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Multi-omics approach reveals mechanisms of proliferation control in cancer

In a recent paper, published in Science, researchers used a multi-omics approach to identify peptides, transcripts, and phosphorylation events and how they change with cell size in melanoma cell lines. This study is one of the first to demonstrate how size and morphology can influence the chemistry of a cancer cell.

Size matters!

The size of eukaryotic cells can vary widely and can affect many processes such as nutrient acquisition and intracellular protein concentration. To keep cell sizes consistent within a population, a checkpoint system can measure the size of individual cells with molecular “rulers” and adjust the cellular division cycle or amount of mass acquired. The concentration of proteins can also be regulated by cell size, in a process known as sub- or super-scaling.

In this study, researchers leveraged the heterogeneity of melanoma cell lines to identify peptides, transcripts, and phosphorylation events that differentially scale with cell size and revealed that size-scaling phenomena in cancer can impact cell cycle progression and intracellular signalling pathways.

The researchers first quantified the morphology of over 17,000 single cells from 11 mouse melanoma cell lines from 3 different genetic backgrounds and investigated the relationship between cell size and clinically relevant oncogenic drivers. Using this data, they defined 3 classes of cells – small, larger, and largest. They then examined the relationship between DNA content and cell size by quantifying the nuclear content of cells, finding that cell size is linearly related to DNA content. This means that as DNA content goes up, cell size goes up as well.

Probing the proteome

To elucidate how molecular drivers regulate cell size, the researchers created a proteomic dataset of over 9000 peptides and identified phosphorylation events on over 4000 peptides. Previous studies have shown that the proteins RB1 and CCND1 are related to cell size control, and have shown that “in normal mammalian cells, cells meet the RB1:CCDN1 set point for proliferation, by synthesizing CCND1 while simultaneously diluting RB1”.

In this study, researchers found that larger cells exhibited a lower mean concentration of RB1, meaning RB1 subscales with size across cell lines. They then quantified the signalling activity upstream of CCND1 by measuring the phosphorylation state of CCND1 transcriptional regulators (CCND1regs). They found again that larger cells exhibited a lower mean concentration of CCND1regs. In other words, the larger the cell, the less proliferative signalling.

The researchers identified other sub- and super-scaling species in their dataset and found that regulators of the G2-M phase checkpoint (which stops cells from undergoing mitosis when damaged intracellular DNA is present), translation, and growth subscale with size across lines. However, proinflammatory proteins, extracellular matrix (ECM) components, regulators of the cytoskeleton, and certain growth factor receptors superscale. The researchers then integrated transcriptomic data and found that scaling is regulated by transcription events (Figure 1A).

Why won’t the cells stop growing?

Despite the sub-scaling in growth regulators and proliferative signalling, larger cells did not have a notably decreased growth rate. To understand why, researchers live-imaged 2 cell lines from each genotype spread across different sizes of cells and quantified the average rate of growth. They found that growth rate actually  increased with cell size (Figure 1B).

The researchers then investigated mTOR signalling and the phosphorylation of upstream regulators in cells of different sizes and discovered that the primary activating phosphorylation sites of mTOR (S2448 and S2481) are under-phosphorylated in larger cells, while some upstream regulators (such as IRS1) are differently phosphorylated across cell sizes. As mentioned before, cytoskeletal peptides were found to be more abundant and phosphorylated in larger cell lines, and cytoskeletal activity driven by receptor tyrosine kinase activation (as indicated by the presence of HER2, SRC, PAK4, ROCK1, VASP, and LIMK1) is up-regulated in larger cell lines. Based on these findings, the authors hypothesize that larger cells may have skewed their RTK signalling towards cytoskeleton reorganization rather than anabolic processes, which may contribute to their increased size and spreading.

The researchers then developed a theoretical model (Figure 1C and D) to demonstrate how the larger cells continued to proliferate. Analysis of cell size variation showed that the cell line G1 distributions were best modelled by a one-growth stage cycle, suggesting a central role for the RB1 scaling observed in these cells. Larger cell lines showed an up-regulated inflammatory response and decreased DNA:cytoplasm ratio, while maintaining high growth rates despite the down-regulation of anabolic pathways and decreased ribosomal mass fractions. The mechanism behind this is unclear and needs further investigation.

The α (mitogenic signaling) and β (growth rate) parameters of the model were best predicted by the expression of the TP53/CDKN1A/CCND1 pathway. High CDKN1A and TP53 mass fractions were mutually exclusive in large, high growth rate cell lines, with one explanation being that increased size provokes stress and up-regulation of TP53 and CDKN1A. However, whether this up-regulation is critical to their proliferation or simply a signature of senescent-like states is not yet clear. More research is needed to further investigate the impact of cell size and morphology on cell chemistry.

Figure 1 ¦ Summary of methods and findings showing how cancer cells increase in size and how proliferation and growth rates continue to scale