Elevated levels of ornithine decarboxylase cooperate with Raf/ERK activation to convert normal keratinocytes into invasive malignant cells

Abstract

Ornithine decarboxylase (ODC) overexpression coupled with activated Ras is fully sufficient to oncogenically transform primary keratinocytes. To determine the Ras effector pathways that represent the minimal essential contribution to full oncogenic transformation in this context, we evaluated the cooperativity of different Ras effector mutants with overexpressed ODC in an in vivo tracheal xenotransplantation assay for epithelial cell invasiveness. Primary keratinocytes, isolated from either K6/ODC transgenic mouse skin (expressing increased ODC) or from normal littermate skin were infected with retrovirus producing an activated RasV12 or partial loss-of-function effector mutants of RasV12 that selectively induce only the Raf/ERK, RalGDS, or the PI3-kinase signaling pathway. Whereas keratinocytes expressing a fully activated RasV12 are not invasive in tracheal xenotransplants, ODC-overexpressing keratinocytes acquire an invasive phenotype with additional expression of either RasV12 or activation of the Raf/ERK pathway. Independent of a mutated ras, elevated levels of ODC activate the Akt/mTOR signaling pathway as well as the Rho/Rac pathway in primary keratinocytes. Thus, Raf/ERK signaling is sufficient to cooperate with increased ODC activity in the conversion of normal keratinocytes to invasive cells. In order to promote invasiveness in keratinocytes, elevated levels of ODC may cooperate with Raf/ERK via activation of the Akt and Rho/Rac signaling pathway.

Targeting polyamine biosynthetic pathway through RNAi causes the abrogation of MCF 7 breast cancer cell line

“…It is well accepted and demonstrated that overexpression of ODC alone or in conjunction with other oncogenes plays a pivotal role in oncogenesis in various cancers [32,33]. Conversely, there is convincing evidence that a low level of ODC activity reduces carcinogenesis [34].…”

“…The hybridization buffer contained a final concentration of 5× Denhardt's solution and 5× SSPE buffer (5× Denhardt's=0.1 % BSA, 0.1 % Ficoll 400, 0.1 % polyvinylpyrolidon MW 40.000; 5× SSPE=0.9 M NaCl, 0.05 M NaH 2 PO 4 .H 2 O, 5 mM EDTA, pH 7.7, all reagents were purchased from Sigma). For hybridization, the cDNA products were synthesized by Revert Aid H-minus first-strand cDNA synthesis Kit (Fermentas, Germany), quantitated by spectrophotometry, and 2 μg of cDNA subjected to 32 P-dCTP labeling (Thermo Scientific DecaLabel DNA Labelling kit, Fermentas, Germany). The probes were mixed with 140 μL of salmon sperm (1 mg/mL), denatured, and placed on ice.…”

Evidence of a Role for Antizyme and Antizyme Inhibitor as Regulators of Human Cancer

“…ODC levels are also upregulated in intraepithelial neoplasias (IENs) – the non-invasive precursors of epithelial cancers. ODC expression is regulated by androgens in the prostate gland, and ODC levels are highly elevated in human prostate cancer (27) and numerous other cancer types (28-30). Furthermore, a meta-analysis of multiple microarray data sets established that the polyamine pathway is often highly altered in human prostate cancer (31).…”

Polyamines and nonmelanoma skin cancer

“…Moreover, skin tumorigenesis in response to Ras activation requires increased polyamine biosynthesis since DFMO and AZ expression can block the spontaneous development of skin papillomas in transgenic mice that express an activated MEK mutant in the basal layer of the epidermis (Feith et al, 2005;Feith et al, 2006). However, activation of Raf or MEK in normal transgenic mouse skin or in primary cultures of keratinocytes does not increase ODC activity and is not sufficient to convert normal keratinocytes to an invasive phenotype (Feith et al, 2005;Hayes et al, 2006). Indeed, conversion of normal keratinocytes to invasive, malignant cells minimally requires activation of the Raf/MEK/ERK signaling pathway and a threshold level of increased ODC activity (Smith et al, 1997;Hayes et al, 2006).…”

“…However, activation of Raf or MEK in normal transgenic mouse skin or in primary cultures of keratinocytes does not increase ODC activity and is not sufficient to convert normal keratinocytes to an invasive phenotype (Feith et al, 2005;Hayes et al, 2006). Indeed, conversion of normal keratinocytes to invasive, malignant cells minimally requires activation of the Raf/MEK/ERK signaling pathway and a threshold level of increased ODC activity (Smith et al, 1997;Hayes et al, 2006). Another requirement may involve a selective susceptibility of a targeted subpopulation of keratinocytes or stem cells within the skin.…”