Christine L. Chaffer

Mesenchymal-to-Epithelial Transition Facilitates Bladder Cancer Metastasis: Role of Fibroblast Growth Factor Receptor-2

Epithelial-to-mesenchymal transition (EMT) increases cell migration and invasion, and facilitates metastasis in multiple carcinoma types, but belies epithelial similarities between primary and secondary tumors. This study addresses the importance of mesenchymal-to-epithelial transition (MET) in the formation of clinically significant metastasis. The previously described bladder carcinoma TSU-Pr1 (T24) progression series of cell lines selected in vivo for increasing metastatic ability following systemic seeding was used in this study. It was found that the more metastatic sublines had acquired epithelial characteristics. Epithelial and mesenchymal phenotypes were confirmed in the TSU-Pr1 series by cytoskeletal and morphologic analysis, and by performance in a panel of in vitro assays. Metastatic ability was examined following inoculation at various sites. Epithelial characteristics associated with dramatically increased bone and soft tissue colonization after intracardiac or intratibial injection. In contrast, the more epithelial sublines showed decreased lung metastases following orthotopic inoculation, supporting the concept that EMT is important for the escape of tumor cells from the primary tumor. We confirmed the overexpression of the IIIc subtype of multiple fibroblast growth factor receptors (FGFR) through the TSU-Pr1 series, and targeted abrogation of FGFR2IIIc reversed the MET and associated functionality in this system and increased survival following in vivo inoculation in severe combined immunodeficient mice. This model is the first to specifically model steps of the latter part of the metastatic cascade in isogenic cell lines, and confirms the suspected role of MET in secondary tumor growth.

Mesenchymal to Epithelial Transition in Development and Disease

Cellular plasticity is fundamental to embryonic development. The importance of cellular transitions in development is first apparent during gastrulation when the process of epithelial to mesenchymal transition transforms polarized epithelial cells into migratory mesenchymal cells that constitute the embryonic and extraembryonic mesoderm. It is now widely accepted that this developmental pathway is exploited in various disease states, including cancer progression. The loss of epithelial characteristics and the acquisition of a mesenchymal-like migratory phenotype are crucial to the development of invasive carcinoma and metastasis. However, given the morphological similarities between primary tumour and metastatic lesions, it is likely that tumour cells re-activate certain epithelial properties through a mesenchymal to epithelial transition (MET) at the secondary site, although this is yet to be proven. MET is also an essential developmental process and has been extensively studied in kidney organogenesis and somitogenesis. In this review we describe the process of MET, highlight important mediators, and discuss their implication in the context of cancer progression.

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.

The Feeding Response to Melanin-Concentrating Hormone Is Attenuated by Antagonism of the NPY Y1-Receptor in the Rat

Melanin-concentrating hormone (MCH) and NPY are orexigenic peptides localized in the lateral hypothalamic area and arcuate nucleus, respectively. Although both NPY- and MCH-containing fibers innervate areas of the hypothalamus implicated in feeding, the extent to which the regulation of appetite is dependent on interactions between these peptides is unknown. Daytime feeding responses to 2 nmol MCH, 1 nmol NPY, or vehicle were investigated in male Sprague Dawley rats previously implanted with intracerebroventricular cannulas. The effects of prior administration of the Y1-receptor antagonists BIBO 3304 (20 nmol) or GR231118 (5 nmol) on these responses were examined. NPY and MCH stimulated food intake relative to vehicle (4 h intake, 5.9 ± 0.7 and 3.6 ± 0.2 g, respectively; P < 0.0001). BIBO 3304 and GR231118 significantly inhibited MCH- induced feeding by 73% (P < 0.01) and 86% (P < 0.01), respectively, at 2 h. Coadministration of NPY and MCH did not increase food intake above that in response to NPY alone;...

Disparate Companions: Tissue Engineering Meets Cancer Research

Recreating an environment that supports and promotes fundamental homeostatic mechanisms is a significant challenge in tissue engineering. Optimizing cell survival, proliferation, differentiation, apoptosis and angiogenesis, and providing suitable stromal support and signalling cues are keys to successfully generating clinically useful tissues. Interestingly, those components are often subverted in the cancer setting, where aberrant angiogenesis, cellular proliferation, cell signalling and resistance to apoptosis drive malignant growth. In contrast to tissue engineering, identifying and inhibiting those pathways is a major challenge in cancer research. The recent discovery of adult tissue-specific stem cells has had a major impact on both tissue engineering and cancer research. The unique properties of these cells and their role in tissue and organ repair and regeneration hold great potential for engineering tissue-specific constructs. The emerging body of evidence implicating stem cells and progenitor cells as the source of oncogenic transformation prompts caution when using these cells for tissue-engineering purposes. While tissue engineering and cancer research may be considered as opposed fields of research with regard to their proclaimed goals, the compelling overlap in fundamental pathways underlying these processes suggests that cross-disciplinary research will benefit both fields. In this review article, tissue engineering and cancer research are brought together and explored with regard to discoveries that may be of mutual benefit.

Abstract 5020: A genome-scale ORF screen reveals an alternative splicing program that regulates mesenchymal and stem-like cell states in breast cancer

Cells residing in mesenchymal state are often associated with stem cell properties. The phenotypic changes from epithelial to mesenchymal cell state, or from non-stem-like to stem-like cell state contribute to tumor heterogeneity and play important roles in tumor initiation, progression and metastasis. To systematically interrogate the modulators of epithelial-to-mesenchymal transition, we performed a genome-scale ORF screen to identify regulators of mesenchymal and stem-like cell states using a barcoded human ORFome expression library in human mammary epithelial cells. In the screen, we used flow cytometric analysis of the CD44 cell surface marker and identified 68 ORFs that can switch cells from CD44 low state to CD44 high state in 7 days. Among these genes, the RNA splicing factors were highly enriched as analyzed by GO terms and Gene set enrichment analysis (GSEA).We employed six different assays for candidate validation: 1) Induction of CD44 cell surface markers; 2) Evaluation of the expression of EMT markers; 3) Test of the ability to form mammospheres; 4) Investigation of the expression during EMT induction; 5) Test of the necessity of these splicing factors for EMT and stem-like states; 6) Examination of the ability to promote tumor formation in vivo. We discovered that QKI and RBFOX1 were both necessary and sufficient to promote EMT and stem-like states. MBNL1, MBNL2 and CELF4 were sufficient to induce some mesenchymal markers. We further investigated the downstream targets of these splicing factors by RNA-sequencing analysis. We found that QKI and RBFOX1 regulated the alternative splicing of genes in 5 functional modules: 1) Cell motility and ECM/cytoskeleton organization; 2) Stem cell fate determination; 3) Oncogenic signaling; 4) Epigenetic targets; 5) cell polarity. Strikingly, using molecular and biochemical assays, we found that QKI and RBFOX1/2 interacted and cooperatively regulated the alternative splicing of a large number of overlapping transcripts, including Filamin B (FLNB). QKI and RBFOX1 induced a shorter isoform via exon skipping. which plays a key functional role in the regulation of EMT. Importantly, the expression of QKI, RBFOX1/2 and the short isoform of FLNB are elevated in basal B type of breast cancer cell lines and in basal-like breast cancer patient samples, the subtype of breast cancer that displays higher degree of mesenchymal and stem-like traits. In conclusion, alternative RNA splicing plays a key role in the regulation of EMT and stem-like cell states. QKI and RBFOX1/2 are both necessary and sufficient to promote EMT and stem-like traits. Alternative splicing of FLNB controlled by QKI and RBFOX1/2 is one of the key downstream targets that regulates EMT. Thus, the molecular targets and mechanism identified in this study may aid in the development of new diagnostic and therapeutic approaches for breast tumors, especially for basal-like breast cancer. Citation Format: Ji Li, Peter Choi, Christine Chaffer, Katherine Labella, Jong Wook Kim, John Doench, Chao Dai, Andrew Giacomelli, Seav Huong Ly, Justin Hwang, Andrew Hong, Nina Ilic, Ole Gjoerup, Matthew Meyerson, Angela Brooks, Robert Weinberg, William Hahn. A genome-scale ORF screen reveals an alternative splicing program that regulates mesenchymal and stem-like cell states in breast cancer [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 5020. doi:10.1158/1538-7445.AM2017-5020