New Frontier In Cancer Biology Research Articles

Cuproptosis: unveiling a new frontier in cancer biology and therapeutics.

Copper plays vital roles in numerous cellular processes and its imbalance can lead to oxidative stress and dysfunction. Recent research has unveiled a unique form of copper-induced cell death, termed cuproptosis, which differs from known cell death mechanisms. This process involves the interaction of copper with lipoylated tricarboxylic acid cycle enzymes, causing protein aggregation and cell death. Recently, a growing number of studies have explored the link between cuproptosis and cancer development. This review comprehensively examines the systemic and cellular metabolism of copper, including tumor-related signaling pathways influenced by copper. It delves into the discovery and mechanisms of cuproptosis and its connection to various cancers. Additionally, the review suggests potential cancer treatments using copper ionophores that induce cuproptosis, in combination with small molecule drugs, for precision therapy in specific cancer types.

Shining a light on the dark proteome: Non-canonical open reading frames and their encoded miniproteins as a new frontier in cancer biology.

In the decades following the discovery that genes encode proteins, scientists have tried to exhaustively and comprehensively characterize the human genome. Recent advances in computational methods along with transcriptomic and proteomic techniques have now shown that historically non-coding genomic regions may contain non-canonical open reading frames (ncORFs), which may encode functional miniproteins or otherwise exert regulatory activity through coding-independent functions. Increasingly, it is clear that these ncORFs may play critical roles in major human diseases such as cancer. In this review, we summarize the history and current progress of ncORF research and explore the known functions of ncORFs and the miniproteins they may encode. We particularly highlight the emerging body of evidence supporting a role for ncORFs and miniproteins contributions in cancer. Finally, we provide a blueprint for high-priority areas of future research for ncORFs in cancer, focusing on ncORF detection, functional characterization, and therapeutic intervention.

Mathematical oncology: A new frontier in cancer biology and clinical decision making: Comment on "Improving cancer treatments via dynamical biophysical models" by M. Kuznetsov, J. Clairambault & V. Volpert.

Mathematical oncology: A new frontier in cancer biology and clinical decision making: Comment on "Improving cancer treatments via dynamical biophysical models" by M. Kuznetsov, J. Clairambault & V. Volpert.

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 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 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 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 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 A40: Expression of cancer-associated fibroblast-related proteins differs between invasive lobular carcinoma and invasive ductal carcinoma

Abstract Purpose: Cancer-associated fibroblasts (CAFs) are classified into various functional subtypes such as fibroblast activation protein-α (FAP-α), fibroblast specific protein-1 (FSP-1), platelet-derived growth factor receptor-α (PDGFR-α), and PDGFR-β. In this study, we compared the expression of CAF-related proteins in invasive lobular carcinoma (ILC) with those in invasive carcinoma of no special type (NST), and assessed the implications of the differences observed. Methods: Using tissue microarrays of 104 ILC and 524 invasive carcinoma (NST) cases, immunohistochemistry for CAF-related proteins [podoplanin, prolyl 4-hydroxylase, FAP-α, FSP-1/S100A4, PDGFR-α, PDGFR-β, and chondroitin sulfate proteoglycan (NG2)] was conducted. Results: In invasive carcinoma (NST), tumor cells expressed a high level of PDGFR-α, whereas ILC tumor cells expressed high levels of podoplanin, prolyl 4-hydroxylase, FAP-α, and FSP-1/S100A4. In stromal cells of invasive carcinoma (NST), high expression levels of prolyl 4-hydroxylase, PDGFR-α, and NG2 were observed, whereas ILC stromal cells expressed high levels of FAP-α, FSP-1/S100A4, and PDGFR-β. In ILC, tumoral FSP-1/S100A4 positivity was associated with higher Ki-67 labeling index (p = 0.010) and non-luminal A type cancer (p = 0.014). Stromal PDGFR-α positivity was associated with lymph node metastasis (p = 0.011). On survival analysis of entire cases, tumoral FSP-1/S100A4 positivity (p = 0.001) and stromal podoplanin positivity (p = 0.035) were associated with shorter disease-free survival; only tumoral FSP-1/S100A4 positivity (p = 0.001) was associated with shorter overall survival. Conclusion: In ILC, the expression of FAP-α and FSP-1/S100A4 was higher in both tumor and stromal cells than that observed in invasive carcinoma (NST). These results indicate that CAFs are a potential target in ILC treatment. Citation Format: Lee YuKyung, Kim Eun-Sol, Koo Ja Seung. Expression of cancer-associated fibroblast-related proteins differs between invasive lobular carcinoma and invasive ductal carcinoma. [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 A40.

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.

Abstract PR03: NRF2 promotes tumor maintenance by modulating mRNA translation in pancreatic cancer

Abstract Pancreatic Ductal Adenocarcinoma (PDA) is the 4th leading cause of cancer death in the USA. Lethality of PDA is largely ascribed to poor drug delivery and augmented cell survival pathways. We previously found that oncogenic Kras expression induced an important regulator of redox control, the transcription factor Nuclear factor erythroid-derived 2-like 2, Nfe2l2/Nrf2. Expression of Nrf2-dependent proteins is critical for neutralizing or eliminating toxicants and to maintain cellular redox homeostasis. The NRF2 transcriptional program is believed to protect neoplastic cells from oxidative stress, and may confer the resistance of these cells to chemotherapy. Thus, antagonizing NRF2 effector functions represents an attractive therapeutic strategy. Activation of Nrf2 leads to alterations in cellular redox levels, to which cysteine residues are particularly reactive. Redox modifications on reactive cysteines may regulate the activity of their corresponding protein, rendering these proteins as candidate redox-sensitive effectors of NRF2. To decipher changes in the cysteine proteome, we devised a highly sensitive proteomic method that combines a selectively cleavable cysteine-reactive affinity tag to enrich for and identify reduced cysteines, with amine-reactive isobaric tags for relative and absolute quantification of the total proteome. Using this approach, we identified cysteines on translational regulatory proteins to be explicitly oxidized in Nrf2-deficient, Kras mutant cells. Both cap-dependent and -independent mRNA translation was impaired in Nrf2-deficient pancreatic cancer cells, and can be rescued upon supplementation with antioxidants. In addition to stimulating translation through maintaining the reduced state of specific cysteine residues, redox regulation by Nrf2 also promotes EGFR autocrine signaling through AKT in KRAS mutant cells to fuel cap-dependent translation initiation. These functions converge to promote global protein synthesis in PDA. As a consequence, combined inhibition of AKT signaling and glutathione synthesis hampered the survival of PDA cells in vitro and in vivo, presenting a new opportunity for therapeutic intervention. This abstract is also being presented as Poster B31. Citation Format: Iok In Christine Chio, Seyed Mehdi Jafarnejad, Mariano Ponz-Sarvise, Youngkyu Park, Keith Rivera, Wilhelm Palm, Daniel Ohlund, Molly Hammell, Howard Crawford, Edward Schmidt, Craig Thompson, Darryl Pappin, Nahum Sonenberg, David Tuveson. NRF2 promotes tumor maintenance by modulating mRNA translation in pancreatic 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 PR03.

Abstract B23: Inhibition of ribosomal RNA synthesis as a new therapeutic approach to treat advanced prostate cancer

Abstract Background: Prostate epithelium is exquisitely sensitive to the overexpression of the proto-oncogene MYC which causes neoplastic transformation. Indeed, MYC protein is almost universally overexpressed in metastatic castration-resistant prostate cancer (CRPC) making targeting MYC an attractive option for treating advanced stage disease. Unfortunately, the development of therapeutic agents directly targeting MYC has been largely unsuccessful, thus emphasizing the need to indirectly target MYC activity through inhibition of downstream cellular processes it regulates. One of the main effects of MYC in cancer cells is to accelerate proliferative growth via stimulation of high levels of ribosome biogenesis. Accordingly, the control of protein synthesis rate has emerged as the “Achilles' heel” of a wide array of tumors. MYC also regulates and cooperates with PIM kinases to increase the activity of the eIF4F translation initiation complex and MYC-driven tumors are addicted to eIF4E. Here, we investigate the efficacy of a single and dual approach targeting ribosome biogenesis and function to treat prostate cancer (PC). Experimental design: We employed numerous models of PC, including a novel CRPC patient derived xenograft system, which showed the pre-clinical efficacy of therapies that combine to target MYC directed signaling to the ribosome. The inhibition of ribosomal RNA (rRNA) synthesis with CX-5461, a potent, selective and orally bioavailable inhibitor of RNA polymerase I (Pol I) transcription has been successfully exploited therapeutically, but only in models of hematological malignancy. CX-5461 and CX-6258, a pan-PIM kinase inhibitor, were tested alone and in combination in PC cell lines, in Hi-MYC and PTEN-deficient mouse models and in patient derived xenografts (PDX) of metastatic tissue obtained from a castration-resistant PC patient. Results: CX-5461 inhibited anchorage-independent growth and induced cell cycle arrest in PC cell lines at nanomolar concentrations. Oral administration of 50 mg/kg CX-5461 induced p53 expression and activity and reduced proliferation (Ki-67) and invasion (loss of ductal actin) in Hi-MYC tumors, but not in PTEN null (low MYC) tumors. While 100 mg/kg CX-6258 showed limited effect alone, its combination with CX-5461 further suppressed proliferation and dramatically reduced large invasive lesions in both models. This rational combination strategy significantly inhibited proliferation and induced cell death in PDX of PC. Conclusion: Our results demonstrate preclinical efficacy of targeting the ribosome at multiple levels and provide a new approach for the treatment of PC. In addition, a key conclusion of our study is that the androgen receptor (AR) presence or activity has no significant impact on the therapeutic activity of our novel combination therapy. Therefore, we believe our new exciting combination therapy could be used in the clinic in combination with current anti-androgens or as salvage therapy in multi-drug resistant CRPC. Citation Format: Richard J. Rebello, Eric Kusnadi, Don P. Cameron, Helen B. Pearson, Analia Lesmana, Jennifer R. Devlin, Denis Drygin, Ashlee K. Clark, Laura Porter, John Pedersen, Shahneen Sandhu, Gail P. Risbridger, Richard B. Pearson, Ross D. Hannan, Luc Furic. Inhibition of ribosomal RNA synthesis as a new therapeutic approach to treat advanced prostate 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 B23.

Abstract A47: Immunogenic cancer cell death induced by high hydrostatic pressure and hyperthermia: Calreticulin exposure pathway

Abstract Immunogenic cell death (ICD) is a type of cell death induced by various chemotherapeutics, physical modalities such as photodynamic therapy, radiotherapy, severe hyperthermia (sHT) or high hydrostatic pressure (HHP). HHP and sHT are able to trigger a specific antitumor immune response and induce the rapid translocation of calreticulin (CRT) to the plasma membrane surface (ecto-CRT). Ecto-CRT on dying tumor cells represents an “eat me” signal for dendritic cells as well as a major molecular determinant that makes the difference between immunogenic and non-immunogenic cell death. Nevertheless, the mechanisms implicated in CRT translocation after HHP treatment is unknown. Inducers of ICD trigger the ER stress response, Reactive Oxygen Species (ROS) generation and Ca2+ release. Kinetics of ecto-CRT was assessed in lung A549 and ovarian OV90 tumor cell lines. CRT localization in sHT- and HHP-treated tumor cells was tracked by endosomal staining (caveolin, clathrin, early and late endosomes) and visualized by confocal microscopy. For detection of CRT translocation pathway, the tumor cells were pretreated with selected cytoskeletal inhibitors, inhibitor of intracellular transport, ER stress activator or small interfering RNA followed by sHT and HHP treatment. The cells were analyzed by flow cytometry and confocal microscopy. Plasma membrane fraction and endosomes were isolated and analyzed by western blotting. Our results indicate that the ROS - PERK - eIF2α - caspase 2 - caspase 8 - caspase 8-mediated cleavage of the ER protein BAP31 is central for the perception of HHP-driven cell death as immunogenic. On the other hand our first results indicate that none of these processes are involve in exposure of CRT on cell surface in sHT treated tumor cells. We observed that sHT treatment does not lead to caspase 8 activation and Bap31 cleavage. However, we showed that translocation of CRT to the cell surface is dependent on endoplasmic reticulum-sessile kinase PERK and presence of Bax/Bak proteins. CRT colocalization with clathrin in tumor cells after HHP and particularly with early endosomes was detected. Surprisingly, we also did not observe colocalization with any endosomes in sHT treated tumor cells. Further, inhibition of microtubule-dependent transport with Nocodazole D results in decrease of CRT transport on cell surface in HHP treated tumor cells. Similar but less significant effect was seen when Brefeldin A was used to inhibit anterograde transport in both treatments. Inhibition of actin polymerization by Cytochalasin D did not significantly affect CRT translocation. In conclusion, PERK seems to play a central role in the exposure of CRT upon induction of ICD. However, up-stream and downstream signaling cascades differ depending the modality used to induce ICD, such as HHP or sHT. Citation Format: Sarka Vosahlikova, Irena Adkins, Linda Urbanova, Nada Hradilova, Lenka Sadilkova, Radek Spisek, Irena Moserova. Immunogenic cancer cell death induced by high hydrostatic pressure and hyperthermia: Calreticulin exposure pathway. [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 A47.

Abstract A31: eIF4F links translation to energy stress response in cancer

Abstract Protein synthesis is one of the most energy consuming process in the cell. Oncogenic kinases (e.g. EGFR/HER2, BCR/ABL and BRAF) play a central role in reprogramming translation and energy metabolism in neoplasia, whereby cancer cells must provide sufficient ATP to support increased levels of protein synthesis required for neoplastic growth. The downstream mechanisms that link translational machinery and energy homeostasis in cancer, however, remain largely unknown. We found that widely used anti-diabetics (biguanides) abrogate adaptations to EGFR/HER2 inhibitor-induced energetic stress, which results in synergistic anti-neoplastic effects both in vitro and in vivo. In turn, breast cancer cells in which 4E-BP1/2 expression was abrogated by CRISPR were partially resistant to the combination of EGFR/HER2 inhibitors and biguanides. This was paralleled by the inability of the drugs to inhibit the eIF4F complex assembly and translation of mRNAs encoding important metabolic regulators including those involved in serine biogenesis (PHGDH, PSAT1) and one carbon metabolism (MTHFD1L). Comparable results were observed when BRAF and BCR/ABL inhibitors were combined with biguanides, which suggests that translational regulation of metabolic genes via the mTORC1/4E-BE/eIF4E pathway plays a major role in energy stress response in cancer. Together our findings demonstrate that the eIF4F complex is an important mediator of metabolic adaptation in response to the combination of biguanides and clinically-used kinase inhibitors and suggest that the efficiency of such anti-cancer strategies are dependent on the integrity of the translation initiation machinery. Citation Format: Laura Hulea, Marie Cargnello, Simon-Pierre Gravel, Young Im, Shannon McLaughlan, Yunhua Zhao, Jenna Ching, Yutian Cai, Ola Larsson, Michael Ohh, Josie Ursini-Siegel, Julie St-Pierre, Michael Pollak, Ivan Topisirovic. eIF4F links translation to energy stress response 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 A31.