8. These findings demonstrate a spatial and temporal association between pulmonary expression of inflammatory cytokines and acute lung pathology, supporting the hypothesis that cytokines contribute to inflammatory lung injury in BPP.

In bovine alveolar macrophages (BAMs), exposure to leukotoxin (Lkt) and endotoxin (LPS) from Pasteurella haemolytica results in expression of inflammatory cytokine genes and intracellular calcium ([Ca2+]i) elevation. Leukotoxin from P. haemolytica interacts only with leukocytes and platelets from ruminant species. Upregulation of cytokine genes in different cells by LPS involves activation of the transcription factor NF-kappaB (NF-kappaB), resulting in its translocation from the cytoplasm to the nucleus. Using immunocytochemical staining and confocal imaging, we studied whether NF-kappaB activation represents a common mechanism for the expression of multiple cytokine genes in BAMs (Lkt-susceptible cells) stimulated with Lkt and LPS. Bovine pulmonary artery endothelial cells and porcine alveolar macrophages were used as nonsusceptible cells. The role of Ca2+ and tyrosine kinases in NF-kappaB activation and inflammatory cytokine gene expression was studied, since an inhibitor of tyrosine kinases attenuates LPS-induced [Ca2+]i elevation in BAMs. The results are summarized as follows: (a) Lkt induced NF-kappaB activation and [Ca2+]i elevation only in BAMs, while LPS effects were demonstrable in all cell types; (b) chelation of [Ca2+]i blocked NF-kappaB activation and IL-1beta, TNFalpha, and IL-8 mRNA expression; and (c) tyrosine kinase inhibitor herbimycin A blocked expression of all three cytokine genes in BAMs stimulated with Lkt, while only the expression of IL-1beta was blocked in BAMs stimulated with LPS. We conclude that cytokine gene expression in BAMs requires NF-kappaB activation and [Ca2+]i elevation, and Lkt effects exhibit cell type- and species specificity.

The clinical development of selective RAF and MEK inhibitors has transformed the treatment of the ∼50% of melanoma patients whose tumors harbor BRAF mutations. However, a substantial percentage of these patients fail to respond to therapy, and most responses are partial and short-lived. Although multiple mechanisms of resistance have been identified in BRAF-mutant melanoma, no clinically useful biomarkers have been established to predict which patients are most likely to demonstrate sensitivity or resistance to RAF or MEK inhibitors. We found that suppression of TORC1 activity in response to RAF or MEK inhibitors, as measured by decreased phosphorylation of ribosomal protein-S6 (P-S6), was a functional biomarker that effectively predicted sensitivity in BRAF-mutant melanoma cell lines in vitro and in mouse tumor xenografts. In sensitive melanomas, TORC1 and P-S6 were suppressed in response to RAF or MEK inhibitors, but in resistant melanomas, TORC1 activity was maintained, in some cases despite robust suppression of MAPK signaling by these inhibitors. In mouse models, suppression of TORC1 after MAPK inhibition was necessary for induction of apoptosis and tumor response in vivo. Notably, in paired biopsies obtained from patients with BRAF-mutant melanoma before treatment and after initiation of RAF inhibitor therapy, P-S6 suppression was associated with significantly improved progression-free survival [HR 0.19, 95% CI 0.01-0.84, p=0.03]. Finally, we found that changes in P-S6 in patients’ tumor cells could be readily monitored in real-time by multiplexed, quantitative immunofluorescence microscopy of serial fine-needle aspiration biopsies obtained from patients before and during the first 2 weeks of RAF inhibitor therapy. This approach provides a minimally-invasive means of rapidly monitoring the efficacy of treatment, before changes in tumor volume are apparent by traditional radiographic imaging. Together, these results establish suppression of P-S6 after initiation of RAF inhibitor therapy as a robust potential functional biomarker to guide the treatment of patients with BRAF-mutant melanoma, and present a powerful methodology for monitoring changes in potentially any signaling pathway in response to targeted therapies in patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C137. Citation Format: Ryan B. Corcoran, S. Michael Rothenberg, Aaron N. Hata, Anthony C. Faber, Adriano Piris, Rosalynn M. Nazarian, Ronald D. Brown, Jason T. Godfrey, Daniel Winokur, John Walsh, Mari Mino-Kenudson, Shyamala Maheswaran, Jeffrey Settleman, Jennifer A. Wargo, Keith T. Flaherty, Daniel A. Haber, Jeffrey A. Engelman. Rapid assessment of TORC1 suppression as a functional biomarker predicting responsiveness to RAF and MEK inhibitors in BRAF-mutant melanoma patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C137.

Circulating tumor cells (CTCs) are present at extraordinarily low levels in the blood of patients with non-hematological cancers, but they provide a non-invasive source of cancer cells for potential diagnostic applications, as well as for understanding the process of blood-borne metastasis. We have developed and tested three generations of microfluidic devices for the isolation of CTCs, each with further improvements in automation, processing throughput, and downstream analytical capabilities. The CTC-iChip allows high throughput and high efficiency depletion of normal leukocytes through a microfluidic magnetophoresis device, revealing untagged CTCs. The unbiased isolation of CTCs irrespective of tumor epitopes, together with their presentation in a format where they can be readily manipulated, provides unique opportunities for their detailed molecular characterization. We have previously shown that rising or declining numbers of CTCs are well correlated with tumor progression or therapeutic response; that they can be used to monitor the prevalence of known clinically-relevant mutations; and that changes in their composition can be followed longitudinally during the course of targeted cancer therapies. Epithelial-to-mesenchymal transition (EMT) is evident among CTCs and evolves dynamically during the course of response or progression following cancer therapy. The heterogeneity of CTC populations is evident in single cell imaging analyses and sequencing studies, with implications for understanding the complexity of metastatic progression and therapeutic responses. Taken together, the application of novel bioengineering technologies to the isolation and analysis of CTCs across multiple different cancer types offers a unique window into the properties of cancer cells that invade into the bloodstream and give rise to metastases. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL06-04. Citation Format: Daniel A. Haber, Shyamala Maheswaran, Lecia Sequist, Min Yu, Emre Ozkumur, David Ting, David Miyamoto, Richard Lee, Ajay Shah, Aditya Bardia, Shannon Stott, Mehmet Toner. Molecular characterization of circulating tumor cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PL06-04.

Local and systemic activation of coagulation is frequently associated with bacterial sepsis. The coagulopathy is due, at least in part, to expression of tissue factor (TF) by monocytes and macrophages. The purpose of this study was to evaluate the expression of procoagulant activity by bovine alveolar macrophages, leukocytes and platelets, and to determine the relative potency of three chemical inhibitors of TF expression (pentoxifylline, retinoic acid, and cyclosporin A). Bovine alveolar macrophages were stimulated with lipopolysaccharide (LPS) derived from Pasteurella haemolytica or recombinant bovine tumour nervous factor (TNF) and dose- and time-dependent effects on TF expression were studied. LPS and TNF induced TF expression in alveolar macrophages and LPS treatment of whole blood induced TF expression in mononuclear cells. Neutrophils and platelets also expressed procoagulant activity, but this activity was not inhibited by anti-bovine TF monoclonal antibody. Pentoxifylline (40 mumol/L), retinoic acid (0.01 mmol/L) and cyclosporin A (0.08 mumol/L) inhibited TF expression when added concurrently with LPS or TNF, but not when added 4 h after stimulation. TF mRNA was not detected in unstimulated alveolar macrophages by Northern blot analysis. In contrast, exposure to LPS or TNF for 6 h induced marked expression of TF mRNA, which was inhibited by treatment with pentoxifylline, retinoic acid and cyclosporin A. Expression of TNF by alveolar macrophages stimulated with LPS was also inhibited by these compounds. Our results indicate that procoagulant activity expressed by alveolar macrophages and monocytes is associated with expression of TF, whereas procoagulant activity expressed by neutrophils and platelets is not. The concentrations of pentoxifylline and retinoic acid necessary for inhibition of TF expression in vitro may not be achievable in vivo owing to their toxic effects. However, the in vitro concentration of cyclosporin A that inhibited TF expression did not exceed the plasma concentration observed in humans, and therefore may be useful for inhibition of TF expression in vivo.

Epithelial mesenchymal transition (EMT) has been postulated to contribute to the dissemination of cancer cells, but supporting histopathological evidence is limited. We used a microfluidic device to isolate circulating tumor cells (CTCs), combined with multiplex RNA-in-situ hybridization (ISH) and RNA sequencing, to quantify and characterize EMT in breast CTCs. Whereas only rare cells in the primary tumor expressed both mesenchymal and epithelial markers, such biphenotypic as well as purely mesenchymal cells were enriched among CTCs, across all histological subtypes of breast cancer. Analysis of treatment responses in 10 patients suggested an association of mesenchymal CTCs with disease progression. In an index patient followed longitudinally, fluctuation in epithelial and mesenchymal states was observed as a function of initial response and subsequent resistance to therapy. Mesenchymal markers were predominant in clusters of tumor cells, many of which had adherent platelets. Finally, RNA sequencing of CTC clusters identified EMT-related signatures, including FOXC1, a known transcriptional regulator of EMT. Together, these data support a role for EMT in the blood-borne dissemination of breast cancer and point to the dynamic nature of this cell fate change. Citation Format: Min Yu, Aditya Bardia, Ben Wittner, Shannon Stott, Malgorzata Smas, David Ting, Steven Isakoff, Jordan Ciciliano, Marissa Wells, Ajay Shah, Kyle Concannon, Maria Donaldson, Lecia Sequist, Elena Brachtel, Dennis Sgroi, Jose Baselga, Sridhar Ramaswamy, Mehmet Toner, Daniel Haber, Shyamala Maheswaran. Circulating tumor cells in human breast cancer exhibit dynamic changes in epithelial and mesenchymal composition. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A75.

Epithelial to mesenchymal transition (EMT) has been postulated to contribute to the migration and dissemination of cancer cells, but supporting histopathological evidence is limited. We used a microfluidic device to isolate circulating tumor cells (CTCs), combined with multiplex fluorescent RNA-in-situ hybridization (ISH) and RNA sequencing, to quantify and characterize EMT in breast cancer cells within the bloodstream. Whereas only rare (0.1–10%) cells in the primary tumor expressed both mesenchymal and epithelial markers, such biphenotypic as well as purely mesenchymal cells were enriched among CTCs, across all histological subtypes of breast cancer. Analysis of the therapy response in 8 patients suggest an association of mesenchymal CTCs with disease progression. In an index patient followed longitudinally, fluctuation in epithelial and mesenchymal states was observed as a function of initial response and subsequent resistance to therapy. Mesenchymal markers were predominant in clusters of tumor cells, many of which had adherent platelets. Finally, RNA sequencing of mesenchymal CTC clusters identified TGF-B and other EMT-related signatures, which were absent from more epithelial CTCs. FOXC1, a known regulator of EMT, was abundantly expressed in mesenchymal CTCs and was detectable within localized regions of the primary breast tumor. Together, these data support a role for EMT in the blood-borne dissemination of breast cancer and point to the dynamic nature of this cell fate change. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-14.

Corrigendum: RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis

4566 Background: Cabo (XL184) is an oral inhibitor of MET and VEGFR2. In a randomized discontinuation trial of cabo 100mg daily, 76% of men with CRPC and bone metastases had partial or complete resolution of bone scan lesions as early as week (wk) 6. However, treatment was limited by adverse events (AEs), with dose reductions in 51% of patients (pts), and discontinuations in 10%. The current study was designed to determine the efficacy and tolerability of cabo at lower starting doses. Methods: An adaptive response scheme was used to determine the lowest active daily cabo dose among dose levels +1 (60mg), 0 (40mg), and -1 (20mg). The primary endpoint was wk 6 bone scan response (BSR) assessed with an automated FDA 510(k) approved computer-aided detection system. A ≥30% decrease in total bone scan lesion area (BSLA) was defined as a response. The first cohort was treated at dose level 0. The number of responses (≥8 vs.

Serial radiographic imaging is the primary means for determining the efficacy of cancer treatments. However, a long time interval is required before changes are appreciated, and the direct effects of therapy on tumor cells cannot be measured. We have utilized highly multiplexed, quantitative immunofluorescence microscopy to monitor multiple signaling proteins simultaneously in direct tumor biopsies and circulating tumor cells obtained serially from patients with BRAF- and PIK3CA-mutant solid cancers undergoing treatment with agents that target these mutated oncogenes. Changes in signaling that accurately predict subsequent clinical response can be determined from small numbers (∼100) of tumor cells and are apparent within days of the first dose of therapy, and weeks before radiographic determination of response. Preliminary results suggest that therapeutic efficacy (or lack thereof) is correlated with both the degree and rapidity of downregulation of tumor cell signaling. The ability to measure the effects of therapy directly in patient-derived tumor cells may permit real time information about efficacy (or lack thereof) to be rapidly incorporated into the decision to continue an ongoing therapy or to switch to a new one, or in the identification of patient populations most likely to respond to a novel class of agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2446. doi:1538-7445.AM2012-2446