Cancer Aetiology

Translation is a key process in a wide range of cancers, being significantly altered to drive its initiation and progression. Practically all oncogenic signalling pathways (including RAS–MAPK, PI3K–AKT–mTOR, MYC and WNT–ß-catenin) are implicated in translational reprogramming and many oncogenes (including KRAS, PTEN, APC, and EGFR) also regulate signalling pathways targeting translation.  It is a process that often differs from its transcriptional counterpart, and thus alternative tools are needed to gain the necessary insights. Translatomic tools such as ribosome profiling specifically targets translating ribosomes, identifying crucial features of translation which allow researchers to decipher the intricacies and details of this process.

The following papers exemplify how ribosome profiling is illuminating the crucial role translation plays in cancer development and progression.

The translational landscape of mTOR signalling steers cancer initiation and metastasis

Nature, 2012; 485(7396):55-61
Hsieh, A.C., Liu, Y., Edlind, M.P., Ingolia, N.T., Janes, M.R., Sher, A., Shi, E.Y., Stumpf, C.R., Christensen, C., Bonham, M.J. and Wang, S

The mammalian target of rapamycin (mTOR) kinase is a regulator of protein synthesis that links nutrient sensing to cellular growth. However, in many cancers it is significantly dysregulated, with its hyperactivity leading to rapid cell growth. Yet, the downstream translational targets of gene expression that may direct cancer progression are poorly characterised. In this paper, the authors utilise ribosome profiling in conjunction with pharmacological inhibition of mTOR in cancer cell lines to establish a map of translationally controlled mTOR target genes.

Key Findings

  • Inhibition of mTOR via PP242 led to decreases of ~150 target genes at the translational level, compared to rapamycin inhibition. Of these genes, 89% of them had an either pyrimidine-rich translational element (PRLE) and/or a 5’ terminal oligopyrimide (TOP) tract.
  • mTOR-sensitive genes stratify into functional categories that may promote tumourigenesis, such as cell invasion, cell proliferation, metastasis, and metabolism. Using a novel mTOR inhibitor (INK128), the authors were able to demonstrate a distinct reduction in the translational expression (but not transcriptional) of the mTOR-sensitive invasion signature (including the genes YB1, MTA1, vimentin, and CD44).
  • Inhibition of 4E-BP1, a downstream effector of mTOR, demonstrated its involvement in the regulation of key genes involved in this invasion signature, and subsequent cancer cell invasion. 
  • It was found that INK128 treatment in an in vivo mouse model of prostate cancer returned phosphorylation levels of 4EBP1 and p70S6K1/2 back to wild type levels. Importantly, this had a real effect on higher level cancer metrics, drastically reducing the levels of cancer cell invasion and the formation of distant metastases. 

Implications

This research has far reaching consequences in cancer biology, with mTOR hyperactivation common in a wide range of cancers. Analysis of the translational landscape as a consequence of mTOR hyperactivation opens up new potentialities for therapeutics in this area, as demonstrated by the authors with the use of a novel inhibitor, displaying promising anti-oncogenic properties at an in vivo level.

Ribosome profiling analysis identified a KRAS-interacting microprotein that represses oncogenic signalling in hepatocellular carcinoma cells

Science China Life Sciences, 2019; 63(4):529-542
Xu, W., Deng, B., Lin, P., Liu, C., Li, B., Huang, Q., Zhou, H., Yang, J. and Qu, L

Long non-coding RNAs (lncRNAs) were previously believed to play no role in peptide formation. However, recent advancements in gene expression technologies have allowed greater insights into these types of RNAs. Here, the authors utilise ribosome profiling to analyse translational efficiencies and coding potential of a variety of lncRNAs in a hepatocyte-derived carcinoma cell line, in an effort to identify potential microproteins involved in hepatocellular carcinoma (HCC).

Key Findings

  • Researchers uncovered roughly 350 ORFs within approximately 130 transcripts that were previously thought of as being lncRNAs. Most of these (~63%) were annotated as small ORFS (sORFs), predicted to encode microproteins (<100 amino acids in length). 
  • Researchers identified KRASIM (KRAS interacting microprotein, KRAS being a protein involved in the RAS pathway, which is implicated in cancer development), a novel micropeptide encoded by a sORF within a previously thought of lncRNA transcript. This peptide was highly conserved across species and had an especially high translational efficiency within this HCC cell line.
  • The KRASIM protein, as well as the mRNA encoding it, was found to be significantly downregulated in HCC tissue samples compared to paired healthy tissue samples. Furthermore, KRASIM gain- and loss-of-function experiments in carcinoma cell lines demonstrated that this protein is associated with suppression of proliferation and colony formation.
  • It was discovered that the KRASIM protein interacted strongly with a number of GTPases, most significant among them being KRAS. Overexpression of KRASIM lead to decreased levels of KRAS in HCC cells, while reduced expression had the opposite effect. Increased KRASIM expression also led to decreased ERK pathway activity, a known oncogenic pathway.
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Implications

This study sheds light on the regulatory mechanisms involved in oncogenic signalling in HCC and opens up new avenues for potential therapeutics. It also demonstrates ribosome profiling’s ability to identify significant effector molecules, opening up fertile ground for discovery of important regulatory mechanisms.

Ribosome profiling reveals a functional role for autophagy in mRNA translational control

Communications Biology, 2020; 3(1):338
Goldsmith J, Marsh T, Asthana S, Leidal AM, Suresh D, Olshen A, Debnath J

Autophagy is an important process in cells, acting to recycle cellular components. It is particularly linked to the nutritional state of the cell, being transcriptionally and post-translationally upregulated during nutrient starvation. Translation is also heavily linked to this cellular state, as a lack of amino acids has subsequent impact on protein synthesis. However, it is unknown if autophagy has direct effects on translation. In this paper, the authors investigate the effects of autophagy on translation through the use of ribosome profiling.

Key Findings

  • In a mammalian cell line, it was found that abolishment of autophagy (through knockdown of ATG12) does not impact on the rate of translation, in contrast to similar experiments carried out in Saccharomyces cerevisiae.
  • Loss of autophagy did not appear to have any impact on amino acid levels, at least in the short-term, which may also explain its lack of an impact on mTORC1 activity.
  • It appears that autophagy loss does have a negative impact on translation of a specific set of mRNA transcripts, namely those involved in DNA repair and cell cycle control, including BRCA2 transcripts.
  • Regarding BRCA2 transcripts, it was found that the 5’ UTR of BRCA2 mRNAs is a locus of control, with reduced interaction between these mRNAs and RNA-binding proteins, such as the helicase eIF4A1, in autophagy-inhibited cells. 

Implications

This research has particularly strong implications for breast cancer research, as mutations in the BRCA2 gene has been strongly implicated in the development of this cancer. It opens up the possibility of targeting autophagy pathways for the treatment of this cancer going forward.  

Cancer2-scaled

RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer

Nature, 2014; 513(7516):65-70
Wolfe, A.L., Singh, K., Zhong, Y., Drewe, P., Rajasekhar, V.K., Sanghvi, V.R., Mavrakis, K.J., Jiang, M., Roderick, J.E., Van der Meulen, J. and Schatz, J.H.

Activation of translational machinery is known to contribute to malignant cancers. Indeed, multiple oncogenic pathways, such as RAS, ERK, and AKT, converge on mTORC1 and the eIF4E binding protein, driving oncogenesis. eIF4A, a member of the translational eIF4F complex, is an RNA helicase needed in the initiation of mRNA translation and altered eIF4A activity has been implicated in the transformation and progression of cancerous cells. In this study, the authors utilise ribosome profiling, in combination with the eIF4A-inhibiting drug Silvestrol, to explore the key characteristics of eIF4A-dependant mRNA transcripts and their role in cancer.

Key Findings

  • In a mouse model of T-cell acute lymphoblastic leukaemia (T-ALL), retroviral expression of eIF4A1 and eIF4E lead to similar levels of leukaemia expression as seen with the known oncogenes NOTCH1 and pTEN. eIF4A1 and eIF4E are also required to maintain 4E-BP1-driven leukaemia cells.
  • In vivo Silvestrol treatment was effective against xenografted T-ALL cells and delayed tumour growth in mice. 
  • Silvestrol treatment of a T-ALL cell line led to significant alterations in translational efficiency (TE) of a number of genes, with there being downregulation of 281 mRNAs and upregulation of 190 mRNAs.
  • Among the mRNAs with a reduced TE, there was a significant increase in the length of their 5’ UTRs compared to the average length. There was also a striking enrichment of the (CGG)4 motif. These motifs also correlated with the presence of G-quadruplex secondary structures, indicating these may play an important role in eIF4A binding. 
  • Many the genes translationally impacted by Silvestrol treatment, including MYC, NOTCH1, MYB, and ETS1 are known to be involved in oncogenic processes.

Implications

This paper sheds light on the mechanisms behind eIF4A binding leading to the overexpression of several key oncogenes and the ability of Silvestrol to counteract these effects. The authors also identified a 5’UTR structural domain which confers translational sensitivity to eIF4A and which may be targeted in future therapies.  As such, this research may provide an interesting basis for investigation of more cost-effective analogs, such as CR-31-B, in cancer research.

Hallmarks and determinants of oncogenic translation revealed by ribosome profiling in models of breast cancer

Translational Oncology, 2020; 13(2):452-470
Vaklavas C, Blume SW, Grizzle WE

Cancer is associated with significant changes in gene expression, leading to rapid cellular growth and malignancies. Analysis of gene expression has been well studied at the transcriptional level, however, research into the translational changes has been comparatively under-studied. Given that translation is a crucial nexus in the development and progression of cancer, insights into this area are of particular importance. In this paper, the authors utilise ribosome profiling to explore the hallmarks of translational expression in breast cancer.

Key Findings

  • It was found that the median ribosome occupancy was higher in malignant cells compared to healthy human mammalian epithelial cells (HMECs), with malignant cells also exhibiting greater variance in translational efficiency (TE), especially under stress conditions.
  • Authors found that, across multiple cancerous cell lines, genes involved in transcriptional regulation, signal transduction, cell adhesion, and translation itself are those most preferentially upregulated at the translational level.
  • There exists a number of distinctly overrepresented motifs in the 5’ UTRs of transcripts found to be translationally upregulated in cancer cell lines. Of the RNA-binding proteins that could most strongly associate with these motifs, eIF4B and SRSF1 ranked highest.
  • Under stress conditions, SRSF1 prioritises the translation of the MYC gene. Its operation is cell cycle dependant, with it likely operating as a splicing factor in the G0 and G1 phases, while translocating to the cytoplasm and acting as an internal ribosome entry site trans-acting factor in the S, G2 and M phases.
  • The MCF10A cell line (used to approximate healthy cells), while aligning with HMECs (another commonly used cell line to approximate healthy cells) in relation to principal component analysis of RNA reads, approached a profile more consistent with a triple-negative cancer cell line when judged with reads isolated from ribosomes.

Implications

Here, pathways, molecules, and molecular characteristics of oncogenic processes have been identified, across a number of breast cancer cell lines, significantly expanding the knowledge regarding translation in this disease. Importantly, researchers call into question the use of the MCF10A cell line for approximating healthy cells, with its translational profile partially overlapp

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