Regulation of Human Cripto-1 Expression

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Human Cripto-1 (CR-1) is an oncofetal protein. It is a modulator of embryogenesis and oncogenesis. It is involved in multiple signaling pathways, which are essential for cellular transformation, metastasis and angiogenesis. It is expressed in embryonic cells but is absent in adult tissues. However, CR-1 is overexpressed in several types of cancers. Though extensive work has been done to understand the role of CR-1 in oncogenesis and embryonic development, there is limited information on the control of expression of CR-1. In the present work, we have focused on transcriptional control and cellular heterogeneity in expression of CR-1. Based on the existing literature, we hypothesized that CR-1 may control its own expression through an autoregulatory pathway involving Nodal/ALk4/SMAD2/3. We have used human glioblastoma cell line, U-87 MG as a model cellular system for our studies. Expression of CR-1 is low in this cell line. We show that treatment with exogenous recombinant CR-1 induces expression of endogenous CR-1 both at mRNA level and at protein level in U-87 MG cells. Experiments were performed to investigate the dose-dependent and time-dependent behavior of such induction. We have confirmed that such an increase in CR-1 is not due to any change of mRNA stability and does not involve expression of Cripto-3, a pseudogene. Using inhibitor-based assays, we show that CR-1 mediated induction of CR-1 indeed involves Alk4/SMAD2 pathway. During embryonic development, CR-1 is involved in pattern formation. Embryonic pattern formation requires heterogeneous gene expression, with some cells expressing a pattern forming gene at very high level and some having very low expression of the same. Transcriptional networks involving positive feedback often give rise to such cellular heterogeneity. We looked into the single cell expression of CR-1 using flow cytometry. We observed that induction of CR-1 expression by recombinant CR-1 leads to a heterogeneous population, with only a minority subpopulation expressing CR-1. Size of the minority population and level of CR-1 expression in these cells, increases with increase in inducing signal. For further exploration of this heterogeneity, we have analyzed the noise in expression of CR-1 in this heterogeneous population. Heterogeneity in gene expression in tumor cells is believed to limit chemotherapy and associated with emergence of drug resistance. We have investigated the expression of multi drug resistance protein 1 (MDR1). We observed that MDR1 is expressed only in a minority population of cells.
Supervisor: Biplab Bose