The orphan tyrosine kinase LMTK3 has been identified as a potential biomarker of response to endocrine therapy in patients with breast cancer, as well as a possible new therapeutic target. Scientists claim the protein plays a key role in regulating estrogen receptor α (ERα) activity; in breast cancer patients high nuclear LMTK3 expression levels are associated with significantly shorter disease-free and overall survival times. In addition, knocking down the LMTK3 gene in breast cancer cell lines was found to significantly boost the anticancer effects of tamoxifen.
The Imperial College, London-based team that carried out the research reports its findings in Nature Medicine, in a paper titled “Kinome screening for regulators of the estrogen receptor identifies LMTK3 as a new therapeutic target in breast cancer.”
Resistance of ERα-positive breast cancer to tamoxifen, aromatase inhibitors, and fulvestrant is a common occurrence, and in vitro evidence has implicated phosphorylation of ERα in the development of endocrine resistance, explains Justin Stebbing, M.D., at the department of surgery and cancer at Imperial College’s Hammersmith Hospital Campus, and colleagues.
The researchers carried out a whole human kinome siRNA screen to try and identify kinases that might regulate ERα activity. Narrowing down the pool of resulting candidates by evaluating their effects on the expression of genes on which ERα acts, they found that silencing LMTK3 consistently inhibited the expression of estrogen-regulated genes, whereas transfecting LMTK3 in ERα-positive breast cancer cells lines had the opposite effects. ERα is encoded by the ESR1 gene, and LMTK3 knockdown was shown to reduce expression of ESR1 mRNA.
LMTK3 is one of a family of LMTK serine-threonine-tyrosine kinases. Subsequent studies by Dr. Stebbing’s team showed that knocking down LMTK3, but not LMTK1 or LMTK2, inhibited the growth of human ERα-positive cell lines, but had no effect on ERα-negative cells. Knocking down LMTK3 reduced levels of ERα in breast cancer cell lines by 80%. Interestingly, ERα half-life was reduced after LMTK3 knockdown, while LMTK3 overexpression stabilized ERα.
To further investigate the potential involvement of LMTK3 in the development of tamoxifen resistance, the team analyzed the effects of LMTK3 silencing in tamoxifen-resistant cell lines. Addition of LMTK3 siRNA to the cells increased the growth inhibitory effects of tamoxifen, and the expected elevated levels of phosphorylated ERα and its major oncogenic co-activator amplified in breast cancer were decreased. In addition, LMTK3 was found to be essential for E2-induced growth, as silencing of LMTK3 impeded cell proliferation in the presence of E2. LMTK3 expression was downregulated by E2 and upregulated in response to tamoxifen, revealing a feedback loop between LMTK3 and ERα.
To confirm their findings in vivo, the researchers then injected an LMTK3 siRNA into established human breast tumors grown in nude mice. In vivo bioluminescence imaging of the xenografted tumors showed that loss of LMTK3 protein expression led to a significant decrease in tumor growth.
Analysis of cells taken from breast cancer patients suggested that two intronic polymorphisms in the LMTK3 gene were associated with the risk of developing tumor recurrence. The results of multivariate analysis suggested the combination of both polymorphisms represented an independent prognostic factor for both disease-free survival and overall survival.
By looking at the effects of LMTK3 on the expression of proteins already known to regulate ERα, including GATA3, FOXO3, and FOX M1, the researchers derived a potential mechanism by which the tyrosine kinase impacts on ERα in breast cancer. In this model LMTK3 acts by decreasing the leads to increased binding of forkhead box O3 (FOXO3) to the ESR1 promoter.
Interestingly, comparing the DNA sequences of the LMTK3 genes in humans and chimpanzees found substantial differences, despite the fact that related genes are very similar between the two species. The reasons behind this evolutionary divergence in the LMTK3 gene are unclear, the researchers note, but it is interesting that chimpanzees are not susceptible to estrogen receptor-positive breast cancer. It thus appears that whatever selective pressures were responsible for evolution of the human version of the gene have been associated with a trade-off that in parallel increased human susceptibility to breast cancer.