Translational Upregulation of an Individual p21Cip1 Transcript Variant by GCN2 Regulates Cell Proliferation and Survival under Nutrient Stress.

Multiple transcripts encode for the cell cycle inhibitor p21(Cip1). These transcripts produce identical proteins but differ in their 5' untranslated regions (UTRs). Although several stresses that induce p21 have been characterized, the mechanisms regulating the individual transcript variants and their functional significance are unknown. Here we demonstrate through (35)S labeling, luciferase reporter assays, and polysome transcript profiling that activation of the Integrated Stress Response (ISR) kinase GCN2 selectively upregulates the translation of a p21 transcript variant containing 5' upstream open reading frames (uORFs) through phosphorylation of the eukaryotic translation initiation factor eIF2α. Mutational analysis reveals that the uORFs suppress translation under basal conditions, but promote translation under stress. Functionally, ablation of p21 ameliorates G1/S arrest and reduces cell survival in response to GCN2 activation. These findings uncover a novel mechanism of p21 post-transcriptional regulation, offer functional significance for the existence of multiple p21 transcripts, and support a key role for GCN2 in regulating the cell cycle under stress.

Identification of a long non-coding RNA-associated RNP complex regulating metastasis at the translational step.

Long non-coding RNAs (lncRNAs) are a novel class of regulatory genes that play critical roles in various processes ranging from normal development to human diseases such as cancer progression. Recent studies have shown that lncRNAs regulate the gene expression by chromatin remodelling, transcription, splicing and RNA decay control, enhancer function, and epigenetic regulation. However, little is known about translation regulation by lncRNAs. We identified a translational regulatory lncRNA (treRNA) through genome-wide computational analysis. We found that treRNA is upregulated in paired clinical breast cancer primary and lymph-node metastasis samples, and that its expression stimulates tumour invasion in vitro and metastasis in vivo. Interestingly, we found that treRNA downregulates the expression of the epithelial marker E-cadherin by suppressing the translation of its mRNA. We identified a novel ribonucleoprotein (RNP) complex, consisting of RNA-binding proteins (hnRNP K, FXR1, and FXR2), PUF60 and SF3B3, that is required for this treRNA functions. Translational suppression by treRNA is dependent on the 3'UTR of the E-cadherin mRNA. Taken together, our study indicates a novel mechanism of gene regulation by lncRNAs in cancer progression.

The oncogene HER2/neu (ERBB2) requires the hypoxia-inducible factor HIF-1 for mammary tumor growth and anoikis resistance.

ERBB2, a receptor tyrosine kinase amplified in breast cancer, is a well established regulator of tumor growth in vivo and anoikis resistance leading to disruption of architecture in three-dimensional mammary epithelial acinar structures in vitro. ERBB2 promotes anoikis resistance by maintaining signaling pathways and by rescuing metabolic defects and thus inhibiting accumulation of deleterious reactive oxygen species. Recent evidence suggests that hypoxia, via hypoxia-inducible factors (HIFs), can inhibit anoikis; thus, we hypothesized that HIF-1 may play a role in ERBB2-mediated anoikis resistance and oncogenesis. Indeed, tumors isolated from MMTV-Neu mice contain elevated HIF-1α levels and tumor cells created from MMTV-Neu mice harboring deletion of Hif1α alleles reduced primary tumor growth in vivo. ERBB2 overexpressing cancer cells stabilize HIF under normoxic conditions and require HIF-1 for ERBB2-mediated anchorage-independence, three-dimensional culture growth and anoikis resistance. HIF-1 reduction in ERBB2 cells was associated with induction of the pro-anoikis protein BIM and decreased ERK and AKT signaling during cell detachment. ERBB2-mediated inhibition of metabolic defects, including decreased reactive oxygen species generation in suspension, required HIF-1 expression that was critical for ERBB2-mediated oncogenesis. Gene expression profiling of hypoxic three-dimensional acinar structures identified a number of genes elevated in response to hypoxia that are known ERBB2 targets, suggesting that hypoxic conditions and ERBB2 overexpression share both phenotypic and genetic components via HIF-1 regulation. Thus, our data demonstrate that ERBB2 requires HIF-1 for tumor growth and suggest that HIF is a major downstream regulator of ERBB2 that protects cells from anoikis and metabolic stress caused by decreased matrix adhesion.

Association of autotaxin and lysophosphatidic acid receptor 3 with aggressiveness of human breast carcinoma.

In vitro and in vivo experimental studies have demonstrated the role of lysophosphatidic acid (LPA) signaling in tumor proliferation, invasiveness, and metastasis. Among LPA receptors, the overexpression of LPA receptor 3 (LPAR3) in transgenic mice has resulted in the highest rate of breast cancer metastasis. Our goal is to evaluate the LPA-producing enzyme autotaxin and LPAR3 as potential therapeutic targets in breast cancer patients. The expression of autotaxin and LPAR3 was examined by immunohistochemical analysis of 87 invasive human breast carcinomas. Carcinomas were more frequently positive for autotaxin and LPAR3 (24.4 and 43 %, respectively) compared to adjacent normal breast tissue (6.1 and 2.9 %, respectively). Increased stromal autotaxin expression was found in 16.3 % of the tumors. LPAR3 overexpression was associated with less differentiated tumors, human epidermal growth factor receptor 2 expression, and absence of progesterone receptors. The luminal type A carcinomas showed the lowest frequency of autotaxin and LPAR3 expression. Strong desmoplastic stromal reaction was more frequent among the carcinomas with autotaxin-positive tumor cells or autotaxin-positive stroma. Patients with carcinomas overexpressing LPAR3 in epithelial cells or autotaxin in stromal cells were more likely to have larger tumors, nodal involvement, and higher stage disease. Autotaxin overexpression in tumor cells also correlated with tumor size and clinical stage. Our data indicate that the increased expression of LPAR3 and autotaxin in human breast cancer is associated with tumor aggressiveness. They also suggest that LPA mediates tumor metastatic ability and peritumoral desmoplastic reaction through autocrine-paracrine mechanisms. A substantial portion of breast cancer patients might benefit from autotoxin/LPA receptor-targeted therapies.

Translational repression during chronic hypoxia is dependent on glucose levels.

Translation is often repressed in cell lines that are exposed to hypoxic conditions (0.5% - 1.5% O2) but this repression requires prolonged exposure (> 16 h). We report here that prolonged exposure to hypoxia results in the depletion of glucose from the media and that the loss of glucose correlates with the shut down in translation. Furthermore, we show that the addition of glucose or reoxygenation restores translation in hypoxic PC3 cells. This indicates that both glucose depletion and hypoxia are required for translational repression. We also show that eIF2alpha phosphorylation is reversed by glucose addition. Moreover, we present data that strongly indicate that eIF2alpha phosphorylation as well as the translational inhibition that occurs when cells are grown under conditions of glucose depletion and hypoxia is pancreatic eIF2alpha kinase (PERK) independent. We believe this is the first report to show that glucose depletion is required for translational repression under hypoxic conditions and that this explains why prolonged exposure to hypoxia is required for this inhibition. Since the physiological conditions that lead to tumor hypoxia would also likely lead to reduced glucose levels, understanding the interplay of glucose and hypoxia in regulating tumor metabolism will provide important information on the growth and development of solid tumors.

Iodine alters gene expression in the MCF7 breast cancer cell line: evidence for an anti-estrogen effect of iodine.

The protective effects of iodine on breast cancer have been postulated from epidemiologic evidence and described in animal models. The molecular mechanisms responsible have not been identified but laboratory evidence suggests that iodine may inhibit cancer promotion through modulation of the estrogen pathway. To elucidate the role of iodine in breast cancer, the effect of Lugol's iodine solution (5% I(2), 10% KI) on gene expression was analyzed in the estrogen responsive MCF-7 breast cancer cell line. Microarray analysis identified 29 genes that were up-regulated and 14 genes that were down-regulated in response to iodine/iodide treatment. The altered genes included several involved in hormone metabolism as well as genes involved in the regulation of cell cycle progression, growth and differentiation. Quantitative RT-PCR confirmed the array data demonstrating that iodine/iodide treatment increased the mRNA levels of several genes involved in estrogen metabolism (CYP1A1, CYP1B1, and AKR1C1) while decreasing the levels of the estrogen responsive genes TFF1 and WISP2. This report presents the results of the first gene array profiling of the response of a breast cancer cell line to iodine treatment. In addition to elucidating our understanding of the effects of iodine/iodide on breast cancer, this work suggests that iodine/iodide may be useful as an adjuvant therapy in the pharmacologic manipulation of the estrogen pathway in women with breast cancer.

Bone-metastatic potential of human prostate cancer cells correlates with Akt/PKB activation by alpha platelet-derived growth factor receptor.

Prostate adenocarcinoma metastasizes to the skeleton more frequently than any other organ. An underlying cause of this phenomenon may be the ability of bone-produced factors to specifically select disseminated prostate cancer cells that are susceptible to their trophic effects. Platelet-derived growth factor (PDGF), a potent mitogen for both normal and tumor cells, is produced in several tissues including bone, where it is synthesized by both osteoblasts and osteoclasts. Here, we show that PDGF causes a significantly stronger activation of the Akt/PKB survival pathway in bone-metastatic prostate cancer cells compared to nonmetastatic cells. Normal prostate epithelial cells and DU-145 prostate cells, originally derived from a brain metastasis, are not responsive to PDGF. In contrast, epidermal growth factor stimulates Akt to the same extent in all prostate cells tested. This difference in PDGF responsiveness depends on the higher expression of alpha-PDGFR in bone-metastatic compared to nonmetastatic prostate cells and the lack of alpha-PDGFR expression in normal and metastatic prostate cells derived from tissues other than bone. Thus, alpha-PDGFR expression might identify prostate cancer cells with the highest propensity to metastasize to the skeleton.

Hypoxia suppression of Bim and Bmf blocks anoikis and luminal clearing during mammary morphogenesis.

Proper adhesion to extracellular matrix is critical for epithelial cell survival. Detachment from matrix signals results in apoptosis, referred to as anoikis. Selective apoptosis of cells that become detached from matrix is associated with the formation of a lumen in three-dimensional mammary epithelial acinar structures in vitro. Because early breast cancer lesions such as carcinoma in situ, characterized by ducts exhibiting lumens filled with cells, are often associated with hypoxic markers, we sought to examine the role of hypoxia in anoikis and lumen formation in mammary epithelial cells. Here, we show that hypoxic conditions inhibit anoikis and block expression of proapoptotic BH3-only family members Bim and Bmf in epithelial cells. Hypoxia-mediated anoikis protection is associated with increased activation of the epidermal growth factor receptor-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase (Erk) kinase pathway and requires the hypoxia-activated transcription factor. Consistent with these data, hypoxic conditions inhibit luminal clearing during morphogenesis in human mammary epithelial acini when grown in three-dimensional cultures and are associated with decreased expression of Bim and Bmf as well as Erk activation. We show that hypoxia regulates specific cell survival pathways that disrupt tissue architecture related to clearing of luminal space during mammary morphogenesis and suggest that hypoxia-mediated anoikis resistance may contribute to cancer progression.

Activation of proteasome by insulin-like growth factor-I may enhance clearance of oxidized proteins in the brain.

The insulin-like growth factor type 1 (IGF-I) plays an important role in neuronal physiology. Reduced IGF-I levels are observed during aging and this decrease may be important to age-related changes in the brain. We studied the effects of IGF-I on total protein oxidation in brain tissues and in cell cultures. Our results indicate that in frontal cortex the level of oxidized proteins is significantly reduced in transgenic mice designed to overproduce IGF-I compared with wild-type animals. The frontal cortex of IGF-I-overproducing mice exhibited high chymotrypsin-like activity of the 20S and 26S proteasomes. The proteasome can also be activated in response to IGF-I in cell cultures. Kinetic studies revealed peak activation of the proteasome within 15 min following IGF-I stimulation. The effects of IGF-I on proteasome were not observed in R(-) cells lacking the IGF-I receptor. Experiments using specific kinase inhibitors suggested that activation of proteasome by IGF-I involves phosphatidyl inositol 3-kinase and mammalian target of rapamycin signaling. IGF-I also attenuated the increase in protein carbonyl content induced by proteasome inhibition. Thus, appropriate levels of IGF-I may be important for the elimination of oxidized proteins in the brain in a process mediated by activation of the proteasome.

Depletion of the poly(C)-binding proteins alphaCP1 and alphaCP2 from K562 cells leads to p53-independent induction of cyclin-dependent kinase inhibitor (CDKN1A) and G1 arrest.

The alpha-globin poly(C)-binding proteins (alphaCPs) comprise an abundant and widely expressed set of K-homolog domain RNA-binding proteins. alphaCPs regulate the expression of a number of cellular and viral mRNAs at the levels of splicing, stability, and translation. Previous surveys have identified 160 mRNAs that are bound by alphaCP in the human hematopoietic cell line, K562. To explore the functions of these alphaCP/mRNA interactions, we identified mRNAs whose levels are altered in K562 cells acutely depleted of the two major alphaCP proteins, alphaCP1 and alphaCP2. Microarray analysis identified 27 mRNAs that are down-regulated and 14 mRNAs that are up-regulated in the alphaCP1/2-co-depleted cells. This alphaCP1/2 co-depletion was also noted to inhibit cell proliferation and trigger a G(1) cell cycle arrest. Targeted analysis of genes involved in cell cycle control revealed a marked increase in p21(WAF) mRNA and protein. Analysis of mRNP complexes in K562 cells demonstrates in vivo association of p21(WAF) mRNA with alphaCP1 and alphaCP2. In vitro binding assays indicate that a 127-nucleotide region of the 3'-untranslated region of p21(WAF) interacts with both alphaCP1 and alphaCP2, and co-depletion of alphaCP1/2 results in a marked increase in p21(WAF) mRNA half-life. p21(WAF) induction and G(1) arrest in the alphaCP1/2-co-depleted cells occur in the absence of p53 and are not observed in cells depleted of the individual alphaCP isoforms. The apparent redundancy in the actions of alphaCP1 and alphaCP2 upon p21(WAF) expression correlates with a parallel redundancy in their effects on cell cycle control. These data reveal a pivotal role for alphaCP1 and alphaCP2 in a p53-independent pathway of p21(WAF) control and cell cycle progression.

Identification of mRNAs that continue to associate with polysomes during hypoxia.

Tumors must adapt to the hypoxic environment in order to grow beyond a benign microscopic mass. In addition to transcriptional activation mediated by HIF-1alpha, hypoxia has also been reported to inhibit translation. The degree of translational inhibition is dependent on the duration as well as the severity of the hypoxic insult. Anoxia (<0.02% O(2)) seems to have a more rapid and dramatic effect on translation as compared to hypoxia. We show here that prolonged hypoxia dramatically and reversibly inhibits translation in PC-3 cells. We also found that mTOR is inactivated and eIF-2alpha is phosphorylated during hypoxic treatment but only the eIF-2alpha phosphorylation correlates with the translational repression. We further used polysome analysis and microarray technology to analyze the impact of this translational repression on gene expression. We found that 33 mRNAs were refractory to this translational repression and that there was no correlation between mRNA induction and the ability to recruit ribosomes during hypoxia. We also found that ribosomal protein encoding mRNAs are more sensitive to this translational repression as compared to the majority of mRNAs. Although other reports have analyzed the effect of translation inhibition on gene expression under anoxic conditions, we believe that this is the first report in hypoxic cells. Our results show that the translational repression that occurs during hypoxia does impact gene expression in the highly transformed prostate cancer cell line, PC-3.