Translational Control Of Cancer Research Articles

Targeting Translational Control in Cancer Using Small Molecule Activators of PP2A

BackgroundCancer cells rely on hyperactive protein synthesis to reprogram the proteome to sustain the transformed phenotype. Thus, aberrant protein translation has emerged as a promising therapeutic target. Cap‐dependent translation is an exquisitely regulated process that requires the assembly of the eIF4F translation initiation complex. The rate limiting component of the eIF4F complex is eIF4E, which binds to the 5’ cap structure of mRNAs to initiate translation. Cap‐dependent translation is kept in check by the translation repressor 4E‐BP1; in its active hypophosphorylated form, 4E‐BP1 sequesters eIF4E to prevent translation initiation. Cancer cells use several mechanisms to bypass 4E‐BP1 translational suppres­sion, including hyperactivation of mTORC1 for hyperphos­phorylation and inactivation of 4E‐BP1; inactivation of the 4E‐BP1 phosphatase, PP2A; and profound suppression of 4E‐BP1 expression. We have found that the expression and activity of 4E‐BP1 can be restored in tumor cells using novel small molecule acti­vators of PP2A (SMAPs), a class of compounds that activate selected PP2A tumor suppressors and are being ac­tively developed as anticancer agents (Leonard et al. Cell, 2020). The objective of this study was to investigate the mechanism(s) by which SMAPs lead to restoration of the expression and activity of 4E‐BP1 in cancer cells for inhibition of hyperactive protein translation and tumor suppression.HypothesisBased on our finding that SMAPs promote PP2A‐dependent transcriptional activation of the 4E‐BP1 gene, we hypothesize that PP2A activation modulates the levels and/or activity of key transcription factors that regulate 4E‐BP1 gene expression.Results/DataWe have found that SMAPs (e.g., lead compound DT‐061) upregulate 4E‐BP1 mRNA and protein in tumor cells by inducing transcription of the 4E‐BP1 gene. Importantly, SMAPs promote the accumulation of active 4E‐BP1, capable of suppressing cap‐dependent translation in cancer cells. SMAPs promote PP2A‐dependent loss of Snail/Slug and upregulation of ATF4, transcription factors that have been implicated in negative and posi­tive control of 4E‐BP1 gene ex­pression, respectively. Snail downregulation was prevented by proteasome inhibition, indicating that Snail loss is due to proteasomal degradation. Use of GSK‐3β and PKD1 inhibitors excluded canonical mechanisms of Snail degradation in the effects of SMAPs. siRNA knockdown experiments are underway to explore the role of changes in Snail and ATF4 in SMAP‐induced upregulation of 4E‐BP1. Ongoing studies are also examining the mechanisms of SMAP/PP2A regulation of Snail/Slug and ATF4 in tumor cells.ConclusionsSMAPs promote PP2A‐dependent proteasomal degradation of Snail/Slug protein via a non‐canonical mechanism, as well as upregulation of ATF4. We propose that these effects relieve transcriptional repression of 4E‐BP1 by Snail/Slug while promoting 4E‐BP1 transcription by ATF4, leading to a robust increase in 4E‐BP1 levels. 4E‐BP1 translation repressive activity is ensured by PP2A‐induced 4E‐BP1 dephosphorylation. Thus, SMAPs offer a powerful tool for therapeutic targeting of aberrant protein translation in cancer.

Regulation of eIF2α by RNF4 Promotes Melanoma Tumorigenesis and Therapy Resistance

Among the hallmarks of melanoma are impaired proteostasis and rapid development of resistance to targeted therapy that represent a major clinical challenge. However, the molecular machinery that links these processes is unknown. Here we describe that by stabilizing key melanoma oncoproteins, the ubiquitin ligase RNF4 promotes tumorigenesis and confers resistance to targeted therapy in melanoma cells, xenograft mouse models, and patient samples. In patients, RNF4 protein and mRNA levels correlate with poor prognosis and with resistance to MAPK inhibitors. Remarkably, RNF4 tumorigenic properties, including therapy resistance, require the translation initiation factor initiation elongation factor alpha (eIF2α). RNF4 binds, ubiquitinates, and stabilizes the phosphorylated eIF2α (p-eIF2α) but not activating transcription factor 4 or C/EBP homologous protein that mediates the eIF2α-dependent integrated stress response. In accordance, p-eIF2α levels were significantly elevated in high-RNF4 patient-derived melanomas. Thus, RNF4 and p-eIF2α establish a positive feed-forward loop connecting oncogenic translation and ubiquitin-dependent protein stabilization in melanoma.

Abstract 3910: Tetrandrine inhibits signal transduction through PI3K/Akt/mTOR and PERK/eIF2á pathways to target aberrantly activated cap-dependent translation in cancer cells

Abstract The PI3K/Akt/mTOR pathway plays a key role in the control of cap-dependent mRNA translation and is also an important driver of tumor progression. Owing to mutations in PTEN across pan cancer, this pathway is activated in several solid tumors. mTOR signaling enhances tumor growth by stimulating both the global translation rates and the translation of a selected subset of sensitive transcripts, including mRNAs, encoding oncogenes, growth factors, anti-apoptotic factors - c-Myc, ornithine decarboxylase, Cyclin D1, VEGF, IGF, BCL-2, and survivin in several solid tumors. The foregoing data demonstrate that abnormal translational control in cancer, implemented by oncogenic signaling via mTOR and other pathways, may represent an attractive therapeutic target. Tetrandrine (Tet) a bis-benzylisoquinoline alkaloid with broad anticancer activity attracts increasing attention lately. However, the underlying molecular mechanisms were not studied systematically. In the present studies, we evaluated the effects of Tet on aberrantly activated cap-dependent translation and on its principal regulator - Akt / mTOR signaling pathway in cancer cells for the first time in any system. The anti-proliferative effects of Tet was evaluated in several cancer cell lines (LNCaP, 22Rv1, BXPC3, MiaPaCa2, A498, UOK262 HCC827, etc) representing many solid tumors. Using capped mRNA reporter construct, we evaluated the effect of Tet on translation in 22Rv1 and LNCaP PC cell cultures. We found that Tet was not able to inhibit translation of mRNA on the ribosomes per se in the in vitro translation system. However, Tet was operational in LNCaP and 22Rv1 cells, inhibiting cap-dependent translation rapidly and efficiently during the first few hours. A subsequent study of the polysomal distribution in LNCaP and 22Rv1 cells showed that Tet mainly inhibited the initiation phase of translation. Importantly, the translation-inhibiting effect of Tet correlated with its growth-suppressive activity in concentration and time-dependent manner. Our study of the PI3K/Akt signaling showed that Tet inhibited signaling to mTOR and its downstream effectors in PC cells. This explains the inhibition of cap-dependent mRNA translation and the suppression of cancer growth. Collectively, presented results show a new, previously unknown “translation side” of the anticancer activity of Tet. This novel insight may help with potential therapeutic applications of Tet in the treatment of cancer in general and prostate cancer in particular. Citation Format: Sergey Slepenkov, Praveen Jaiswal, Sweaty Koul, Hari K. Koul. Tetrandrine inhibits signal transduction through PI3K/Akt/mTOR and PERK/eIF2á pathways to target aberrantly activated cap-dependent translation in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3910.

Translation acrobatics: how cancer cells exploit alternate modes of translational initiation.

Recent work has brought to light many different mechanisms of translation initiation that function in cells in parallel to canonical cap-dependent initiation. This has important implications for cancer. Canonical cap-dependent translation initiation is inhibited by many stresses such as hypoxia, nutrient limitation, proteotoxic stress, or genotoxic stress. Since cancer cells are often exposed to these stresses, they rely on alternate modes of translation initiation for protein synthesis and cell growth. Cancer mutations are now being identified in components of the translation machinery and in cis-regulatory elements of mRNAs, which both control translation of cancer-relevant genes. In this review, we provide an overview on the various modes of non-canonical translation initiation, such as leaky scanning, translation re-initiation, ribosome shunting, IRES-dependent translation, and m6A-dependent translation, and then discuss the influence of stress on these different modes of translation. Finally, we present examples of how these modes of translation are dysregulated in cancer cells, allowing them to grow, to proliferate, and to survive, thereby highlighting the importance of translational control in cancer.

PO-133 Hoxa9 transcription factor: a potential novel therapeutic target in renal cell carcinoma (RCC)

IntroductionDysregulation of mRNA translational control in cancer leads to cell transformation, metabolic reprogramming and angiogenesis. eIF6 is an oncogenic translation factor, which regulates the initiation phase of translation acting on 60S availability in the cytoplasm and controlling active 80S complex formation. eIF6 activation is mTORC1-independent and driven by PKCβ mediated phosphorylation on Ser235. An increment of eIF6 expression is reported in several cancer cell lines and human tumours, due to amplification or overexpression. In mice, eIF6 haploinsufficiency blocks Myc-driven lymphomagenesis. Intriguingly, high levels of PKC and eIF6 are found in T-cell lymphomas. In particular, in Anaplastic Large Cell Lymphoma (ALCL) eIF6 is overexpressed and hyperactivated.Material and methodsHere, we aimed to define the role of eIF6 phosphorylation in NPM-ALK mediated T-cell lymphomagenesis, combining multidisciplinary studies on murine and cellular models. We used a conditional eIF6SAKI mouse model in which Ser235 is replaced by an Ala.Results and discussionsFirst, we addressed the effect of eIF6 mutated protein expression in all tissues: homozygosity is lethal after gastrulation while heterozygous mice are viable but resistant to NPM-ALK driven lymphomagenesis. Then, we investigated the role of Ser235 phosphorylation specifically in T-cell lineage, crossing eIF6SAKI mice with CD4-Cre mice. Physiological T-cell development and subsets composition are not affected by the eIF6 mutated protein. In cancer, eIF6SA/SA CD4-Cre NPM-ALK mice have a significant increase in survival time, compared to wt with a delay in the appearance of lymphoma up to 6 months. Histological analysis and ex vivo cultures confirm the delay in disease development. eIF6SA/SA CD4-Cre NPM-ALK thymocytes are smaller respect to wt counterparts and show a striking senescence-like phenotype in vitro. Similarly, in vitro generated eIF6SA/SA MEFs show a markedly reduced proliferation and increased SA β-gal positivity. This phenotype is completely rescued by transducing eIF6 wild-type, but not by eIF6SA. Currently, we are investigating the molecular mechanisms by which eIF6 phosphorylation affects ALK-induced malignancy and whether it may modulate premature cell senescence, thus establishing an effective barrier to T-cell lymphomagenesis.ConclusionOur work demonstrates for the first time that eIF6 phosphorylation plays an essential role in mammals development, cell homeostasis and is rate-limiting for T-cell lymphomagenesis in vivo.

Translational Control in Cancer.

The translation of messenger RNAs (mRNAs) into proteins is a key event in the regulation of gene expression. This is especially true in the cancer setting, as many oncogenes and transforming events are regulated at this level. Cancer-promoting factors that are translationally regulated include cyclins, antiapoptotic factors, proangiogenic factors, regulators of cell metabolism, prometastatic factors, immune modulators, and proteins involved in DNA repair. This review discusses the diverse means by which cancer cells deregulate and reprogram translation, and the resulting oncogenic impacts, providing insights into the complexity of translational control in cancer and its targeting for cancer therapy.

PO-130 SER235 residue drives eIF6 oncogenic activity in NPM-ALK induced T cell lymphomagenesis

Introduction Dysregulation of mRNA translational control in cancer leads to cell transformation, metabolic reprogramming and angiogenesis. eIF6 is an oncogenic translation factor, which regulates the initiation phase of translation acting on 60S availability in the cytoplasm and controlling active 80S complex formation. eIF6 activation is mTORC1-independent and driven by PKCβ mediated phosphorylation on Ser235. An increment of eIF6 expression is reported in several cancer cell lines and human tumours, due to amplification or overexpression. In mice, eIF6 haploinsufficiency blocks Myc-driven lymphomagenesis. Intriguingly, high levels of PKC and eIF6 are found in T-cell lymphomas. In particular, in Anaplastic Large Cell Lymphoma (ALCL) eIF6 is overexpressed and hyperactivated. Material and methods Here, we aimed to define the role of eIF6 phosphorylation in NPM-ALK mediated T-cell lymphomagenesis, combining multidisciplinary studies on murine and cellular models. We used a conditional eIF6 SA KI mouse model in which Ser235 is replaced by an Ala. Results and discussions First, we addressed the effect of eIF6 mutated protein expression in all tissues: homozygosity is lethal after gastrulation while heterozygous mice are viable but resistant to NPM-ALK driven lymphomagenesis. Then, we investigated the role of Ser235 phosphorylation specifically in T-cell lineage, crossing eIF6 SA KI mice with CD4-Cre mice. Physiological T-cell development and subsets composition are not affected by the eIF6 mutated protein. In cancer, eIF6 SA/SA CD4-Cre NPM-ALK mice have a significant increase in survival time, compared to wt with a delay in the appearance of lymphoma up to 6 months. Histological analysis and ex vivo cultures confirm the delay in disease development. eIF6 SA/SA CD4-Cre NPM-ALK thymocytes are smaller respect to wt counterparts and show a striking senescence-like phenotype in vitro . Similarly, in vitro generated eIF6 SA/SA MEFs show a markedly reduced proliferation and increased SA β-gal positivity. This phenotype is completely rescued by transducing eIF6 wild-type, but not by eIF6 SA . Currently, we are investigating the molecular mechanisms by which eIF6 phosphorylation affects ALK-induced malignancy and whether it may modulate premature cell senescence, thus establishing an effective barrier to T-cell lymphomagenesis. Conclusion Our work demonstrates for the first time that eIF6 phosphorylation plays an essential role in mammals development, cell homeostasis and is rate-limiting for T-cell lymphomagenesis in vivo .

Translational control in the tumor microenvironment promotes lung metastasis: Phosphorylation of eIF4E in neutrophils

The translation of mRNAs into proteins serves as a critical regulatory event in gene expression. In the context of cancer, deregulated translation is a hallmark of transformation, promoting the proliferation, survival, and metastatic capabilities of cancer cells. The best-studied factor involved in the translational control of cancer is the eukaryotic translation initiation factor 4E (eIF4E). We and others have shown that eIF4E availability and phosphorylation promote metastasis in mouse models of breast cancer by selectively augmenting the translation of mRNAs involved in invasion and metastasis. However, the impact of translational control in cell types within the tumor microenvironment (TME) is unknown. Here, we demonstrate that regulatory events affecting translation in cells of the TME impact cancer progression. Mice bearing a mutation in the phosphorylation site of eIF4E (S209A) in cells comprising the TME are resistant to the formation of lung metastases in a syngeneic mammary tumor model. This is associated with reduced survival of prometastatic neutrophils due to decreased expression of the antiapoptotic proteins BCL2 and MCL1. Furthermore, we demonstrate that pharmacological inhibition of eIF4E phosphorylation prevents metastatic progression in vivo, supporting the development of phosphorylation inhibitors for clinical use.

Abstract A12: Role of eIF5-mimic protein 1 (5MP1) for translational control in cancer

Abstract In eukaryotic translation initiation, both the m7G-capped mRNA and the small ribosomal subunit (SSU) are activated through eukaryotic initiation factors bound to Met-tRNAIMet, which allows formation of the 48S ribosome pre-initiation complex (PIC) with the small subunit attached to the mRNA 5' end. The PIC subsequently scans for a start codon, upon which it forms the ribosome initiation complex, ready for the polypeptide elongation, with the large subunit joined together. eIF5 is a crucial component of the multi-initiation factor complex (MFC) involved in the SSU activation and subsequent start codon selection by the 48S complex through its GTPase activating function (GAP). Paradoxically, however, overexpression turns eIF5 into an inhibitor of translation and decreases the accuracy of translation initiation, thereby allowing non-AUG initiation. Translation regulatory protein termed eIF5-mimic protein (5MP) exists in nearly all the eukaryotes except in nematodes, yeasts and some protozoans. Humans encode two of its copies, 5MP1 and 5MP2. It interacts with eIF2 and eIF3, major MFC components, and induces translation of ATF4 mRNA with regulatory uORFs. ATF4 is a pro-oncogenic transcription factor whose expression endorses tumor survival during stress conditions (hypoxia and nutritional deprivation) encountered by cancer cells in their development and metastasis. Moreover, preliminary study using yeast as a model system suggests that the universal role of 5MP in eukaryotic translation initiation is to increase the accuracy of translation initiation through inhibiting mis-initiation caused by excessive amount of eIF5. Here we examined the role of 5MP1 and 5MP2 in cultured human cells and found that 5MP1 expression suppresses non-AUG initiation of a luciferease reporter gene that occurs normally at a low level or at a higher level due to eIF5 overexpression, depending on its ability to bind the PIC. Furthermore, ribosome profiling studies in human cells show that expression of 5MP1 by itself inhibits non-AUG initiation of endogenous genes, such as GUG-initiated NAT1/eif4g2 and CUG-initiated c-Myc. These results indicate that 5MP in general suppresses non-AUG initiation through competing with eIF5 that occurs free of the ribosome. In support of the role of 5MP in cancer progression, 5MP1 knockdown and 5MP2 knockdown reduce the tumorigenicity of fibrosarcoma and salivary mucoepidermoid carcinoma, respectively. To test the model that 5MP promotes tumorigenicity through enhancing ATF4 expression, we examined correlation between eIF5 or 5MP expression and that of ATF4 target genes, using cancer genomics database. 5MP2 and eIF5 approximately correlated with the same subset of ATF4 targets in certain cancer types, but 5MP1 correlated with more ATF4 targets in more cancer types. These results not only confirm that eIF5 and 5MP promote tumorigenesis through up-regulating ATF4, but also suggest that 5MP1 has a greater role in cancer by unknown mechanism. Related to this point, we found a good correlation between 5MP1 and c-Myc expression in most types of cancers, and moreover c-Myc appears to be physically associated with 5MP1 promoters. Furthermore, in breast cancer high levels of 5MP1 are associated with poor prognosis, whereas high eIF5 levels correlate with more positive clinical outcomes. These results together suggest that, although up-regulation of ATF4 by eIF5 or 5MP is important, 5MP1 specially may play a major role in c-Myc-driven tumors, wherein a wider variety of ATF4 target genes are activated through 5MP1-ATF4 axis. The contrasting roles of 5MP1 and eIF5 in patient prognosis and accurate initiation suggests that suppression of non-AUG initiation may favor carcinogenesis. Collectively, future studies are warranted to establish the role of 5MP1 in cancer. Citation Format: Chelsea Moore, Sarah Gillaspie, Ji Wan, Eric Aube, Abbey Anderson, Chingakham Singh, Michael Witcher, Ivan Topisirovic, Shu-Bing Qian, Katsura Asano. Role of eIF5-mimic protein 1 (5MP1) for translational control in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A12.

Abstract A49: Transcription factor upregulation after mTOR inhibition by Torin1 induces growth factor receptor expression

Abstract Introduction: mTOR (mammalian target of rapamycin) integrates signals from cellular nutrient status and growth factors, to regulate cell growth, proliferation, and metabolism. mTOR is a part of the PI3K-Akt-mTOR signalling pathway, the components of which are frequently mutated in human cancers. Various inhibitors of mTOR activity have been developed to target this pathway, including rapamycin and its analogs sirolimus, everolimus, temsirolimus. These inhibitors are currently approved for the treatment of certain cancers, and under investigation in clinical trials for the treatment of others. Despite displaying initial effectiveness in delaying tumor progression, mTOR inhibitors eventually lose efficacy and patients relapse due to development of resistance. Due to the regulation of cap-dependent translation by mTOR, treatment of cells with mTOR inhibitors inhibits general protein translation. However, a subset of proteins exhibit increased translation in response to mTOR inhibitors. We postulate that these proteins that exhibit upregulation in response to mTOR inhibition play a role in conferring mTOR inhibitor resistance. In this study, we aim to characterize the landscape of proteins that are upregulated in response to mTOR inhibition, in order to identify pathways that contribute to resistance. We use a second-generation mTOR inhibitor Torin1, which targets both mTORC1 and mTORC2 complexes. Methods: In order to study newly translated proteins after mTOR inhibition by Torin1 on a proteomic scale, we combined the methods of click chemistry with SILAC (stable isotopic labelling of amino acids in cell culture) and tandem mass spectrometry. The translatome was studied through click-pulse-SILAC experiments conducted at 2 hours and 24 hours post Torin1 treatment in MEFs (mouse embryonic fibroblasts) as well as PC3 prostate carcinoma cells. The total proteome of MEF and PC3 cells 24 hours after Torin1 treatment was studied using SILAC and tandem mass spectrometry. Proteomics data processing and pathway enrichment analysis was performed using the Perseus software, and upregulated proteins were subject to analysis by RNA silencing, qPCR, and click chemistry. Results: Translatome analysis after Torin1 treatment showed general translation inhibition as expected, except for a small subset of proteins with increased translation. Increased translation of growth factor receptors was observed 24 hours post-Torin1 treatment, and of many transcription factors 2 hours post-treatment. Total proteome analysis 24 hours post-treatment revealed an increase in total protein levels of many of these growth factor receptors as well as transcription factors. qPCR experiments revealed increased transcripts levels of these growth factor receptors as early as 2 hours post-treatment, suggesting transcriptional upregulation. Through cross-referencing our 2 hour translatome data with genome-wide ChIP-seq datasets accessed through Cscan, we determined candidate transcription factors that may be responsible for upregulating the expression of growth factor receptors. Knockdown studies suggest that some of these transcription factors are indeed responsible for the upregulation of growth factor receptors. Conclusion: mTOR inhibition by Torin1 suppresses general translation, but many transcription factors escape this suppression and exhibit increased expression. These transcription factors induce expression of growth factor receptors, consequently enhancing their signaling. These studies identify an axis which potentially reduces the efficacy of mTOR inhibitors in cancer therapy, as well as targets that may require simultaneous inhibition. Citation Format: Tianqing T. Yang, Gian L. Negri, Anders Kristensen, Poul H. B. Sorensen. Transcription factor upregulation after mTOR inhibition by Torin1 induces growth factor receptor expression. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A49.

Abstract B11: Oncogenic KRAS regulates 4E-BP1, a repressor of cap-dependent translation, independently of growth factor activity

Abstract Oncogenic mutations in K-Ras often occur at high frequencies in human cancer resulting in the sustained activation of the ERK1/2 mitogen-activated protein kinase (MAPK) signaling pathway. However, the unique dependencies on upstream or downstream oncogenic signaling amongst the mutant K-Ras proteins remains elusive and often masked by the remaining wild-type allele, other Ras isoforms, or additional secondary genetic alterations. Thus, we employed an isogenic mouse embryonic fibroblast (MEFs) system devoid of H-, N-, and K-Ras alleles and reconstituted with a single Ras isoform to examine the dependencies of growth factors on K-Ras mediated control of translational regulation. Although oncogenic K-Ras G12D and G12V mutant MEF lines proliferated at twice the rate of K-Ras4b wild-type MEF cells, growth factor deprivation led to a cytostatic effect on the proliferation of all MEF lines. Analysis of signaling downstream of K-Ras revealed that phosphorylation of 4E-BP1, eIF4E, and ERK1/2 occurred in a growth factor-independent manner, which stood in contrast to the substantive effects of growth factor inhibition on AKT, p70S6K, and ribosomal protein S6 phosphorylation. Moreover, growth factor deprivation resulted in a marginal decrease of global protein synthesis with negligible effects on cap-dependent translation initiation in all K-Ras MEF cell lines. Taken together, these data suggest that oncogenic K-Ras signaling regulates 4E-BP1 phosphorylation independently of growth factor and mTORC1/2 effects. Citation Format: Jillian M. Silva, Rachel K. Bagni, Davide Ruggero, Frank McCormick. Oncogenic KRAS regulates 4E-BP1, a repressor of cap-dependent translation, independently of growth factor activity. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B11.

Abstract A22: Hypoxia-mediated translational control of hypoxia inducible factor 1 alpha through aldolase A feedback regulation in lung carcinogenesis

Abstract Lung cancer cells grow in-situ during low-oxygen situation and usually results in invasion and is frequently recurrent after therapy. However, the oxygen-mediated translation control of the metabolic events in lung cancer remains largely unknown. By using the integrated transcriptomics data and the shRNA library against glycolytic enzymes, here we identified that Aldolase A (ALDOA) up-regulation is highly correlated with migratory/invasive activity of lung cancer cells and poorer clinical outcomes. Significantly, knockdown of Aldolase A inhibited in vitro and in vivo migratory/invasive activity of lung cancer cell line with high ALDOA expression. Complementary, enforced overexpression of exogenous ALDOA promoted migration/invasion. Furthermore, microarray and Ingenuity pathway analyses reveal that ALDOA-driven lung cancer metastasis likely recruits Hypoxia-inducible factor 1 alpha (HIF1α)-associated signaling pathway. There were no significance changes at the mRNA level of HIF1α in ALDOA two-way models. However, HIF1α protein was found stabilized in hypoxic or normoxic condition through ALDOA overexpression to decrease prolyl hydroxylase (PHD) activity for hydroxylation-form of HIF1α to activate downstream genes including CXCR4, MMP9 in lung carcinogenesis. Interesting, HIF1α also was found to bind hypoxia-response element (HRE) domain of ALDOA to activate its transcription and form a positive feedback loop. IHC staining results further demonstrate that Aldolase A and HIF1α expression is positively correlated in clinical lung cancer tissues and the signature of high-level Aldolase A and HIF1α; expression strongly associates with worst survival probability in lung cancer patients. Our results not only unveil molecular mechanism by which Aldolase A promotes lung cancer metastasis via inducing the HIF1α activity but also provide a novel therapeutic strategy to combat malignant lung cancer via targeting ALDOA. Citation Format: Yu-Chan Chang, Michael Hsiao. Hypoxia-mediated translational control of hypoxia inducible factor 1 alpha through aldolase A feedback regulation in lung carcinogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A22.

Abstract A39: Expression of DNA methylation-related proteins in metastatic breast cancer

Abstract Purpose: To investigate the expressions of methylation-related proteins (5-methylcytosine [5-meC] and DNA [cytosine-5-]-methyltransferase 1 [DNMT1]) in metastatic breast cancers of variable sites and their associations with clinicopathologic factors. Methods: A total of 126 metastatic breast cancers (31, 36, 11, and 48 bone, brain, liver, and lung metastases, respectively) were made into tissue microarrays, and immunohistochemical staining was performed for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER-2), Ki-67, 5-meC, and DNMT1. Molecular classification was made on the basis of the immunohistochemical staining results of ER, PR, HER-2, and Ki-67, as luminal A, luminal B, HER-2-positive, and triple-negative breast cancers. Results: Methylation-related proteins were differentially expressed based on the metastatic sites. Tumoral and stromal 5-meC showed the lowest expression in bone metastases (P < 0.001), while tumoral DNMT1 showed the lowest expression in bone metastases and the highest expression in brain metastases (P < 0.001). Expression of DNMT1 correlated with ER-negativity (P = 0.004), PR-negativity (P = 0.011), HER-2-positivity (P = 0.016), higher Ki-67 labeling indices (P = 0.016), and non-luminal A type (P = 0.017). Univariate analysis revealed that DNMT1-positivity was associated with shorter overall survival in patients with bone (P = 0.017) and lung metastases (P = 0.028). Conclusion: In metastatic breast cancer, methylation-related proteins are differentially expressed according to the metastatic sites. Tumoral and stromal 5-meC showed the lowest expression in bone metastases, while tumoral DNMT1 expression was low in bone metastases and the highest in brain metastases. Citation Format: Lee Yukyung, Kim Eun-Sol, Koo Ja Seung. Expression of DNA methylation-related proteins in metastatic breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A39.

Abstract B39: Asparagine drives translational adaptation of cancer cells to glutamine deficit

Abstract Most proliferating cells consume exogenous glutamine to fuel biosynthesis and cell growth, which leads to its marked depletion from the microenvironment. Glutamine provides carbon and nitrogen for a wide array of biomolecules, among which are non-essential amino acids, glucosamine and nucleotides; in addition, carbon backbone of glutamine is used as a preferred anaplerotic input into the TCA cycle. However, as recent in vivo metabolomic studies indicate, a number of tumor types are capable of utilizing alternative anaplerotic routes and, in fact, synthesize glutamine de novo. In addition, select cancer cell lines are able to proliferate in the absence of exogenous glutamine in culture medium. However, the metabolic and regulatory underpinnings that enable the cellular adaptation to glutamine deficit and de novo glutamine production need further investigation. In this work, we identified the non-essential amino acid asparagine as essential for the survival and proliferation of cancer cells in the absence of exogenous glutamine supply. Our data indicate that asparagine, though structurally related to glutamine, is not catabolized in the same manner. Instead, asparagine acts by restoring protein translation, which becomes profoundly compromised in the absence of glutamine. This, in turn, enables the post-transcriptional adaptive upregulation of glutamine synthetase (GLUL), which is required for glutamine-independent growth. Taken together, these findings show that the cellular adaptation to glutamine-limiting conditions relies upon the availability of asparagine, which may represent an important therapeutic vulnerability. Citation Format: Natalya N. Pavlova, Ji Zhang, Craig B. Thompson. Asparagine drives translational adaptation of cancer cells to glutamine deficit. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B39.

Abstract B44: Protein interactions involving LARP1 in chemotherapy resistant ovarian cancer cells

Abstract Ovarian cancer (OC) is the most lethal gynecological malignancy accounting for 152,000 deaths annually worldwide. Due to the lack of a validated diagnostic tool, patients usually present with disseminated disease and the majority will develop resistance to platinum chemotherapy. Resensitizing OC to cisplatin-based chemotherapy is an unmet clinical need. The RNA-binding protein LARP1 is highly expressed in OC and is a post-transcriptional regulator of cell survival and tumorigenesis. LARP1 depletion using RNA interference (RNAi) restores platinum sensitivity in cisplatin-resistant OC cell lines and shows a synergistic anti-tumour effect with cisplatin. We explored the effect of this resensitization on the efficiency of protein translation using the Surface Sensing of Translation (SUnSET) method. Whilst cisplatin-induced genotoxic stress attenuated de novo protein synthesis in platinum sensitive OVCAR3 cells, this was not observed in platinum-resistant OVCAR8 cells where residual protein synthesis was maintained. However this residual translation was completely abolished after RNAi to LARP1 suggesting that, in the OVCAR8 cells, LARP1 maintains protein synthesis during genotoxic stress. Although LARP1 is known to bind Poly A binding protein (PABP) we questioned whether other interacting partners might also regulate this response. Using immunoprecipitation followed by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) in OVCAR3 and OVCAR8 cells before and after cisplatin treatment, we identified PABPC1, PABPC4 and YB-1 as strong interactors with LARP1. By western blotting, Duolink Proximity Ligation Assay and Immunofluorescence we then confirmed these candidates as being complexed with LARP1. We are currently elucidating the functional role of these interactions on the LARP1-mediated genotoxic stress response with the overall aim of identifying therapeutic strategies to overcome platinum-resistance in ovarian cancer. Citation Format: Chara Stavraka, Manuela Mura, Roman Fischer, Marie-Laetitia Thezenas, Sadaf Ghaem Maghami, James Chettle, Laki Buluwela, Benedikt Kessler, Sarah P. Blagden. Protein interactions involving LARP1 in chemotherapy resistant ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B44.

Abstract B10: Inhibiting eIF4F-mediated adaptive translatome reprogramming is a salvage strategy for targted melanoma therapy

Abstract BRAFV600E mutation is observed in ~50% of melanomas. Once melanomas have progressed with acquired resistance to BRAF-targeted therapy, mutational heterogeneity presents a major challenge. While enormous effort has gone into understanding the molecular events in mutational acquired resistance, not much attention has been attributed to what happens during therapy phase when patients still respond to BRAF-targeted treatment. It has been hypothesized that initally drug-tolerant cells without bona fide mutational resistance may survive by cellular adaptations and undergo evolution over time towards acquired genetic mutational resistance. We defined the nature of eIF4F-mediated translatome reprogramming and its role during the early phase of targeted melanoma therapy before acquired mutational resistance developed. To explore the possibility that melanoma cells can develop resistance via drug-tolerant evolution (versus pre-existing mutation), we cultured over 1000 small pools (5000 cells each) of parental A375 cells (BRAFV600E) in the presence of vemurafenib and cobimetinib and monitored the emergence of resistant clones. 99.7% of the wells contained a small number of surviving, drug-tolerant cells after 3 weeks of drug exposure. Strikingly, to further demonstrate adaptive drug-tolerant cells exist as a mechanism of resistance evolution, we established A375 subclones derived from single cells to eliminate possible pre-existing mutational resistant cells. Indeed, in five independent single-cell subclones, no early resistant colonies emerged after 3 weeks of drug treatment. These drug-tolerant cells are transiently resistant to re-exposure of anti-BRAF or anti-MEK treatment, and the cells drifted back to the similar sensitivity compared to parental cells after ~9 days of drug-free culture. These drug-tolerant cells are instead more sensitive to eIF4F inhibition compared to parental cells, indicating that eIF4F plays a central role in reprogramming adaptive translatome of drug-tolerant cells. To study the eIF4F activity at single cell level, we developed a single cell image analysis pipeline by using proximity ligation assay and automatic image analysis. By cell image machine learning, we identified five types of eIF4F localization in a large population of melanoma cells. With the classification model, we classified each single drug-tolerant cell into one of the five localization type. Rather than modifying global translation rate in drug-tolerant cells, we revealed that cell-to-cell heterogeneity of the subcellular localization of eIF4F complex discriminates the parental and drug-tolerant cells. This indicates that differential localized eIF4F might initiate the translation of different subsets of genes in drug-tolerant cells. Particularly, the localization pattern of eIF4F is also dynamic. Consistent with the sensitivity of the drug-tolerant cells, the localization pattern of eIF4F drifted back to the similar distribution of parental cells after ~9 days of drug-free culture. Thus, we delineated a novel level of adaptive regulation of gene expression at translational level during the course of targeted melanoma therapy, which might provide a general mechanism of network rewiring during tumor evolution. This translational controlled network reprogramming is likely to be missed if one looks merely at the genetic alone, we thus stress the need to break the current “glass ceiling” of relying solely on genomic dataset analysis to advance cancer therapies. Note: This abstract was not presented at the conference. Citation Format: Shensi Shen, Caroline Robert, Stephan Vagner. Inhibiting eIF4F-mediated adaptive translatome reprogramming is a salvage strategy for targted melanoma therapy. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B10.

Abstract A42: Role of cellular and extracellular Par-4 expression in tumor formation and drug sensitivity

Abstract Breast cancer is a complex and multifactorial disease, making their categorization an extremely demanding task, especially with regard to therapeutic response and disease progression. Understanding the biological basis of formation and tumor progression of breast cancer is very important for the development of new, more specific and effective therapeutic strategies. Experimental evidences indicate that the pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator, also known as Par-4, Prostate Apoptosis Response-4) plays an important role in the selective induction of apoptosis in a wide variety of cancer cells. Par-4 acts both in intrinsic and extrinsic pathway of apoptosis and studies have shown that systemic expression of Par-4 confers resistance to the development of spontaneous tumors in mice. Furthermore, the recombinant Par-4 injected intravenously in mice inhibited tumor growth and metastasis. Previous data from our group demonstrated that reduced nuclear expression of Par-4 is associated with poor prognosis in breast cancer. However, little is known regarding the Par-4's role in tumorigenesis of breast cancer. In the present study, we sought to evaluate the effects of suppression of Par-4 in proliferation, survival, and chemosensitivity of breast cancer cells in vitro and in vivo. MDA-MB-231 breast cancer cells were transfected with different sequence of the pRS vector containing the shRNA to silencing the Par-4. Western Blot technique was used to characterize the clones silenced for Par-4. Using fluorescence-based microplate assay and flow cytometry we evaluated the effect of reduced expression of Par-4 in the rate of cell proliferation and cell cycle. In our results, no difference was observed in the proliferation rates and in the phases of cell cycle between MDA-MB-231 cells with reduced expression of Par-4 in comparison with the control cells. On the other hand, by flow cytometry we have found that decreased expression of Par-4 reduces the sensitivity of the MDA-MB-231 cells to treatment with chemotherapeutic agents as docetaxel and 5-Fluorouracil. Interestingly, by flow cytometry with Annexin and PI, we have observed a significant reduction in the percentage of late apoptosis in MDA-MB-231 cells with reduced expression of Par-4 in comparison with the control cells after 24 hours of treatment with docetaxel (19.50% and 32.64%, p<0.05). Furthermore, MDA-MB-231 cells with reduced expression of Par-4 also showed reduced activation of caspase-7. To confirm if the effect of reduced expression of Par-4 also extends to in vivo experiments, MDA-MB-231 cells transfected with silencing vector for Par-4 or the empty vector were injected into the left flank of Balb/c NUDE animals. Although preliminary, our data indicate that mice injected with breast cancer cells with reduced expression of Par-4 develop tumours twice as fast as mice injected with the control cells. Our findings could be of particular interest in to highlight the important role of Par-4 in breast tumour formation and in the modulation of chemiosensitivity. Supported by FAPESP and CNPq. Citation Format: Lourival A. Oliveira Filho, Maria José Carlini, Maria Aparecida Nagai. Role of cellular and extracellular Par-4 expression in tumor formation and drug sensitivity. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A42.

Abstract A24: Translation control of tumor metastasis via FSTL1 binding with SPP1/OPN to modulated cytoskeleton remodeling

Abstract Follistatin-like protein 1 (FSTL1) plays a critical role in lung organogenesis. However, the prognostic significance and functional consequence of FSTL1 downregulation remains unclear in lung cancer. Here we show that the reduced levels of FSTL1 are widely detected in tumors compared to normal tissues and reflect a poor clinical outcome in non-small cell lung cancer, especially lung adenocarcinoma. Moreover, we found that FSTL1 expression negatively correlates with the metastatic potential of lung cancer cells in vitro and in vivo. Whereas the antibody-based neutralization of extracellular FSTL1 enhances the cellular migration/invasion ability, the inclusion of recombinant FSTL1 protein diminishes the metastatic capacity of lung cancer cells in vitro and in vivo. Notably, the administration of FSTL1 was capable of effectively preventing the metastatic progression of lung cancer cells in an orthotropic animal model. The in vitro protein-protein interaction (PPI) assay demonstrated that FSTL1 directly binds to the pro-form of secreted phosphoprotein 1 (SPP1), also named osteopontin (OPN), and thereby restrains the proteolytic activation of SPP1/OPN, which leads to the inactivation of integrin/CD44-associated signaling cascades and actin cytoskeleton. Runx2 and osterix transcription factors are required for the expression of SPP1/OPN. Thus, FSTL1 compromised the SPP1/OPN related translation control activity through PPI mechanism to reverse the phenotype in lung cancer. The signature of combining low-level FSTL1 with high-level SPP1 predicts a worse prognosis in lung cancer patients. This study is the first to document the PPI between FSTL1 and SPP1/OPN and our findings provide a novel and effective strategy to combat SPP1/OPN-driven metastatic cancers with FSTL1 downregulation. Citation Format: Jean Chiou, Yuan-Feng Lin, Michael Hsiao. Translation control of tumor metastasis via FSTL1 binding with SPP1/OPN to modulated cytoskeleton remodeling. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A24.

Abstract IA03: mTORC1/S6K1: Regulation of RNA biogenesis, protein synthesis, and cell metabolism

Abstract The mTOR Complex 1 (mTORC1) signaling pathway has evolved to sense and respond to cellular energy status, nutrient availability and surrounding oxygen concentrations. In addition, mTORC1 signaling can be further activated by mitogen- and hormone-stimulated kinases including Akt, ERK and RSK, and suppressed by mTORC1 and its effector S6K1, via a variety of negative feedback loops. This incredible integration of extracellular and intracellular cues translates into carefully regulated outputs that control cell growth, motility and survival. A major mechanism through which mTOR regulates downstream processes is via the regulation of gene expression, mRNA processing, protein synthesis and protein degradation. In an mTOR phosphoproteomic screen, we and others identified SR Protein Kinase 2 (SRPK2) as a potential downstream effector of mTORC1. I will discuss a novel mechanism by which SRPK2 is regulated and contributes to mTORC1-dependent regulation of lipid metabolism via the regulation of mRNA processing. Citation Format: John Blenis, Gina Lee, Jamie Dempsey, Christina England. mTORC1/S6K1: Regulation of RNA biogenesis, protein synthesis, and cell metabolism. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA03.

Abstract PR02: A novel 40S complex involved in the regulation of 5' TOP mRNA stability: A role in mTOR hyperactivated tumors?

Abstract The ability of ribosomes to translate genetic information into protein is a hallmark of evolution. However, during cancer progression, tumors often hijack and boost ribosome biogenesis in their favor, which they rely on for high rates of proliferation. In eukaryotes four distinct ribosomal RNAs (rRNAs) and approximately 80 different ribosomal proteins (RPs) are assembled into either the small 40S or large 60S subunit, while more than 300 additional factors participate in the complex process of ribosome biogenesis. Moreover, with the rise of multicellular organisms in higher eukaryotes a hierarchical translational control mechanism of cellular RP mRNAs has evolved. This mechanism has been put into place to guarantee that the building blocks of ribosomes are preferentially translated and integrated with those of the transcriptionally regulated rRNAs, in order to endow the cell with adequate protein synthetic capacity in different tissues upon cellular demand. In higher metazoans this translational mechanism is regulated through a cis regulatory element at the transcriptional start site of RP mRNAs, termed a 5'-terminal oligonucleotide pyrimidine (5' TOP) sequence. We previously demonstrated that 5' TOP mRNA expression at the translational levels is controlled by mTORC1 signaling, a central nexus of cellular growth and proliferation that is often exploited by tumor cells. Although it is thought that 5' TOP translation is mediated by mTORC1, new findings have suggested that their stability also may be selectively controlled by the 5' TOP element. Here we show that the 40S ribosomal subunit, in concert with RNA binding protein LARP1, take on this responsibility, through forming a novel complex with 5' TOP mRNAs. Importantly, we show that the repertoire of 5' TOPs mRNAs recognized by this new 40S complex encompasses categories of transcripts not only belonging to RP and translational factor mRNAs, but also to previously unidentified 5' TOPs involved in distinct cellular processes. LARP1 overexpression, like increased ribosome biogenesis is associated with many aggressive cancers. Thus the functional interaction between LARP1, the 40S subunit and 5' TOPs constitutes a cellular reservoir of RP mRNAs and other factors that can be translationally mobilized or suppressed upon cellular growth demands, particularly in tumors where mTOR signaling is hyperactivated. This abstract is also being presented as Poster A27. Citation Format: Antonio Gentilella, Francisco D. Morón-Duran, Guilherme Z. Rocha, Ferran Riaño-Canalias, Joffrey Pelletier, Albert Tauler, Sara Kozma, George Thomas. A novel 40S complex involved in the regulation of 5' TOP mRNA stability: A role in mTOR hyperactivated tumors? [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr PR02.