CANCER AND PHOSPHATIDYLINOSITOL 3-KINASE (PI3K)
Both RTKs and GPCRs can activate PI3K enzymes that are made up of a catalytic subunit (p100α, β or γ) with different regulatory subunits (p85s). PI3Ks phosphorylate the 3 position on the inositol ring of phosphatidylinositol (PI) to produce phosphatidylinositol 3, 4, 5- trisphosphate (PtdIns(3,4,5)P3) to which a number of pleckstrin homology domain proteins bind, leading to the activation of AKT kinases (Fig. 1).
2. Signal integration by an integrin scaffold protein.
In addition, activating point mutations in mTOR have been detected in several cancers (lung, skin, ovary, bowel, brain and kidney). mTOR signalling is also enhanced by GOLPH3, an oncoprotein that is amplified in a variety of human cancers. These multiple roles suggest that mTORC2 may emerge as an important target for cancer therapy.
During autophagy, the action of a PI3K and beclin-1 (also known as an autophagy-related gene (Atg6)) initiates the formation of autophagosomes. Beclin-1 is another member of the BH3 family that is released from the interaction with BCL2 proteins in the stress response. It is, in fact, a haploinsufficient tumour suppressor with a role in both autophagy and apoptosis.
Autophagy is emerging as a complex factor in cancer in that, for some types at least, it can suppress initiation but may also provide an essential support for tumour progression. Thus mice with disrupted autophagic genes (Atg5 and Atg7) develop benign growths that do not become malignant, consistent with a normal role for autophagy of preventing tissue damage and cancer progression. Heterozygous deletion of beclin-1 also causes tumours in a variety of tissues although these do become malignant, probably because beclin has other activities in addition to its autophagic role. The evidence that autophagy actually promotes tumourigenesis is particularly strong for pancreatic cancers for which, in both genetic mouse models and xenografts of human tumour cells, inhibition of autophagy prevents growth and causes regression. This is presumed to be due to an increase in reactive oxygen species and DNA damage, together with decreased oxidative phosphorylation, as a response to inhibition. Autophagy plays a prominent role in tumours with activating mutations in RAS (prevalent in lung, pancreatic and colon cancers), as indicated by the high basal autophagy in a cell from such cancers. In these aggressive tumours, the autophagic response sustains the level of mitochondrial function necessary for growth, a requirement that has been termed ‘autophagy addiction’ by Eileen White and her colleagues.
The tumour necrosis factor receptor (TNFR) family respond to cell death signals by promoting apoptosis in a variety of cells. The activating ligands are trimeric complexes that interact with three monomeric TNFR chains. Activation of these cell-surface receptors by their ligands induces apoptotic death in many cell types (Fig. 3).
3. The apoptosis pathway.
Remarkably, however, in tumour cells that express CD95 (also known as FAS and APO1), CD95 can act in an autocrine manner (i.e. it binds to receptors on the same type of cell from which it is secreted) to promote tumour development. This can happen because the intracellular signal pathways activated by CD95L/CD95 depend on the cell type.
The expression of TRAIL (tumour necrosis factor-related apoptosis inducing ligand) high on some tumour cells (e.g. from colorectal carcinoma) and monoclonal antibodies that selectively activate TRAIL show promise as an inducing tumour cell apoptosis.
Consistent with the importance of the JAK-STAT signalling in hematopoietic cell development, activating mutations in JAK2 and JAK3 occur in some leukaemias and in myelofibrosis (abnormal proliferation of marrow cells) and the resultant constitutive effect is presumed to drive abnormal cell proliferation. A selective inhibitor of JAKs has been developed (INCB018424) that gives sustained remission of myelofibrosis. An inactivating mutation in JAK1 has been identified in a rare smooth muscle tumour: this appears to inactivate interferon-γ signalling and thus promote evasion of the immune system by tumour cells. In addition, over-expression of members of the STAT family has been detected in a variety of tumours, indicating the impact that aberrant JAK-STAT signalling may have.
The WNT family are secreted morphogenic ligands that are involved in development. Amplification or over-expression of several WNTs has been detected in a variety of cancers and reduced expression of specific WNT receptors (members of the Frizzled family of GPCRs) has also been reported (e.g. Frizzled 5 in some prostate carcinomas). In the canonical WNT pathway (Fig. 4) a complex of casein kinase 1, glycogen synthase kinase 3, axin and the APC (adenomatous polyposis coli) protein regulate the fate of β-catenin – whether it is ubiquitylated or released to activate transcription of key cell proliferation genes (notably MYC).
4. WNT signalling.
The majority of mutations identified in APC inactivate the gene product by truncation and, because APC is involved in mitosis, this promotes chromosome instability. Mutant forms of APC are unable to associate with β-catenin and hence cannot down-regulate its transactivation function. Colorectal tumours with normal APC generally have mutations in β-catenin, the latter being the most frequently mutated gene in colorectal cancer. Hence the loss of APC promotes anomalously high expression of genes driving cell proliferation, an alternative way of inducing genetic instability. APC/β-catenin complexes also mediate signal transduction from E-cadherin cell surface adhesion proteins. The prolyl isomerase PIN1 regulates β-catenin turnover and subcellular localisation by interfering with its interaction with APC. Inhibition of PIN1 induces apoptosis but it is strongly over-expressed in a subset of human tumours and this increases the transcription of β-catenin target genes. Depending on whether AKT is attached to the plasma membrane or located in the nucleus it can exert opposing effects on β-catenin, repressing its transcriptional activity when nuclear.
In addition to the WNT system, activating mutations occur in a number of other GPCR pathways (e.g. in the thyroid stimulating hormone receptor in thyroid tumours) and in Gα subunits (e.g. in Gαs in pituitary adenomas and in Gαi in ovarian tumours). Gq subunit coupling to phospholipase Cβ activates the protein kinase C (PKC) family, of which there are about ten isoforms. Mutations have been identified in each of the PKC isozymes and they appear to be tumours and glioblastomas. These enzymes, therefore, number among the ~150 of the human genome kinase complement of 539 that can be mutated in cancer. As we have seen, kinase mutations are almost always activating, i.e. they confer gain-of-function. However, the PKC family and some other kinases (e.g. MAPK2K4 and DAPK3) are exceptional in that the mutations are inhibitory, that is, the normal form of these enzymes acts as a tumour suppressor. This means of course that PKC inhibitors will not be useful therapeutic agents.