Julie B. Sneddon

Gene Expression Signature of Fibroblast Serum Response Predicts Human Cancer Progression: Similarities between Tumors and Wounds

Cancer invasion and metastasis have been likened to wound healing gone awry. Despite parallels in cellular behavior between cancer progression and wound healing, the molecular relationships between these two processes and their prognostic implications are unclear. In this study, based on gene expression profiles of fibroblasts from ten anatomic sites, we identify a stereotyped gene expression program in response to serum exposure that appears to reflect the multifaceted role of fibroblasts in wound healing. The genes comprising this fibroblast common serum response are coordinately regulated in many human tumors, allowing us to identify tumors with gene expression signatures suggestive of active wounds. Genes induced in the fibroblast serum-response program are expressed in tumors by the tumor cells themselves, by tumor-associated fibroblasts, or both. The molecular features that define this wound-like phenotype are evident at an early clinical stage, persist during treatment, and predict increased risk of metastasis and death in breast, lung, and gastric carcinomas. Thus, the transcriptional signature of the response of fibroblasts to serum provides a possible link between cancer progression and wound healing, as well as a powerful predictor of the clinical course in several common carcinomas.

Bone morphogenetic protein antagonist gremlin 1 is widely expressed by cancer-associated stromal cells and can promote tumor cell proliferation

Although tissue microenvironments play critical roles in epithelial development and tumorigenesis, the factors mediating these effects are poorly understood. In this work, we used a genomic approach to identify factors produced by cells in the microenvironment of basal cell carcinoma (BCC) of the skin, one of the most common human cancers. The global gene expression programs of stromal cell cultures derived from human BCCs showed consistent, systematic differences from those derived from nontumor skin. The gene most consistently expressed at a higher level in BCC tumor stromal cells compared with those from nontumor skin was GREMLIN 1, which encodes a secreted antagonist of the bone morphogenetic protein (BMP) pathway. BMPs and their antagonists are known to play a crucial role in stem and progenitor cell biology as regulators of the balance between expansion and differentiation. Consistent with the hypothesis that BMP antagonists might have a similar role in cancer, we found GREMLIN 1 expression in the stroma of human BCC tumors but not in normal skin in vivo. Furthermore, BMP 2 and 4 are expressed by BCC cells. Ex vivo, BMP inhibits, and Gremlin 1 promotes, proliferation of cultured BCC cells. We further found that GREMLIN 1 is expressed by stromal cells in many carcinomas but not in the corresponding normal tissue counterparts that we examined. Our data suggest that BMP antagonists may be important constituents of tumor stroma, providing a favorable microenvironment for cancer cell survival and expansion in many cancers.

Predicting a local recurrence after breast-conserving therapy by gene expression profiling

IntroductionTo tailor local treatment in breast cancer patients there is a need for predicting ipsilateral recurrences after breast-conserving therapy. After adequate treatment (excision with free margins and radiotherapy), young age and incompletely excised extensive intraductal component are predictors for local recurrence, but many local recurrences can still not be predicted. Here we have used gene expression profiling by microarray analysis to identify gene expression profiles that can help to predict local recurrence in individual patients.MethodsBy using previously established gene expression profiles with proven value in predicting metastasis-free and overall survival (wound-response signature, 70-gene prognosis profile and hypoxia-induced profile) and training towards an optimal prediction of local recurrences in a training series, we establish a classifier for local recurrence after breast-conserving therapy.ResultsValidation of the different gene lists shows that the wound-response signature is able to separate patients with a high (29%) or low (5%) risk of a local recurrence at 10 years (sensitivity 87.5%, specificity 75%). In multivariable analysis the classifier is an independent predictor for local recurrence.ConclusionOur findings indicate that gene expression profiling can identify subgroups of patients at increased risk of developing a local recurrence after breast-conserving therapy.

The macrophage-stimulating protein pathway promotes metastasis in a mouse model for breast cancer and predicts poor prognosis in humans

A better understanding of tumor metastasis requires development of animal models that authentically reproduce the metastatic process. By modifying an existing mouse model of breast cancer, we discovered that macrophage-stimulating protein promoted breast tumor growth and metastasis to several organs. A special feature of our findings was the occurrence of osteolytic bone metastases, which are prominent in human breast cancer. To explore the clinical relevance of our model, we examined expression levels of three genes involved in activation of the MSP signaling pathway (MSP, MT-SP1, and MST1R) in human breast tumors. We found that overexpression of MSP, MT-SP1, and MST1R was a strong independent indicator of both metastasis and death in human breast cancer patients and significantly increased the accuracy of an existing gene expression signature for poor prognosis. These data suggest that signaling initiated by MSP is an important contributor to metastasis of breast cancer and introduce an independent biomarker for assessing the prognosis of humans with breast cancer.

Self-renewal of embryonic-stem-cell-derived progenitors by organ-matched mesenchyme

One goal of regenerative medicine, to use stem cells to replace cells lost by injury or disease, depends on producing an excess of the relevant cell for study or transplantation. To this end, the stepwise differentiation of stem cells into specialized derivatives has been successful for some cell types, but a major problem remains the inefficient conversion of cells from one stage of differentiation to the next. If specialized cells are to be produced in large numbers it will be necessary to expand progenitor cells, without differentiation, at some steps of the process. Using the pancreatic lineage as a model for embryonic-stem-cell differentiation, we demonstrate that this is a solvable problem. Co-culture with organ-matched mesenchyme permits proliferation and self-renewal of progenitors, without differentiation, and enables an expansion of more than a million-fold for human endodermal cells with full retention of their developmental potential. This effect is specific both to the mesenchymal cell and to the progenitor being amplified. Progenitors that have been serially expanded on mesenchyme give rise to glucose-sensing, insulin-secreting cells when transplanted in vivo. Theoretically, the identification of stage-specific renewal signals can be incorporated into any scheme for the efficient production of large numbers of differentiated cells from stem cells and may therefore have wide application in regenerative biology.

The contribution of niche-derived factors to the regulation of cancer cells.

In normal adult tissues, paracrine signals that derive from the stem cell niche, or microenvironment, play an important role in regulating the critical balance between activity and quiescence of stem cells. Similarly, evidence has emerged to support the hypothesis that signals derived from the microenvironment regulate cancer cells in an analogous manner. We recently reported that in basal cell carcinoma of the skin and in diverse other solid tumors, fibroblasts that comprise the tumor cell niche are, indeed, molecularly distinct from those that comprise the normal stroma. In particular, we found evidence suggesting that expression of secreted BMP antagonists by tumor-associated stromal cells may promote self-renewal of tumor stem cells in vivo. This chapter describes methods for identifying and evaluating the molecular signals that derive from fibroblasts in human tumors.

Brief Report: VGLL4 Is a Novel Regulator of Survival in Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) are maintained in a self‐renewing state by an interconnected network of mechanisms that sustain pluripotency, promote proliferation and survival, and prevent differentiation. We sought to find novel genes that could contribute to one or more of these processes using a gain‐of‐function screen of a large collection of human open reading frames. We identified Vestigial‐like 4 (VGLL4), a cotranscriptional regulator with no previously described function in hESCs, as a positive regulator of survival in hESCs. Specifically, VGLL4 overexpression in hESCs significantly decreases cell death in response to dissociation stress. Additionally, VGLL4 overexpression enhances hESC colony formation from single cells. These effects may be attributable, in part, to a decreased activity of initiator and effector caspases observed in the context of VGLL4 overexpression. Additionally, we show an interaction between VGLL4 and the Rho/Rock pathway, previously implicated in hESC survival. This study introduces a novel gain‐of‐function approach for studying hESC maintenance and presents VGLL4 as a previously undescribed regulator of this process. Stem Cells 2013;31:2833–2841

Defining epithelial cell dynamics and lineage relationships in the developing lacrimal gland

The tear-producing lacrimal gland is a tubular organ that protects and lubricates the ocular surface. The lacrimal gland possesses many features that make it an excellent model in which to investigate tubulogenesis, but the cell types and lineage relationships that drive lacrimal gland formation are unclear. Using single-cell sequencing and other molecular tools, we reveal novel cell identities and epithelial lineage dynamics that underlie lacrimal gland development. We show that the lacrimal gland from its earliest developmental stages is composed of multiple subpopulations of immune, epithelial and mesenchymal cell lineages. The epithelial lineage exhibits the most substantial cellular changes, transitioning through a series of unique transcriptional states to become terminally differentiated acinar, ductal and myoepithelial cells. Furthermore, lineage tracing in postnatal and adult glands provides the first direct evidence of unipotent KRT5+ epithelial cells in the lacrimal gland. Finally, we show conservation of developmental markers between the developing mouse and human lacrimal gland, supporting the use of mice to understand human development. Together, our data reveal crucial features of lacrimal gland development that have broad implications for understanding epithelial organogenesis. Summary: Single-cell sequencing combined with a battery of molecular tools reveals novel features of cellular dynamics and differentiation during lacrimal gland development in mice.

Stem Cell Therapies for Treating Diabetes: Progress and Remaining Challenges

Restoration of insulin independence and normoglycemia has been the overarching goal in diabetes research and therapy. While whole-organ and islet transplantation have become gold-standard procedures in achieving glucose control in diabetic patients, the profound lack of suitable donor tissues severely hampers the broad application of these therapies. Here, we describe current efforts aimed at generating a sustainable source of functional human stem cell-derived insulin-producing islet cells for cell transplantation and present state-of-the-art efforts to protect such cells via immune modulation and encapsulation strategies.

VGLL4 is a Novel Regulator of Survival in Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) are maintained in a self‐renewing state by an interconnected network of mechanisms that sustain pluripotency, promote proliferation and survival, and prevent differentiation. We sought to find novel genes that could contribute to one or more of these processes using a gain‐of‐function screen of a large collection of human open reading frames. We identified Vestigial‐like 4 (VGLL4), a cotranscriptional regulator with no previously described function in hESCs, as a positive regulator of survival in hESCs. Specifically, VGLL4 overexpression in hESCs significantly decreases cell death in response to dissociation stress. Additionally, VGLL4 overexpression enhances hESC colony formation from single cells. These effects may be attributable, in part, to a decreased activity of initiator and effector caspases observed in the context of VGLL4 overexpression. Additionally, we show an interaction between VGLL4 and the Rho/Rock pathway, previously implicated in hESC survival. This study introduces a novel gain‐of‐function approach for studying hESC maintenance and presents VGLL4 as a previously undescribed regulator of this process. Stem Cells 2013;31:2833–2841