Cyclin E1 and cyclin E2 in ER+ breast cancer: prospects as biomarkers and therapeutic targets.

Cyclin E1 is one the most promising biomarkers in estrogen receptor positive (ER+) breast cancer for response to the new standard of care drug class, CDK4/6 inhibitors. Because of its strong predictive value, cyclin E1 expression may be used in the future to triage patients into potential responders and non-responders. Importantly, cyclin E1 is highly related to cyclin E2, and both cyclin E1 and cyclin E2 are estrogen target genes that can facilitate anti-estrogen resistance and can be highly expressed in breast cancer. However cyclin E1 and E2 are often expressed in different subsets of patients. This raises questions about whether the expression of cyclin E1 and cyclin E2 have different biological drivers, if high expressing subsets represent different clinical subtypes, and how to effectively develop a biomarker for E-cyclin expression. Finally, several pan-CDK inhibitors that target cyclin E-CDK2 activity have reached Phase II clinical trials. In this review, we outline the data identifying that different cohorts of patients have high expression of cyclins E1 and E2 in ER+ cancer and address the implications for biomarker and therapeutic development.

Functional characterization of cancer-associated Gab1 mutations.

Grb2-associated binder 1 (Gab1) is a docking protein that transduces signals from a variety of tyrosine kinases, including Met and the epidermal growth factor receptor (EGFR). Although the related protein Gab2 is strongly implicated in human cancer, a role for Gab1 has been less clear. However, a screen for gene mutations in breast cancer identified two somatic mutations in Gab1, Y83C and T387N. In this paper we describe the functional characterization of these Gab1 mutants. MCF-10A immortalized mammary epithelial cells overexpressing Gab1 Y83C and T387N exhibited a more elongated, fibroblastic phenotype compared with wild-type Gab1 controls. Expression of Gab1 or the mutants promoted epidermal growth factor (EGF)-independent proliferation in monolayer culture to a similar degree. However, in Matrigel culture, both mutants enhanced the formation of acini exhibiting an aberrant, branched morphology. In addition, expression of the mutants modestly increased Erk activation. The two mutants also enhanced branching morphogenesis in a different mammary epithelial cell line, HC11. To gain further insights into the mechanism of action of these mutations, we mapped Gab1 phosphorylation sites by mass spectrometry. This detected phosphorylation of T387 but ;not Y83. Cellular stimulation with EGF or hepatocyte growth factor (HGF) led to a transient, or sustained, induction of T387 phosphorylation, respectively. As T387 corresponds in position to Gab2 T391, which suppresses Gab2 signaling in a phosphorylation-dependent manner, these data support a model in which the T387N mutation abrogates negative-feedback regulation of Gab1. Interrogation of publically-available databases revealed additional cancer-associated mutations at, or in close proximity to, identified serine/threonine phosphorylation sites in other docking proteins. These data indicate that aberrant Gab1 signaling can directly contribute to breast cancer progression, and that negative feedback sites in docking proteins can be targeted by oncogenic mutations.

The antiproliferative effects of progestins in T47D breast cancer cells are tempered by progestin induction of the ETS transcription factor Elf5.

Prolactin and progesterone act together to regulate mammary alveolar development, and both hormones have been implicated in breast cancer initiation and progression. Here we show that Elf5, a prolactin-induced ETS transcription factor that specifies the mammary secretory cell lineage, is also induced by progestins in breast cancer cells via a direct mechanism. To define the transcriptional response to progestin elicited via Elf5, we made an inducible Elf5 short hairpin-RNA knock-down model in T47D breast cancer cells and used it to prevent the progestin-induction of Elf5. Functional analysis of Affymetrix gene expression data using Gene Ontologies and Gene Set Enrichment Analysis showed enhancement of the progestin effects on cell cycle gene expression. Cell proliferation assays showed a more efficacious progestin-induced growth arrest when Elf5 was kept at baseline levels. These results showed that progestin induction of Elf5 expression tempered the antiproliferative effects of progestins in T47D cells, providing a further mechanistic link between prolactin and progestin in the regulation of mammary cell phenotype.

Wilms' tumor protein 1: an early target of progestin regulation in T-47D breast cancer cells that modulates proliferation and differentiation.

Progesterone regulates the proliferation and differentiation of normal mammary epithelium. In breast cancer cells, progesterone and its synthetic analogs, progestins, induce long-term growth inhibition and differentiation. However, the mechanisms responsible are not fully understood. When T-47D breast cancer cells were treated with the synthetic progestin ORG 2058 (16alpha-ethoxy-21-hydroxy-19-norpregn-4-en-3,20-dione), all isoforms of Wilms' tumor protein 1 (Wt1) mRNA and protein were rapidly downregulated. We reasoned that the decrease in Wt1 levels may contribute to the long-term antiproliferative and differentiative effects of progestins as proliferation and differentiation are known end points of Wt1 action. Consistent with this idea, Wt1 small interfering RNA led to a decrease in S phase and cyclin D1 levels, and increased Oil-Red-O staining, indicating increased lipogenesis. Conversely, overexpression of Wt1 attenuated the decrease in S phase induced by ORG 2058 at 48-96 h. This was accompanied by the sustained expression of cyclin D1 despite progestin treatment, and increased levels of retinoblastoma (Rb) phosphorylation at sites targeted by cyclin D1-Cdk4 (Ser249/Thr252). Wt1 overexpression also attenuated the ORG 2058-mediated increase in fatty acid synthase levels and reduced lipogenesis. Thus, Wt1 downregulation was sufficient to mimic the effects of progestin and was necessary for complete progestin-mediated proliferative arrest and subsequent differentiation. Furthermore, Wt1 overexpression modulated the effects of progestins but not anti-estrogens or androgens. These results indicate that Wt1 is an important early target of progestins that regulates both proliferation and differentiation in breast cancer cells.

Evolution of a molecular switch: universal bacterial GTPases regulate ribosome function.

The GTPases comprise a protein superfamily of highly conserved molecular switches adapted to many diverse functions. These proteins are found in all domains of life and often perform essential roles in fundamental cellular processes. Analysis of data from genome sequencing projects demonstrates that bacteria possess a core of 11 universally conserved GTPases (elongation factor G and Tu, initiation factor 2, LepA, Era, Obg, ThdF/TrmE, Ffh, FtsY, EngA and YchF). Investigations aimed at understanding the function of GTPases indicate that a second conserved feature of these proteins is that they elicit their function through interaction with RNA and/or ribosomes. An emerging concept suggests that the 11 universal GTPases are either necessary for ribosome function or transmitting information from the ribosome to downstream targets for the purpose of generating specific cellular responses. Furthermore, it is suggested that progenitor GTPases were early regulators of RNA function and may have existed in precursors of cellular systems driven by catalytic RNA. If this is the case, then a corollary of this hypothesis is that GTPases that do not bind RNA arose at a later time from an RNA-binding progenitor that lost the capability to bind RNA.