Hanli Fan

Network modeling identifies molecular functions targeted by miR-204 to suppress head and neck tumor metastasis

Due to the large number of putative microRNA gene targets predicted by sequence-alignment databases and the relative low accuracy of such predictions which are conducted independently of biological context by design, systematic experimental identification and validation of every functional microRNA target is currently challenging. Consequently, biological studies have yet to identify, on a genome scale, key regulatory networks perturbed by altered microRNA functions in the context of cancer. In this report, we demonstrate for the first time how phenotypic knowledge of inheritable cancer traits and of risk factor loci can be utilized jointly with gene expression analysis to efficiently prioritize deregulated microRNAs for biological characterization. Using this approach we characterize miR-204 as a tumor suppressor microRNA and uncover previously unknown connections between microRNA regulation, network topology, and expression dynamics. Specifically, we validate 18 gene targets of miR-204 that show elevated mRNA expression and are enriched in biological processes associated with tumor progression in squamous cell carcinoma of the head and neck (HNSCC). We further demonstrate the enrichment of bottleneckness, a key molecular network topology, among miR-204 gene targets. Restoration of miR-204 function in HNSCC cell lines inhibits the expression of its functionally related gene targets, leads to the reduced adhesion, migration and invasion in vitro and attenuates experimental lung metastasis in vivo. As importantly, our investigation also provides experimental evidence linking the function of microRNAs that are located in the cancer-associated genomic regions (CAGRs) to the observed predisposition to human cancers. Specifically, we show miR-204 may serve as a tumor suppressor gene at the 9q21.1–22.3 CAGR locus, a well established risk factor locus in head and neck cancers for which tumor suppressor genes have not been identified. This new strategy that integrates expression profiling, genetics and novel computational biology approaches provides for improved efficiency in characterization and modeling of microRNA functions in cancer as compared to the state of art and is applicable to the investigation of microRNA functions in other biological processes and diseases.

MicroRNA Expression Characterizes Oligometastasis(es)

Background Cancer staging and treatment presumes a division into localized or metastatic disease. We proposed an intermediate state defined by ≤5 cumulative metastasis(es), termed oligometastases. In contrast to widespread polymetastases, oligometastatic patients may benefit from metastasis-directed local treatments. However, many patients who initially present with oligometastases progress to polymetastases. Predictors of progression could improve patient selection for metastasis-directed therapy. Methods Here, we identified patterns of microRNA expression of tumor samples from oligometastatic patients treated with high-dose radiotherapy. Results Patients who failed to develop polymetastases are characterized by unique prioritized features of a microRNA classifier that includes the microRNA-200 family. We created an oligometastatic-polymetastatic xenograft model in which the patient-derived microRNAs discriminated between the two metastatic outcomes. MicroRNA-200c enhancement in an oligometastatic cell line resulted in polymetastatic progression. Conclusions These results demonstrate a biological basis for oligometastases and a potential for using microRNA expression to identify patients most likely to remain oligometastatic after metastasis-directed treatment.

Oligo- and Polymetastatic Progression in Lung Metastasis(es) Patients Is Associated with Specific MicroRNAs

Rationale Strategies to stage and treat cancer rely on a presumption of either localized or widespread metastatic disease. An intermediate state of metastasis termed oligometastasis(es) characterized by limited progression has been proposed. Oligometastases are amenable to treatment by surgical resection or radiotherapy. Methods We analyzed microRNA expression patterns from lung metastasis samples of patients with ≤5 initial metastases resected with curative intent. Results Patients were stratified into subgroups based on their rate of metastatic progression. We prioritized microRNAs between patients with the highest and lowest rates of recurrence. We designated these as high rate of progression (HRP) and low rate of progression (LRP); the latter group included patients with no recurrences. The prioritized microRNAs distinguished HRP from LRP and were associated with rate of metastatic progression and survival in an independent validation dataset. Conclusion Oligo- and poly- metastasis are distinct entities at the clinical and molecular level.

Human Breast Cancer Cell Lines Co-Express Neuronal, Epithelial, and Melanocytic Differentiation Markers In Vitro and In Vivo

Differentiation programs are aberrant in cancer cells allowing them to express differentiation markers in addition to their tissue of origin. In the present study, we demonstrate the multi-lineage differentiation potential of breast cancer cell lines to express multiple neuronal/glial lineage-specific markers as well as mammary epithelial and melanocytic-specific markers. Multilineage expression was detected in luminal (MCF-7 and SKBR3) and basal (MDA-MB-231) types of human breast cancer cell lines. We also observed comparable co-expression of these three cell lineage markers in MDA-MB-435 cells in vitro, in MDA-MB-435 primary tumors derived from parental and single cell clones and in lung metastases in vivo. Furthermore, ectoderm multi-lineage transdifferentiation was also found in human melanoma (Ul-MeL) and glioblastoma cell lines (U87 and D54). These observations indicate that aberrant multi-lineage transdifferentiation or lineage infidelity may be a wide spread phenomenon in cancer.

Time-Course Imaging of Therapeutic Functional Tumor Vascular Normalization by Antiangiogenic Agents

We describe here new technology that enables noninvasive imaging of therapeutic functional normalization of tumor blood vessels by antiangiogenic agents. Noninvasive variable-magnification in vivo-fluorescence imaging as well as fluorescence tomography was used to visualize functional vessel normalization. Changes in the same vessel before and after drug treatment were imaged with high resolution in real time. Differences in vascular responses to the mTOR inhibitor rapamycin and to an anti-VEGF antibody were functionally imaged. Tumor vessel normalization was shown by significantly reduced leakiness and subsequent improved tumor delivery of Paclitaxel-BODPY as well as by normalized morphology. The tumor vascular pool agent, AngioSense750, was retained only in tumors after either anti-VEGF antibody or rapamycin treatment, as visualized by noninvasive fluorescence tomography. The antiangiogenic therapy normalized vessels, which significantly enhanced the antitumor efficacy of paclitaxel because of increased drug penetration throughout the tumor. The optical imaging technology described here is thus a powerful, noninvasive, time-course imaging tool of functional tumor vessel normalization and its therapeutic consequences. Mol Cancer Ther; 10(7); 1173–84. ©2011 AACR.

Abstract 3405: MicroRNA expression characterizes oligometastasis(es)

Background: Cancer staging and treatment presumes a division into localized or metastatic disease. We proposed an intermediate state defined by β5 cumulative metastasis(es), termed oligometastases. In contrast to widespread polymetastases, oligometastatic patients may benefit from metastasis-directed local treatments. However, many patients who initially present with oligometastases progress to polymetastases. Predictors of progression could improve patient selection for metastasis-directed therapy. Methods: Here, we identified patterns of microRNA expression of tumor samples from oligometastatic patients treated with high-dose radiotherapy. Results: Patients who failed to develop polymetastases are characterized by unique prioritized features of a microRNA classifier that includes the microRNA-200 family. We created an oligometastatic-polymetastatic xenograft model in which the patient-derived microRNAs discriminated between the two metastatic outcomes. MicroRNA-200c enhancement in an oligometastatic cell line resulted in polymetastatic progression. Conclusions: These results demonstrate a biological basis for oligometastases and a potential for using microRNA expression to identify patients most likely to remain oligometastatic after metastasis-directed treatment. 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 3405. doi:1538-7445.AM2012-3405

Abstract 1369: Mouse models of clinical oligo- and poly-metastatic progression

We previously proposed a metastatic state defined by a limited number of metastases, termed oligometastasis(es). These limited metastases may be cured by metastasis-directed treatments in contrast to widespread metastatic disease. While many animal models of polymetastases exist, an oligometastasis(es) model is lacking. Here, we report the first mouse xenograft model of oligometastasis(es) employing the MDA-MB-435 human tumor that maintains a stable oligometastatic phenotype in vivo. We also developed an MDA-MB-435 polymetastatic progression model in which the pattern of dissemination induced poly-foci in the lung, or to multiple anatomic sites including lung, heart, muscle, pleura, bone and peritoneal cavity. We performed microRNA expression profiling from distinct lung metastases of oligometastatic and polymetastatic animals. MicroRNA expression distinguished oligometastatic cell lines from those of polymetastatic, and accurately identified oligometastatic patients from a prior clinical study who failed to develop widespread metastases (p=0.005). These findings form the basis for investigating mechanisms underlying the specific metastatic pattern of individual cancers. 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 1369. doi:1538-7445.AM2012-1369

Abstract 1546: Effective co-targeting of tumor and tumor stroma for head and neck cancer treatment: making the right combination

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Successful cancer management has been hindered by the development of inherent or acquired resistance. Increasing evidence suggest that manifestation of comparable resistance phenotype may arise from different combinations of molecular lesions. Signal transduction pathways in cancer cells exhibit remarkable flexibility for re-wiring such that cancer cells of solid tumors are able of altering the targeting receptor tyrosine kinases (RTKs) such as EGFR, or using unaffected and co-expressed RTKs signaling, or manifesting gain-of-function changes of the downstream signaling molecules to sustain tumor growth and progression. However, The potential of a “pathway rewiring” strategy to more efficiently target a deregulated signaling network has not been effectively explored due to the lack of scientific framework. Furthermore, the effort of developing effective agents or combinations that can effectively co-target both cancer cells and tumor stroma is lagging behind the therapeutic development aiming at cancer cell targeting. In this report, we demonstrate the effectiveness of integrating predicative pathway analysis to identify mTOR as a key interacting signaling target for developing a rational “pathway-based” combination to overcome resistance to anti-EGFR therapies in squamous carcinoma of the head and neck (HNC). We show for the first time that HNC cancer cells can effectively exploit the molecular interactions between EGFR and mTOR to confer reciprocal resistance. In addition, network modeling and comparison of xenograft with human tumor specimens identifies the abundant presence of myofibroblasts specifically associated with cancer state. We subsequently show the dependence of the clonogenic survival of myofibroblast cells on mTOR signaling in vitro. In vivo, only concomitant inhibition of EGFR and mTOR signaling via erlotinib and rapamycin, respectively, achieves effective differential and co-targeting of cancer cells and myofibroblast and endothelial cell functions in tumor stroma, leading to tumor regression. Thus, we show a systems’ approach-guided biological investigation has the potential to identify resistance mechanisms that can be explored to design more effective combinations of targeted therapies capable of overcoming resistance via “pathway rewiring” and achieving tumor-tumor stroma co-targeting. Based on these observations, an NIH funded network Phase II human clinical trial, lead by the University of Chicago Cancer Center, is soon underway to evaluate the therapeutic efficacy of this combination therapy. 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 1546.