Yucheng Xu

Cancer cells acquire a drug resistant, highly tumorigenic, cancer stem-like phenotype through modulation of the PI3K/Akt/β-catenin/CBP pathway

Cancer initiation and progression have been attributed to newly discovered subpopulations of self‐renewing, highly tumorigenic, drug‐resistant tumor cells termed cancer stem cells. Recently, we and others reported a new phenotypic plasticity wherein highly tumorigenic, drug‐resistant cell populations could arise not only from pre‐existing cancer stem‐like populations but also from cancer cells lacking these properties. In the current study, we hypothesized that this newfound phenotypic plasticity may be mediated by PI3K/Akt and Wnt/β‐catenin signaling, pathways previously implicated in carcinogenesis, pluripotency and drug resistance. Using GFP expression, Hoechst dye exclusion and fluorescence activated cell sorting (FACS) of cancer cell lines, we identified and tracked cancer stem‐like side populations (SP) of cancer cells characterized by high tumorigenicity and drug resistance. We found that pharmacological inhibition or genetic depletion of PI3K and AKT markedly reduced the spontaneous conversion of nonside population (NSP) cells into cancer stem‐like SP cells, whereas PI3K/Akt activation conversely enhanced NSP to SP conversion. PI3K/AKT signaling was mediated through downstream phosphorylation of GSK3β, which led to activation and accumulation of β‐catenin. Accordingly, pharmacological or genetic perturbation of GSK3β or β‐catenin dramatically impacted conversion of NSP to SP. Further downstream, β‐catenins effects on NSP‐SP equilibrium were dependent upon its interaction with CBP, a KAT3 family coactivator. These studies provide a mechanistic model wherein PI3K/Akt/β‐catenin/CBP signaling mediates phenotypic plasticity in and out of a drug‐resistant, highly tumorigenic state. Therefore, targeting this pathway has unique potential for overcoming the therapy resistance and disease progression attributed to the cancer stem‐like phenotype.

Telomere and Telomerase Therapeutics in Cancer

Telomerase is a reverse transcriptase capable of utilizing an integrated RNA component as a template to add protective tandem telomeric single strand DNA repeats, TTAGGG, to the ends of chromosomes. Telomere dysfunction and telomerase reactivation are observed in approximately 90% of human cancers; hence, telomerase activation plays a unique role as a nearly universal step on the path to malignancy. In the past two decades, multiple telomerase targeting therapeutic strategies have been pursued, including direct telomerase inhibition, telomerase interference, hTERT or hTERC promoter driven therapy, telomere-based approaches, and telomerase vaccines. Many of these strategies have entered clinical development, and some have now advanced to phase III clinical trials. In the coming years, one or more of these new telomerase-targeting drugs may be expected to enter the pharmacopeia of standard care. Here, we briefly review the molecular functions of telomerase in cancer and provide an update about the preclinical and clinical development of telomerase targeting therapeutics.

Reprogramming murine telomerase rapidly inhibits the growth of mouse cancer cells in vitro and in vivo

Telomerase plays a critical role in cancer, prompting the pursuit of various telomerase-based therapeutic strategies. One such strategy, telomerase interference, exploits the high telomerase activity in cancer cells and reprograms telomerase to encode “toxic” telomeres. To date, telomerase interference has been tested in human cancer cells xenografted into mice, an approach that does not recapitulate spontaneous malignancy and offers few insights about host toxicities, because human telomerase is targeted in a mouse host. To address these limitations, we designed and validated two new gene constructs specifically targeting mouse telomerase: mutant template mouse telomerase RNA (MT-mTer) and small interfering RNA against wild-type mouse telomerase RNA (α-mTer-siRNA). Using lentiviral delivery in mouse prostate cancer cells, we achieved α-mTer-siRNA–mediated knockdown of wild-type mTer (80% depletion) and concurrent overexpression of MT-mTer (50-fold). We showed that the two constructs effectively synergize to reprogram murine telomerase to add mutant instead of wild-type telomeric repeats, resulting in rapid telomeric uncapping (5-fold increase in DNA damage foci). This, in turn, led to rapid and significant apoptosis (>90% of cells) and growth inhibition in vitro (90% reduction in viable cell mass) and in vivo (75% reduction in tumor allograft wet weight). In summary, we have shown that mouse cancer cells are vulnerable to direct telomerase interference using novel murine telomerase-targeting constructs; this approach can now be used to study the true therapeutic potential of telomerase interference in mouse spontaneous cancer models. Mol Cancer Ther; 9(2); 438–49

AXIN2 expression predicts prostate cancer recurrence and regulates invasion and tumor growth

Treatment of prostate cancer (PCa) may be improved by identifying biological mechanisms of tumor growth that directly impact clinical disease progression. We investigated whether genes associated with a highly tumorigenic, drug resistant, progenitor phenotype impact PCa biology and recurrence.

Isolation of circulating tumor cells by a magnesium-embedded filter

Circulating tumor cells (CTCs) are rare cancer cells that are shed by tumors into the bloodstream and that can be valuable biomarkers for various types of cancers. However, CTCs captured on the filter could not be released easily using the existing CTC analysis platforms based on size. To address this limitation, we have developed a novel magnesium (Mg)-embedded cell filter for capture, release and isolation of CTCs. The CTC-filter consists of a thin Ebeam-deposited Mg layer embedded between two parylene-C (PA-C) layers with designed slots for filtration and CTC capture. Thin Mg film has proved highly biocompatible and can be etched in saline, PBS and Dulbeccos modified eagle medium (DMEM) etc, properties that are of great benefit to help dissociate the filter and thus release the cells. The finite element method (FEM) analysis was performed on the Mg etching process in DMEM for the structure design. After the filtration process, the filter was submerged in DMEM to facilitate Mg etching. The top PA-C filter pieces break apart from the bottom after Mg completely dissolves, enabling captured CTCs to detach. The released CTC can be easily aspirated into a micropipette for further analysis. Thus, the Mg-embedded cell filter provides a new and effective approach for CTCs isolation from the filter, making this a promising new strategy for cancer detection.

Magnesium-embedded live cell filter for CTC isolation

This paper reports a novel Magnesium-embedded cell filter for Circulating Tumor Cell (CTC) capture, release and isolation. The new and novel feature is the use of thin-film Mg to release the captured CTCs based on the fact that any Cl- containing culture medium can readily etch Mg away [1]. The releasing and the isolation of each individual CTC are demonstrated here. After filtration process, the filter is submerged in PBS to facilitate Mg etching. The top PA-C filter pieces break apart from the bottom after Mg completely dissolves, enabling captured CTC cells to detach from the filter. The released CTC can then be easily aspirated into a micropipette, and then for further, such as, DNA mutation analysis.

Abstract 1717: Orthogonal identification of circulating tumor cells (CTCs) using single cell low pass whole-genome sequencing (WGS) and copy-number alteration (CNA) analysis

Introduction: Presence of circulating tumor cells has prognostic value in multiple malignancies, and molecular analysis of CTCs is currently ongoing in numerous clinical trials. Most CTC enrichment methods rely on standard epithelial and leukocyte markers (CK+CD45-), so recovered cells are assumed to be of epithelial origin but never shown to be bona fide tumor cells. Conversely, atypical cells lacking the characteristic marker profile may not be analyzed, even though they may represent important tumor subpopulations. Here we evaluate a rapid, non-exhaustive, and cost-effective first-pass genomic analysis of individual candidate CTCs. This approach allows efficient upfront CNA-based confirmation that a given cell is of tumor origin, while leaving abundant DNA for deeper subsequent analysis in cells of interest. Methods: Whole peripheral blood of metastatic prostate cancer patients was enriched for CTCs using the CellSearch® system (Janssen Diagnostics) under an IRB-approved protocol, and 5 samples with >5 CTCs were selected for further study. Next, the DEPArray™ v2 system (Menarini Silicon Biosystems) was used to identify and isolate single CTCs (CK+CD45-DAPI+) and paired white blood cells (WBCs; CK-CD45+DAPI+) from the enriched samples. In addition, cells negative for both cytokeratin and CD45 but with characteristic malignant morphology (large with high nuclear-cytoplasmic ratio) were isolated. Recovered single cells were whole-genome amplified with Ampli1™ WGA and quality controlled by Ampli1 QC. Ampli1 LowPass kit was then used to prepare NGS libraries for absolute CNA profiling by low-pass WGS. Results: Thirty-three single CTCs (CK+CD45-DAPI+) and 30 WBCs (CK-CD45+DAPI+), as well as 47 putative CTCs with non-conventional phenotype (CK-CD45-DAPI+) were isolated. Single-cell WGA products with high Genome-Integrity Index (QC score ≥3) were prioritized for CNA analysis. Ampli1 LowPass data demonstrated copy number gains/losses confirming tumor origin of the CK+ cells, while WBCs showed a normal profile. In addition, a portion of the cells having non-conventional phenotype also demonstrated copy number alterations consistent with tumor origin. Discussion: We demonstrate a WGA and low-pass WGS approach on single CTCs sorted from enriched peripheral blood, which offers a dual benefit: i) it allows rapid, non-exhaustive upfront identification of bona fide tumor cells for further study, and ii) it reveals genetic similarities and diversities (vis a vis copy number alteration) across CTCs of classical as well as non-conventional phenotypes, which may better represent clonal diversity. In a clinical setting, this molecular approach may be more effective for reliably identifying and characterizing heterogeneous CTCs, yielding profiles that more accurately reflect disease evolution and inform treatment strategies. Citation Format: Gareth Morrison, Valeria Sero, Yucheng Xu, Jacek Pinski, Sue Ingles, David Quinn, Claudio Forcato, Genny Buson, Chiu-Ho Webb, Kyle Horvath, Aditi Khurana, Gianni Medoro, Suman Verma, Matthew Moore, Philip Cotter, Nicolo Manaresi, Farideh Bischoff, Amir Goldkorn. Orthogonal identification of circulating tumor cells (CTCs) using single cell low pass whole-genome sequencing (WGS) and copy-number alteration (CNA) analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1717. doi:10.1158/1538-7445.AM2017-1717

Abstract 1546: Differential gene expression analysis by NGS of primary tumor xenograft cells, circulating tumor cells, and metastasis cells purified from the same mouse hosts to identify drivers of cancer dissemination

Introduction: Cancer dissemination involves ongoing dynamic transit of tumor cells between primary and metastatic sites via a hematogenous or “liquid phase”. To identify molecular mechanisms related to the liquid phase of cancer dissemination, we developed a tractable mouse xenograft model wherein GFP-labeled pure primary tumor (PT) cells, circulating tumor cells (CTCs) and lung metastasis (LM) cells are isolated and characterized from the same host. Methods: GFP+ MDA-MB-231 human breast cancer cells were inoculated subcutaneously into NOD/SCID/IL2r-γnull (NSG) mice. When a 1cm diameter primary tumor xenograft formed, 500 μl blood were collected by intracardiac puncture, and CTCs were enriched by wbc/rbc depletion (StemCell Tech). PT and LM samples were fluorescence microdissected from the same mouse hosts and disaggregated into single cell suspensions. Using a micromanipulator pipette, matched PT, CTC, and LM samples (100 pure GFP+ cells each) were isolated, lysed for RNA extraction (Ambion), and analyzed using the Ion AmpliSeq Transcriptome Human Gene Expression workflow for library preparation and pooled sequencing (Ion Proton). Sequence data from biologic triplicates was aligned and curated using the Genetrix bioinformatics suite, and candidate genes were further analyzed using Ingenuity Pathway Analysis (IPA). Results: The sequence panel provides gene-level expression information from over 20,000 genes covering > 95% of the RefSeq gene database. Initially, we focused on genes that were up- or down- regulated in CTCs compared with both PT and LM (i.e. differentially expressed in the liquid phase: 402 genes, 184 up-regulated in CTCs, 218 down-regulated in CTCs, fold change range 1.2x-22x). After further filtering for false discovery, 4 up-regulated and 39 down-regulated genes were selected for IPA analysis, which revealed associations with cellular movement pathways (16 genes) and cancer pathways (24 genes). Of these, the top-ranked 12 differentially expressed genes in CTCs relative to PT and LM (9 down-regulated and 3 up-regulated) were selected for validation by qPCR and further functional characterization in the mouse xenograft model. Conclusions: Here we leveraged new techniques for pure CTC capture, AmpliSeq transcriptome amplification and sequencing to identify and validate new candidate drivers of cancer dissemination. This model holds the potential to uncover key mechanisms engaged by cells that leave the primary and metastatic tumor niche and enter the cancer9s liquid phase. Further validation and functional analysis of these candidates in vitro, in vivo and in patient samples may identify new therapeutic targets that specifically disrupt cancer dissemination. Citation Format: Tong Xu, Gareth Morrison, Yucheng Xu, Timothy Triche, Jonathan Buckley, Amir Goldkorn. Differential gene expression analysis by NGS of primary tumor xenograft cells, circulating tumor cells, and metastasis cells purified from the same mouse hosts to identify drivers of cancer 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 1546.

Abstract 3160: Circulating tumor cell enrichment and dielectric manipulation for ultra-pure cell recovery in advanced bladder cancer

Background: Advanced transitional cell carcinoma (TCC) of the bladder is a lethal malignancy with few available biomarkers to guide therapy. Recent evidence suggests that circulating tumor cells (CTCs) are detectable in TCC and may have a prognostic role; however, most studies to date have focused on CTC enumeration. We leveraged novel enrichment and capture technologies to isolate and recover ultra-pure CTC (upCTC) that can be used for single cell molecular analysis. Methods: Patients with metastatic bladder cancer were enrolled under an institutional review board approved pilot study. Blood samples (7.5 ml each) were drawn into EDTA or Cell-Free DNA blood collection tubes (Streck), and preliminary CTC enrichment was performed using one of 3 microfluidic platforms: i) EpCAM-based LiquidBiopsy system (Cynvenio); ii) size/deformability-based ClearCell FX system (Clearbridge); or iii) size/deformability-based Parsortix system (Angle). Enriched CTCs were labeled with Cytokeratin (CK), EpCAM, and CD45 immunofluorescent antibodies and Hoechst nuclear stain. Identification and recovery of upCTCs was performed on a DEPArray v2 system (Silicon BioSystems). Additional experiments using cell lines were performed to gauge the recovery of mRNA from rare cancer cells collected in EDTA vs. preservative tubes. Results: To date, samples were collected from 8 patients of whom 5 (∼63%) had detectable CTCs. All 3 enrichment platforms successfully yielded CTC fractions from which Hoechst+/CK+/CD45- upCTCs were subsequently identified and recovered on the DEPArray v2 system. Additional cells that were Hoechst+/CK-/CD45- were identified and collected, and cells also were gated by integral Hoechst intensity and collected for correlation to aneuploidy. Comparison of mRNA recovery from 10 cancer cells collected into EDTA vs. fixative tubes (CellSave, Cynvenio, Streck Cell-free RNA) demonstrated that gene expression readings by qPCR from rare fixed cells was feasible but associated with variable results, likely due to variable proteinase K reversal of mRNA-protein cross-linkages. Conclusion: We leveraged new techniques to develop and optimize a workflow for identification and collection of upCTCs from bladder TCC patients, and we established that gene expression profiling is also feasible, albeit currently is still best achieved using EDTA collection and not any of the available preservative tubes. Single upCTC molecular profiles (DNA alterations, gene expression) from patient samples generated using these protocols may help to elucidate mechanisms of disease progression and ultimately may advance the management of advanced bladder cancer. Citation Format: Gareth Morrison, Cory Hugen, Tong Xu, Yucheng Xu, Dorff Tanya, David Quinn, Sarmad Sadeghi, Amir Goldkorn. Circulating tumor cell enrichment and dielectric manipulation for ultra-pure cell recovery in advanced bladder cancer. [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 3160.

MP37-19 AXIN2 EXPRESSION PREDICTS PROSTATE CANCER RECURRENCE AND MEDIATES AN INVASIVE, TUMORIGENIC PHENOTYPE

INTRODUCTION AND OBJECTIVES: Better biomarkers are needed in prostate cancer (PCa) to predict disease recurrence and guide optimal therapy. We investigated whether genes associated with a highly tumorigenic, drug resistant, progenitor phenotype impact PCa biology and clinical outcomes in localized disease. METHODS: Radical prostatectomy (RP) specimens (þ/disease recurrence) were analyzed by qRT-PCR to quantify expression of genes associated with self-renewal, drug resistance, and tumorigenicity in prior studies. Univariable and multivariable associations between gene expression and PCa recurrence were confirmed by bootstrap internal validation and by external validation in independent cohorts and in-silico. siRNA knockdown and lentiviral overexpression were used to determine the effect of gene expression on PCa proliferation, invasion and tumor growth. RESULTS: Four candidate genes were differentially expressed in PCa recurrence, and of these, low AXIN2 expression was internally validated. In external cohorts and in silico, low AXIN2 was independently associated with more aggressive PCa, biochemical recurrence, and metastasis-free survival after RP. In vitro and in vivo, low AXIN2 expression was associated with a cancer stem-like cell-surface signature, and siRNA knockdown of AXIN2 resulted in significantly greater invasiveness. Conversely, ectopic overexpression of AXIN2 significantly reduced cell proliferation and tumor growth in mice (Figure). CONCLUSIONS: Low AXIN2 expression was associated with PCa recurrence after RP in our test population as well as in external validation cohorts, and its expression levels in PCa cells significantly impacted invasiveness, proliferation and tumor growth. Based on these novel roles in PCa, AXIN2 may represent a new putative biomarker and potential therapeutic target in this disease.