Search

Circulating tumor cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. While isolation and molecular analysis of these cells is technically challenging, they may identify cellular pathways that contribute to the blood-borne dissemination of cancer cells. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to digital gene expression (DGE) analysis using single molecule RNA sequencing. We identified Wnt2 as enriched in both CTCs and metastatic ascites cells, compared with primary tumors. Expression of Wnt2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. The effect of Wnt2 is correlated with fibronectin upregulation, and it is mediated in part through non-canonical Wnt signaling and suppressed by inhibition of the Map3k7 (Tak1) kinase, an integrator of Wnt, BMP and TGF-β signaling. DGE analysis of human pancreatic CTCs reveals significant enrichment for non-canonical Wnt signaling in 5 of 11 cases, with Wnt2 RNA detectable by in situ hybridization in a subset of CTCs. Identifying CTC-enriched transcripts such as Wnt2 may point to pathways that enhance haematogenous metastasis, thus providing novel therapeutic targets to prevent the distal spread of cancer. 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 5304. doi:1538-7445.AM2012-5304

The tumour suppressor gene WT1 encodes a transcription factor expressed in tissues of the genito-urinary system. Inactivation of this gene is associated with the development of Wilms tumour a pediatric kidney cancer. We show that WT1 is also expressed at high levels in many supportive structures of mesodermal origin in the mouse. We also describe a case of adult human mesothelioma, a tumour derived from the peritoneal lining, that contains a homozygous point mutation within WT1. This mutation, within the putative transactivation domain, converts the protein from a transcriptional repressor of its target sequence to a transcriptional activator. The role of WT1 in normal development thus extends to diverse structures derived from embryonic mesoderm and disruption of WT1 function contributes to the onset of adult, as well as pediatric, tumours.

WT1 is a tumor-suppressor gene expressed in the developing kidney, whose inactivation leads to the development of Wilms tumor, a pediatric kidney cancer. WT1 encodes a transcription factor which binds to the EGR1 consensus sequence, mediating transcriptional repression. We now demonstrate that p53, the product of a tumor-suppressor gene with ubiquitous expression, physically associates with WT1 in transfected cells. The interaction between WT1 and p53 modulates their ability to transactivate their respective targets. In the absence of p53, WT1 acts as a potent transcriptional activator of the early growth response gene 1 (EGR1) site, rather than a transcriptional repressor. In contrast, WT1 exerts a cooperative effect on p53, enhancing its ability to transactivate the muscle creatine kinase promoter.

Despite recent molecular advances, the treatment of localized and metastatic prostate cancer has changed little in recent decades and it remains a major cause of cancer deaths in men. Localized prostate cancer may be either indolent or invasive, without reliable distinguishing markers, and the absence of tumor tissue from patients with metastatic bone disease has hampered understanding of acquired resistance to primary hormonal therapy. Given the predominant hematogenous spread of prostate cancer, circulating tumor cells (CTCs) may provide an indicator of vascular invasiveness, as well as a source of tumor specimens for molecular analysis, but available technologies have been limited by relatively low sensitivity. Huge strides in engineering at smaller scales have lead to the tremendous progress in microfabricated devices to facilitate both applied and basic research. Applying a multi-disciplinary approach, we have developed a microfluidic technology “CTC-Chip” for diagnosis and monitoring of non-hematological cancers. Using the CTC-Chip, we were able to isolate CTCs in patients with both localized and metastatic prostate cancer. In patients with advanced cancer, CTCs were present at a frequency of 34 CTCs/mL (range 14-653), with a profound decline in numbers during response to hormone withdrawal therapy. Molecular markers, including the characteristic TMPRSS2-ERG translocation, are readily identified in CTCs. In patients with early stage, resectable prostate cancer, we have monitored CTC load in circulation following surgical resection of the primary tumor. CTCs were examined the day following surgical resection, at postoperative day 9, and at greater than 3 months, coincident with scheduled postoperative clinical visits. Longitudinal monitoring of CTCs revealed all CTC-positive cases showed disappearance of CTCs following prostate resection, with some having an immediate decline and others having a delayed (>24 hours) response. No evident differences in tumor pathology or operative procedure distinguished patients with a rapid versus delayed postoperative CTC decline. Dual staining of captured CTCs for PSA and Ki67 indicated a broad range in proliferative index of CTCs among patients with different clinical courses. Thus, the quantitative isolation and analysis of CTCs in both localized and metastatic prostate cancer provides novel insights into early hematogenous dissemination and enables molecular analyses of treatment-responsive and -resistant disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1136.

5149 Background: The CTC-Chip reliably detects CTCs in patients with localized and metastatic prostate cancer. In localized disease, CTCs may be indicative of tumor invasiveness. In the metastatic setting, CTCs may be useful in monitoring response to therapy. Molecular analyses of CTCs through this minimally invasive technique may provide insights into disease behavior, whereas sampling of metastatic deposits in bone (the predominant site of prostate metastasis) is not feasible. Methods: Peripheral blood was collected from patients undergoing treatment. CTCs were captured on the CTC-Chip. Quantities were determined using cytokeratin- and PSA-based staining and enumeration. In some cases, molecular analyses for the TMPRSS2:ERG chromosomal translocation were performed using FISH in intact cells or nuclei, or RT-PCR in extracted RNA. Subgroups of patients (n) included: localized disease undergoing prostatectomy (25), and metastatic disease undergoing either androgen deprivation therapy (ADT) (12) or docetaxel chemotherapy (5). Results: PSA-stained CTCs were captured in all patients. In localized disease, CTC quantities fell to undetectable levels 7–14 days after surgery; some patients exhibited an immediate decline while others had a delayed decline (>24 hours). In patients with metastatic disease undergoing either ADT or chemotherapy, CTC quantities generally corresponded with changes in serum PSA and radiographic assessments. Approximately 50% of patients exhibited the TMPRSS2:ERG translocation. Conclusions: These studies are the first to use the highly sensitive CTC-Chip technology to assess dynamic changes in CTCs in patients undergoing prostatectomy, ADT, or chemotherapy. Ongoing larger studies will assess whether patterns of CTC changes predict recurrence or treatment response. Molecular characterization of CTCs may provide new insights into prostate cancer biology, clinical behavior, and therapeutic targets. [Table: see text]

A genomic fragment of Pasteurella haemolytica biotype A coding for a serotype 1-specific agglutinating antigen was used as a probe in a series of hybridization experiments to determine distribution of the fragment in various P. haemolytica serotypes as well as other bacteria. Results showed presence of the fragment in seven out of the 12 serotypes tested, all of which belonged to biotype A. Two other serotypes belonging to biotype A, all three serotypes belonging to biotype T, two Pasteurella multocida isolates and Escherichia coli did not have the fragment in their genome. Thus the expression of the P. haemolytica biotype A serotype 1-specific agglutinating antigen (PHA1SAA) seems to be due to serotype-specific regulation of protein expression rather than to genetic deletion. Differences in methylation of the PHA1SAA-coding fragment was also noted in DpnI and Sau3AI genomic DNA digests from the various serotypes analyzed by Southern blot. However, no apparent correlation was observed between methylation and PHA1SAA expression. E. coli with a recombinant plasmid containing a homologous genomic fragment derived from P. haemolytica serotype 2 also expressed PHA1SAA.

SummaryAccumulating evidence implicates heterogeneity within cancer cell populations in the response to stressful exposures, including drug treatments. While modeling the acute response to various anticancer agents in drug-sensitive human tumor cell lines, we consistently detected a small subpopulation of reversibly “drug-tolerant” cells. These cells demonstrate >100-fold reduced drug sensitivity and maintain viability via engagement of IGF-1 receptor signaling and an altered chromatin state that requires the histone demethylase RBP2/KDM5A/Jarid1A. This drug-tolerant phenotype is transiently acquired and relinquished at low frequency by individual cells within the population, implicating the dynamic regulation of phenotypic heterogeneity in drug tolerance. The drug-tolerant subpopulation can be selectively ablated by treatment with IGF-1 receptor inhibitors or chromatin-modifying agents, potentially yielding a therapeutic opportunity. Together, these findings suggest that cancer cell populations employ a dynamic survival strategy in which individual cells transiently assume a reversibly drug-tolerant state to protect the population from eradication by potentially lethal exposures.PaperClip

Background Homeobox B9 (HOXB9), a transcriptional factor, regulates developmental processes and tumor progression and has recently been recognized as one of important transcriptional factors related to angiogenesis. This study aimed to investigate the role of HOXB9 in tumorigenesis and angiogenesis. Methods We examined the expression of HOXB9 in colorectal cancer using qPCR and in situ hybridization. We also examined the effect of HOXB9 overexpression in colorectal cancer using a proliferation assay, ELISA, a multiplex assay, and xenograft models. The clinical significance of HOXB9 was statistically evaluated in resected specimens. Results HOXB9 was expressed in colorectal cancer specimens. HOXB9 induced angiogenesis and tumor proliferation in vitro, which resulted in high tumorigenicity in vivo and poor overall survival. Bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody, remarkably suppressed tumor proliferation by inhibiting angiogenesis in HOXB9-overexpressing xenografts, and it improved overall survival and provided prolonged progression-free survival in HOXB9-overexpressing patients. A comprehensive multiplex assay of the supernatant of cancer cells co-cultured with human vascular endothelial cells and fibroblasts indicated significantly higher interleukin-6 (IL6) levels than those in the supernatant of monocultured cells. HOXB9 overexpression in clinical specimens was significantly correlated with increased IL6 expression. An IL6-neutralizing antibody inhibited VEGF secretion and tumor proliferation in the co-culture system. Conclusions HOXB9 promotes the secretion of angiogenic factors, including VEGF, to induce tumor proliferation through microenvironmental production of cytokines including IL6 signaling. Moreover, silencing of VEGF or IL6 terminates cytokine release in tumor microenvironment. Thus, HOXB9 and IL6 may be potential biomarkers for bevacizumab treatment.