From yeasts and frogs to humans: A tale of battling cancer by understanding cell cycle progression

We’re campaigning to #KeepResearchCurious: highlighting the importance of curiosity-driven cancer research.

In this series of articles we highlight how fundamental research driven by curiosity can lead to advances in medicine, sometimes in unpredictable ways.

One of the hallmarks of cancer is abnormal cell proliferation. This article tells the story of the history and impact that the discovery of cell cycle regulators had on cancer research and treatment.

Follow the journey of how the discovery of the key cell cycle regulators and the understanding of the mechanisms that govern cell cycle progression enabled the development of anti-cancer therapies.

Uncontrolled cell proliferation is a well-known characteristic of cancer cells. Similarly, the tumorigenic potential of DNA lesions has also been long-established as an ‘enabling characteristic’ for tumour progression [1]. Today we know that cyclin-dependent kinases as gatekeepers of cell cycle progression sit at the crossroads of these hallmarks of cancer and several CDK4/6 inhibitors have been licensed for use in the EU and US in recent years (2015-2021) for the treatment of breast and lung cancer [2, 3]. Two different approaches were taken. In breast cancer, CDK4/6 inhibitors were approved to prevent cancer cell proliferation [4]. Small cell lung cancer (SCLC) cells are mostly RB1-deficient and hence do not arrest with CDK4/6 inhibitors and suffer from the toxic effects of chemotherapy. Therefore, in SCLC Trilaciblib was approved to protect normal cells from chemotherapy [5].

Up until today, these medicines have shown consistent progression-free survival results in breast cancer [4]and several similar approaches show tremendous promise in pre-clinical and clinical studies [6]. However, these life-saving medical advances have not been possible, have we not been able to understand how cells regulate their division, and we owe that to scientific discoveries made almost 30 years before the first CDK inhibitor was approved for clinical use.

Looking for the bigger picture: from cell division to the control of the cell cycle

In the mid-1950s, Howard and Pelc were the first to describe the cell cycle as the 4-step process we know today: the G1 phase (for Gap1), the S phase (for DNA synthesis) and the G2 phase (for Gap 2) as part of the interphase and mitosis, or the M phase. By the mid-‘60s, following Lajtha’s finding that cells can also enter a quiescent stage (G0), the cell cycle was well-defined [7-9]. For sustained cell proliferation cells must grow and undergo chromosome duplication and segregation at the same rate. Eukaryotic cells can separate these two processes. The division of the eukaryotic cell cycle into the 4 phases mentioned above was based mostly on chromosome duplication and segregation. However, specific regulatory mechanisms must dictate a cell when to duplicate its genome and when to separate into daughter cells. This is particularly important to avoid cell duplication when DNA damage is present [10]. The concept of the cell cycle as a succession of a clearly defined set of events that occur in a certain order and cannot progress until the completion of previous steps in the process was well established by the mid-’70s and the existence of a regulatory mechanism that controls the transition between these stages was intuited [11, 12].

In 1971, through experiments performed on frog oocytes arrested in metaphase, Masui proved the existence of a ‘maturation/ mitosis promoting factor’ (MPF) in the cytoplasm [9, 13]. Genetic studies performed in yeast cells (Saccharomyces cerevisiae and Schizosaccharomyces pombe) led to the discovery of the basic machinery of the cell cycle, as well as a connection between DNA damage and the ability of a cell to progress through the cell cycle [12, 14, 15]. The two model organisms were ideal for these studies not only because of their genetic amenability but also because cell cycle and cell growth are well coordinated in both fission and budding yeast, so progression through the cell cycle is easily quantified [10, 16].

Approaching one problem from different angles: the power of using different model organisms and different scientific approaches to answer the same question

Lee Hartwell studied the physiology and morphology of wild-type budding yeast and highlighted the relationship between cell growth, cell division and bud formation in fission yeast (S. cerevisiae). Hartwell also studied a variety of mutant yeast cells and through his experiments, he identified a series of mutant cells that despite being stuck at certain stages of their budding cycle, and hence cell cycle progression, would still grow in size. By isolating the mutant genes Hartwell and his team characterised a plethora of genes involved in cell division, many DNA replication genes, but some regulators of the cell cycle, including an impressive collection of CDC genes. Hartwell identified CDC28 (CDK1) and anticipated its importance postulating this gene determines the commitment of the cell to progress through the cell cycle. However, he did not further investigate its function [10, 17].

Later on, work from biochemists such as Lohka from Masui’s laboratory alongside Maller from Krebs’s laboratory continued to study MPF and in 1988 led to the purification of this factor from Xenopus laevis eggs and the identification of the two comprising proteins, a homologue of the yeast cdc2 protein and what was later shown to be cyclin B, a type of protein described a few years earlier by Hunt in sea urchin eggs and thought to be connected to MPF in some way [9, 18, 19]. It took a few more years for the molecular mechanism of MPF activation and deactivation to be understood and described in the literature [9, 20].

At the time, however, Hartwell’s attention was directed towards understanding the fidelity of cell cycle progression. Hartwell noticed that following DNA damage induced by radiation, many mutants were arrested at specific stages of cell cycle progression. He postulated that surveillance mechanisms must be in place to control cell-cycle progression in response to DNA damage [10, 17]. In 1988 Weinert and Hartwell discovered the RAD9 gene and its role in halting cell-cycle progression at the G2/M transition in S. cerevisiae following DNA damage. Irradiated RAD9 mutant cells were able to continue cell division when their wild-type counterparts would arrest in G2/M virtually showing that mitosis is dependent on completion of DNA replication [12, 21]. Whilst Hartwell was not the first one to describe this phenomenon, his work in the late ‘80s defined the notion of a cell-cycle “checkpoint” stressing that genes that are not essential in progressing through the cell cycle are vital in arresting its progress [10, 17]. Through this, Hartwell’s work also paved the role to understanding and recognising the role of p53, a master regulator of cancer progression [10], but that is a tale for another time.

Two years later, research from Enoch and Nurse (1990) dissociated cell-cycle control from DNA replication by identifying CDC2 (CDK1) as the key regulator for G2/M and G1/S transition in fission yeast (S. pombe) [12, 22, 23]. Inspired by Hartwell’s work, initially, Nurse set out to discover other CDC mutants by following similar approaches, but in S. pombe. In constant growth conditions, division in fission yeast occurs at a fixed cell size. In his experiments, Nurse found by chance some mutants that were smaller than expected after completing cell division, suggesting that cells undergo division before concluding their growth. He identified WEE1 and WEE2 (orthologue of CDC2) as the genes responsible for this phenotype. WEE1 acted in G2 as an inhibitor of mitotic onset and controlled the cell cycle timing of mitosis upstream of WEE2, an activator of mitotic onset that acts in both G1 and G2 [10, 16, 24, 25].

Noticing that similar proteins with essentially the same role were discovered in different organisms, Nurse suspected that cell-cycle progression might be highly conserved throughout eukaryotes and went on to discover a CDC2 orthologue in human cells [9, 26].

Continuing efforts from Hartwell’s, Nurse’s, Hunts’ and several other labs identified several other players involved in the control of cell-cycle transitions and eventually painted a picture of cell cycle regulation and its complexities involving specific molecular machinery for the control of each cell-cycle checkpoint. Lee Hartwell, Sir Paul Nurse and Sir Tim Hunt received the Nobel Prize in Physiology or Medicine in 2001 for their contributions to deciphering the cell cycle machinery. Some intricacies of the molecular regulation of the cell cycle are still discovered today, with contemporary research focusing on understanding the redundancy of kinases involved in this process [9, 27], which gives the cancer research community an exciting prospectus to better target and improve the efficiency of anti-cancer treatments.

From bench to bedside

For more than 30 years now, ever since we started understanding the cell-cycle progression and checkpoint machinery, researchers directed their attention to leveraging the differential expression and activity of cell cycle checkpoint regulators in malignant cells. Flavopiridol was the first CDK inhibitor to reach clinical trials in 1994 following promising effectiveness against several cancer types in pre-clinical studies. However, its clinical implementation, similar to that of most 1^st and 2^nd generation pan-CDK inhibitors came to a halt due to limited effectiveness and/ or severe side effects reported in preliminary clinical trials [6, 28, 29].

Both intense side effects and inconsistent efficacy, which prevented most of these drugs from reaching the clinic, could be attributed to the pan nature of the 1^st and 2^nd generation of CDK inhibitors. More selective inhibition of specific CDKs is therefore crucial.  Currently, cancer research is in an exhilarating era of targeted medicine, and 3^rd generation CDK inhibitors, Abemaciclib, Palbociclib, Ribociclib and Trilaciclib, that act selectively on CDK4/6 have been approved for clinical use [2, 3, 30], revolutionising the treatment of aggressive malignancies of the breast and lung. However, despite the clinical success of CDK4/6 inhibitors, their use is not without drawbacks: differences in the degree of treatment response were noticed in the clinic and patients might acquire resistance to therapy [4].

Another promising research area for the treatment of cancer is represented by the development of checkpoint inhibitors, another class of drugs that target cell-cycle checkpoints, but which, by preventing DNA repair, enhance genomic instability and death of tumor cells. Checkpoint inhibitors showed encouraging results, especially in combination with other therapies such as chemo- and radiotherapy as by preventing DNA repair in G2/M, they promote mitotic catastrophe typical for BRCA deficient cancers [31].

The complete clinical development of these drugs is haltered primarily by the still incomplete understanding of the molecular mechanism underlying cell-cycle progression. Current research efforts are directed towards addressing these issues and include further investigating the mechanism of action of those drugs, as immunological and anti-invasive effects have been recently described following treatment with checkpoint inhibitors. Investigating combinatorial therapy approaches with hormonal therapy, immunotherapy and radiotherapy to improve outcomes and reduce toxicity and the identification of predictive biomarkers for treatment response are other avenues for current and future research in this field [4, 6, 32].

It is perhaps important to note, as with many other important discoveries, that neither Hartwell, Nurse nor Hunt were trying to find a cure for cancer. “Hartwell nor Hunt were pursuing regulators of the chromosome cycle when they embarked on the studies that eventually led them to Cdk1 and its cyclin regulatory subunits” [10]. Yet their discoveries performed in simpler model organisms enabled the accumulation of a body of knowledge that led to a better understanding of tumour progression and novel treatment options for cancer patients.

by Dr. Alexandra Boitor, EACR Scientific Officer

We’d like to thank Marcos Malumbres Martínez (Cancer Biology, Group Leader at CNIO, VHIO, ICREA, and external associated to IRB) for reviewing this article.

Let’s #KeepResearchCurious

To continue to save the lives of future cancer patients we must maintain and increase funding to continue exploring the unexpected and unknown. Support for this type of curiosity-driven research opens the door to treatment, prevention and cures we can’t even imagine yet. This potential for exploration and discovery is what makes our work so vital and promising. That’s why we’re campaigning to #KeepResearchCurious.

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