A Repertoire of MicroRNAs Regulates Cancer Cell Starvation by Targeting Phospholipase D in a Feedback Loop That Operates Maximally in Cancer Cells

Abstract

bWe report a negative feedback loop between the signaling protein phospholipase D (PLD), phosphatidic acid (PA), and a specific set of microRNAs (miRNAs) during nutrient starvation of breast cancer cells. We show that PLD expression is increased in four breast cancer cell lines and that hypoxia, cell overcrowding, and nutrient starvation for 3 to 6 h increase expression even further. However, after prolonged (>12-h) starvation, PLD levels return to basal or lower levels. The mechanism for this is as follows. First, during initial starvation, an elevated PA (the product of PLD enzymatic activity) activates mTOR and S6K, known to inhibit apoptosis, and enhances cell migration especially in post-epithelial-to-mesenchymal transition (post-EMT) cancer cells. Second, continued PA production in later starvation induces expression of PLD-targeting microRNA 203 (miR-203), miR-887, miR-3619-5p, and miR-182, which reduce PLD translation. We provide direct evidence for a feedback loop, whereby PLD induction upon starvation leads to PA, which induces expression of miRNAs, which in turn inhibits PLD2 translation. The physiological relevance for breast cancer cells is that as PA can activate cell invasion, then, due to the negative feedback, it can deprive mTOR and S6K of their natural activator. It can further prevent inhibition of apoptosis and allow cells to survive nutrient deprivation, which normal cells cannot do.M icroRNAs (miRNAs) are short molecules of noncoding RNA, ϳ22 nucleotides in length, and have crucial roles in the regulation of many cellular processes, including development, proliferation, differentiation, apoptosis, and stress response (1, 2). Mature miRNA molecules associate with the Argonaute (Ago1 and Ago2) proteins and the RNA-induced silencing complex (RISC) (3, 4). Active miRNAs regulate expression of their target genes via association of an ϳ7-nucleotide-long stretch seed region with a complementary sequence in the target mRNA located in the 3= untranslated region (UTR). Binding of miRNAs to their target mRNAs along with the RISC complex mediates inhibition of translation initiation (5). miRNA involvement in cancer development and metastasis is the subject of intense research (6-10).Phospholipase D (PLD) has been implicated in cellular signals that suppress apoptosis and contribute to cancer cell survival (11-13). Through cell signaling, elevated PLD activity leads to activation of mammalian target of rapamycin (mTOR), a survival signal often hyperactivated in cancer (14, 15). Elevated PLD activity also subdued the tumor suppressors p53 and protein phosphatase 2A (12). Zheng et al. published a model for enhanced survival and migration signals in the developing tumor (16). In a developing tumor mass, cells inside the mass were subjected to hypoxia and nutrient and growth factor deprivation. It is proposed that cells that respond to stress by elevating PLD protein levels will survive presumably by gaining the ability to migrate. However, very little is known about PLD regulation at gene and protein leve...

D‐series Resolvins activate Phospholipase D in phagocytes during inflammation and resolution

“…We have studied previously post-translational modification of PLD2 transcripts and reported how that is under regulation of certain miRs. 18,22 For macrophages, we selected miR-138 and miR-887 that bind to the 3'UTR of PLD2 ( Figure 5B). The demonstration that transfected miR-138 and miR-887 decrease PLD2 expression in M0 macrophages is shown in Figure 5C,D.…”

“…[15][16][17] PLD is upregulated in response to various cell stressors, such as hypoxia and nutrient starvation. 18,19 Moreover, it has been shown that PLD2 -/mice produce less pro-inflammatory cytokines in a sepsis model. 20 PA is a mitogen and a critical secondary messenger that activates many downstream pathways leading to cell growth and proliferation, vesicle trafficking and cell migration.…”

Somatic mutations in miRNA genes in lung cancer – potential functional consequences of non-coding sequence variants

“…Increased levels of miR-887 were previously reported in endometrial and rectal cancer (77,78). In contrast, other authors have demonstrated that miR-887 may act as a tumor suppressor in prostate cancer, breast cancer and small cell lung cancer (SCLC) (79)(80)(81). We have shown that mutations in miRNA genes and known cancer driver genes are not mutually exclusive, but a substantial number of miRNA gene mutations were identified in samples without mutations in known protein-coding drivers.…”

Resolvin D-series regulates Phospholipase D both during inflammation and resolution by modulating phagocyte functions

“…There could be several reasons for this. We have studied previously post-translational modification of PLD2 transcripts and reported how that is under regulation of certain miRs (22,26). For macrophages, we selected miR-138 and miR-887 that are predicted to bind to the 3'UTR of PLD2 (Fig.…”

“…However, the intracellular mechanism of downstream signaling in macrophages is of interest.Phospholipase D (PLD) is a ubiquitously expressed membrane-associated lipase that hydrolyzes phosphatidylcholine (PC) into free choline and phosphatidic acid (PA) (19)(20)(21). PLD is upregulated in response to various cell stressors, such as hypoxia and nutrient starvation (22,23). Moreover, PLD2 -/mice produced less pro-inflammatory cytokines in a sepsis model (24).…”