Oncogene cooperation and biomarkers

What is required for cancer formation?

A single oncogene is unable to drive cancer formation due to the wide variety of cancer hallmarks - a tumour suppressor must almost always need to be lost. To feed into enough of these hallmarks, an oncogene needs to touch a lot of processes.

What does signalling look like in a cell?

The signalling hierarchy within cells is shaped like an hourglass - there are many different ligands and receptors, narrow downstream networks and kinases/TFs, and then a wide variety of gene and phosphorylation targets. A single ligand causes the expression of lots of different genes, and the array changes over time - the phenotypic change will look different early from late. This is because receptors (mostly RTKs) are signalling hubs that can activate many kinases and TFs (does not just bind one intracellularly), but depending on the ligand, different residues may get phosphorylated therefore different kinases and TFs get implicated etc. TFs are able to bind to a whole array of different genes, leading to a wide variety of responses in cancer.

What is MYC, how is it implicated in cancer and how is it diagnosed?

MYC is a well-studied TF which is a basic helix loop helix. It binds DNA to cause a wide variety of cellular processes such as proliferation, differentiation, apoptosis and metabolism. It forms a heterodimer with another protein called Max, which binds to specific DNA sequences called E-boxes (CACGTG - palindromic). This sequence is found in the promoter of many different genes. MYC has a half-life of 20 mins and is rapidly degraded by the proteasome (MYC is potent) - the phenotype changes over 20 mins. 

This TF is constitutively (gene amplifications, translocations - the TF itself is not changed) and aberrantly expressed in over 70% of human cancers. FISH is a test for gene amplification; fluorescently labelled probes which are homologous to the MYC sequence. FISH is not only used for MYC, but all gene amplifications. 

MYC drives cancer through direct amplification and pathways leading to dysregulation - combination of these is the 70% of cancers value.

• In breast cancer, MYC amplification is found in 15%, PI3K is found in 32.6%, EGFR in 7.7% etc.

What is the signalling pathway for MYC?

The signalling pathway for MYC is RTK -> PI3K <-> RAS -> RAF - > MEK -> ERK which phosphorylates MYC to activate it. PI3K can also signal through AKT1 which inhibits GSK3Beta. Inhibition of GSK3Beta stabilises MYC and vice versa.

How does MYC act normally and in cancer?

In physiological levels of MYC, it binds to its high affinity targets to activate them and then it leaves. In deregulated levels of MYC, the high amount means it also binds to low affinity targets (E-box sequences with a single nucleotide change etc). In doing so, more pathways are activated which may have roles in the different hallmarks of cancer - transcription (E2F), protein biosynthesis, microRNAs, cell adhesion and cytoskeleton (integrin), DNA repair, translation (eIF-2a), metabolism (GLUT1), cell cycle (CDK-cyclins), and signal transduction (Wnt - differentiation, makes cell more invasive etc).

How is MYC amplification treated?

There are many ways to theoretically/clinically decrease MYC activity; inhibition of transcription of MYC (JQ1, THZ1, PC585), inhibition of signalling that travels to MYC (PI3K, AKT and mTOR can be inhibited), destabilisation of the protein (degradation), inhibition of binding to Max.

How does MYC and RAS lead to transformation?

MYC amplification leads to transformation. Mutant RAS leads to transformation. In a lung cancer mouse model, mutant RAS and MYC elevation were tested individually and each caused tumours. Together, tumours still formed but there was no synergy/cooperation. In a lymphoma mouse model, there is cooperation meaning tumours are formed extremely quickly. This is not fully understood. 

Summarise transformation.

In summary, oncogenes drive cancer (amplified, mutations, fusions). To drive transformation, lots of cell processes need to change and it is common that oncogenes do this via causing dysregulated signalling through other oncogenic pathways. This cooperation does not always synergise. 

How does different signalling systems cause cooperation?

Another way in which cooperation between oncogenes is achieved is by having different signalling systems present. For example, mutant RAS causes the synthesis of TGF-alpha which gets released from the cell. This can bind to EGFR via autocrine signalling - EGFR leads to production of RAS, MYC etc as RTKs are signalling hubs. This is important because cancer cells want to activate different branches of signalling pathways (the 2 most common are BRAF which activates MEK, ERK etc, and PI3K which activates AKT, mTOR etc). Downstream of both of these IS MYC. This usually occurs when targeted inhibitors are used to block the other branch over the other.

How does RTK switching cause cooperation?

RTK swapping can also occur. In normal conditions, EGF, MET and VEGF receptors all have their own ligands. Often, mutations in the EGFR and other RTKs cause synergy between other receptors present because downstream of all these are similar pathways. For example, EGFR leads to the production of VGF which binds VEGF, which then causes the production of HGF which binds to MET etc. The ligand production feeds back on itself through the same pathways. Again, this commonly occurs in response to inhibiting one RTK.

How does ligand production cause cooperation?

Producing ligands also gives the opportunity to manipulate the TME by the ligands binding to and altering the surrounding cells. The immune system itself is one of the ways we know about cooperation - in mutated RAS and MYC cells alone there is still high immune surveillance but together there is evasion.

How does heterogeneity cause cooperation?

In a polygenic tumour, there are so many different pathways being activated, autocrine and paracrine signalling etc. This heterogeneity is another way of cooperation - this is why some people respond worse to treatments (monoclonal has best outcome).

What is gene expression profiling and how does it affect patient outcome? Give an example.

Gene expression profiling is a technique used to query the expression of thousands of genes simultaneously. In the context of cancer, gene expression profiling has been used to more accurately classify tumours. This information often has an impact on predicting the patient’s clinical outcome and treatment pathway.

For example, there are 4 main types of breast cancer. HR+ means the tumour cells have receptors for the hormones oestrogen or progesterone, which promote the growth of HR+ tumours. HER2 stands for human epidermal growth factor receptor 2 - HER2+ mens that tumour cells make high levels of HER2. The majority of breast cancers are HR+/HER2-, then HR-/HER2- and HR+/HER2+, then HR-/HER+. About 7% is unknown. 

• HER2 is an RTK which plays a role in cell growth, differentiation and survival. Overexpression leads to more growth and division. HER2+ cancers are typically treated with targeted therapies such as trastuzumab and pertuzumab. 

• Categorisation into subgroups allows doctors to predict the prognosis of patients depending on their mutation.

How is immunohistochemistry used in the clinic?

Gene expression profiling is not done for each patient’s cancer - it has already been done for the majority/all cancer types already. The subgroups are known so in the clinic, immunohistochemistry for ER HER2 and KI-67 (marker of proliferation - the more of this marker the more aggressive the cancer is likely is) is done for breast cancer. This determines the oncogene mutations.

• In IHC, they are classified by their staining pattern. HER2- and ER- is basal-like, HER2- and ER+ and low KI-67 is luminal A, HER2- and ER+ and high KI-67 is luminal B, HER2+ and ER+/- is HER2-like. 

• This can determine treatment: luminal A or B will receive hormone therapy, HER2+ is treated with herceptin, and basal-like is further grouped to determine treatment plan (EGFR [gefitinib or lapatinib], BRCA1 [PARP inhibitors or types of chemotherapy], or C-). 

What a specific gene panels?

In the future, known biomarkers of different aspects of cell behaviour are looked at using specific gene panels. For example, one such assay is oncotype Dx which has just 21 genes and uses RT-PCR to quantify gene expression from fixed tumour tissue. Data was analysed from 3 independent studies with recurrence of breast cancer which revealed that 16 genes (+5 reference genes - should not be changing) correlated with proliferation and endocrine response. There is a formula that quantifies these 16 genes to give a recurrence score - the higher it is, the more chemo a patient should receive because they are more likely to relapse.