Period (PER) protein phosphorylation is a critical regulator of circadian period, yet an integrated understanding of the role and interaction between phosphorylation sites that can both increase and decrease PER2 stability remains elusive. Here, we propose a phosphoswitch model, where two competing phosphorylation sites determine whether PER2 has a fast or slow degradation rate. This mathematical model accurately reproduces the three-stage degradation kinetics of endogenous PER2. We predict and demonstrate that the phosphoswitch is intrinsically temperature sensitive, slowing down PER2 degradation as a result of faster reactions at higher temperatures. The phosphoswitch provides a biochemical mechanism for circadian temperature compensation of circadian period. This phosphoswitch additionally explains the phenotype of Familial Advanced Sleep Phase (FASP) and CK1ε(tau) genetic circadian rhythm disorders, metabolic control of PER2 stability, and how drugs that inhibit CK1 alter period. The phosphoswitch provides a general mechanism to integrate diverse stimuli to regulate circadian period.

Circadian rhythms are intrinsic ~24 hour cycles that regulate diverse aspects of physiology, and in turn are regulated by interactions with the external environment. Casein kinase 1 delta (CK1δ, CSNK1D) is a key regulator of the clock, phosphorylating both stabilizing and destabilizing sites on the PER2 protein, in a mechanism known as the phosphoswitch. CK1δ can itself be regulated by phosphorylation on its regulatory domain, but the specific sites involved, and the role this plays in control of circadian rhythms as well as other CK1-dependent processes is not well understood. Using a sensitized PER2::LUC reporter assay, we identified a specific phosphorylation site, T347, on CK1δ, that regulates CK1δ activity towards PER2. A mutant CK1δ T347A was more active in promoting PER2 degradation. This CK1δ regulatory site is phosphorylated in cells in trans by dinaciclib- and staurosporine-sensitive kinases, consistent with their potential regulation by cyclin dependent and other proline-directed kinases. The regulation of CK1δ by site-specific phosphorylation via the cell cycle and other signaling pathways provides a mechanism to couple external stimuli to regulation of CK1δ-dependent pathways including the circadian clock.

Oncogenic mutations in the RAS family of small GTPases are commonly found in human cancers and they promote tumorigenesis by altering gene expression networks. We previously demonstrated that Casein Kinase 1α (CK1α), a member of the CK1 family of serine/threonine kinases, is post-transcriptionally upregulated by oncogenic RAS signaling. Here, we report that the CK1α mRNA contains an exceptionally long 5′-untranslated region (UTR) harbouring several translational control elements, implicating its involvement in translational regulation. We demonstrate that the CK1α 5′-UTR functions as an IRES element in HCT-116 colon cancer cells to promote cap-independent translation. Using tobramycin-affinity RNA-pulldown assays coupled with identification via mass spectrometry, we identified several CK1α 5′-UTR-binding proteins, including SFPQ. We show that RNA interference targeting SFPQ reduced CK1α protein abundance and partially blocked RAS-mutant colon cancer cell growth. Importantly, transcript and protein levels of SFPQ and other CK1α 5′-UTR-associated RNA-binding proteins (RBPs) are found to be elevated in early stages of RAS-mutant cancers, including colorectal and lung adenocarcinoma. Taken together, our study uncovers a previously unappreciated role of RBPs in promoting RAS-mutant cancer cell growth and their potential to serve as promising biomarkers as well as tractable therapeutic targets in cancers driven by oncogenic RAS.

Autophagy is a fundamental cellular homeostasis mechanism known to play multifaceted roles in the natural history of cancers over time. It has recently been shown that autophagy also mediates the crosstalk between the tumor and its microenvironment by promoting the export of molecular payloads such as non-coding RNA (ncRNAs) via LC3-dependent Extracellular Vesicle loading and secretion (LDELS). In turn, the dynamic exchange of exosomal ncRNAs regulate autophagic responses in the recipient cells within the tumor microenvironment (TME), for both tumor and stromal cells. Autophagy-dependent phenotypic changes in the recipient cells further enhance tumor growth and metastasis, through diverse biological processes, including nutrient supplementation, immune evasion, angiogenesis, and therapeutic resistance. In this review, we discuss how the feedforward autophagy-ncRNA axis orchestrates vital communications between various cell types within the TME ecosystem to promote cancer progression.

While we made great strides in the early detection of a handful of cancers, many other cancers are still detected at fairly later stages, thus hindering the deployment of effective surgical or therapeutic intervention to change their dismal clinical outcomes. The arduous journey of cancer cells from the primary tumor to colonize distant secondary organs or tissues begins with their ability to activate or deactivate various cellular processes at will, including the autophagy machinery. In this review, we discuss how circulatory cancer cells from primary tumors could selectively mobilize different subsets of microRNAs (miRNAs) to enable autophagic recycling of nutrients during their search for secondary sites to colonize and to disable such cell survival programs once they have successfully established at distant organs or tissues. We also discuss how this new miRNA-autophagy-metastasis axis can be targeted by the emerging RNA Medicine toolkit.