Loss of human Scribble cooperates with H-Ras to promote cell invasion through deregulation of MAPK signalling

Abstract

Activating mutations in genes of the Ras-mitogenactivated protein kinase (MAPK) pathway occur in approximately 30% of all human cancers; however, mutation of Ras alone is rarely sufficient to induce tumour development. Scribble is a polarity regulator recently isolated from a Drosophila screen for events that cooperate with Ras mutation to promote tumour progression and cell invasion. In mammals, Scribble regulates directed cell migration and wound healing in vivo; however, no role has been identified for mammalian Scribble in oncogenic transformation. Here we show that in human epithelial cells expressing oncogenic Ras or Raf, loss of Scribble promotes invasion of cells through extracellular matrix in an organotypic culture system. Further, we show that the mechanism by which this occurs is in the regulation of MAPK signalling by Scribble. The suppression of MAPK signalling is a highly conserved function of Scribble as it also prevents Raf-mediated defects in Drosophila wing development. Our data identify Scribble as an important mediator of MAPK signalling and provide a molecular basis for the observation that Scribble expression is decreased in many invasive human cancers.

Identification of Novel Ras-Cooperating Oncogenes in Drosophila melanogaster: A RhoGEF/Rho-Family/JNK Pathway Is a Central Driver of Tumorigenesis

“…Matrigel invasion assay for mammalian MCF10A cells: Parental MCF10A cell lines were retrovirally co-infected with JNK1a1, MKK4, and MKK7 overexpression constructs and H-Ras V12cherry selected with puromycin, sorted for GFP/cherry on a FACSVantage SE-DiVa flow cytometer (Becton Dickinson, Franklin Lakes, NJ), and maintained as previously described (Dow et al 2008). MCF10A derivative cell lines stably expressing candidate genes were quantified for invasive phenotypes in 3D organotypic cultures as previously described (Dow et al 2008) using growth factor-reduced Matrigel (BD Biosciences, Franklin Lakes, NJ) and the standard overlay method (Debnath et al 2003;Dow et al 2008).…”

“…MCF10A derivative cell lines stably expressing candidate genes were quantified for invasive phenotypes in 3D organotypic cultures as previously described (Dow et al 2008) using growth factor-reduced Matrigel (BD Biosciences, Franklin Lakes, NJ) and the standard overlay method (Debnath et al 2003;Dow et al 2008). After 7 days in culture, individual acini were classified as "normal" acini, defined as those with a contiguous acini boundary with no cellular extensions, or "invasive," defined as acini with disorganized boundary structures showing cellular protrusions or cellular spikes invading into the surrounding matrix.…”

“…To explore the cooperation of JNK with activated Ras, we utilized MCF10A normal breast epithelial cells grown in 3D matrigel cultures. MCF10A cells form acini in matrigel; however, upon low-level expression of activated Harvey-Ras (HaRas V12 ) the lumens become filled with cells and with the concomitant knockdown of cell polarity regulators, such as hScrib, cells form invasive clusters (Dow et al 2008)-thus this system is a useful model with which to examine cooperative tumorigenesis.…”

Aberrant Overexpression of the Cell Polarity Module Scribble in Human Cancer

“…2 Experimental evidence, first collected in Drosophila melanogaster 3 and later extended to mammalian cells, 4 implicates Scrib as a general regulator of directional cell motility, largely via the assembly of multiprotein complexes and the activation of small GTPase signaling at the leading edge of migrating cells. 5,6 Accordingly, loss-of-function Scrib mutants 7 or depletion of Scrib 4 has disrupted apical-basal polarity 8 and has cooperated with oncogenic signals 9 to enhance tumor cell migration, invasion, and survival. 10 Although these data have prompted a model that Scrib may function in an evolutionary-conserved pathway of tumor suppression, 1,3 its role in human cancer, in vivo, has remained vehemently debated.…”

Computational investigation of intrinsic and extrinsic mechanisms underlying the formation of carcinoma

“…3(D) four distinct experimental morphologies to highlight that the computationally developed architectures have their experimental equivalent. These include the non-tumorigenic MCF10A (Debnath et al, 2003) cells that form normal hollow acini; MCF10A-Ras-Scrible (Dow et al, 2008) mutants producing non-stabilized spheroids without detectable lumen; MCF10A-Mek2-DD (Reginato et al, 2005) mutant forming multiacinar structures of irregular shapes and RWPE-1/rA1 (Inokuchi et al, 2009) mutant producing acini with partially filled lumen. However, without detailed time lapse of the development of these structures, we cannot claim that the computational acini are direct equivalents as we have no means of quantifying the computationally determined thresholds (at least from a single static image).…”