In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

The role of autophagy in oncogenesis remains ambiguous, and mechanisms that induce autophagy and regulate its outcome in human cancers are poorly understood. The maternally imprinted Ras-related tumor suppressor gene aplasia Ras homolog member I (ARHI; also known as DIRAS3) is downregulated in more than 60% of ovarian cancers, and here we show that re-expression of ARHI in multiple human ovarian cancer cell lines induces autophagy by blocking PI3K signaling and inhibiting mammalian target of rapamycin (mTOR), upregulating ATG4, and colocalizing with cleaved microtubule-associated protein light chain 3 (LC3) in autophagosomes. Furthermore, ARHI is required for spontaneous and rapamycin-induced autophagy in normal and malignant cells. Although ARHI re-expression led to autophagic cell death when SKOv3 ovarian cancer cells were grown in culture, it enabled the cells to remain dormant when they were grown in mice as xenografts. When ARHI levels were reduced in dormant cells, xenografts grew rapidly. However, inhibition of ARHI-induced autophagy with chloroquine dramatically reduced regrowth of xenografted tumors upon reduction of ARHI levels, suggesting that autophagy contributed to the survival of dormant cells. Further analysis revealed that autophagic cell death was reduced when cultured human ovarian cancer cells in which ARHI had been re-expressed were treated with growth factors (IGF-1, M-CSF), angiogenic factors (VEGF, IL-8), and matrix proteins found in xenografts. Thus, ARHI can induce autophagic cell death, but can also promote tumor dormancy in the presence of factors that promote survival in the cancer microenvironment.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel constructed from two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBD) and a regulatory (R) domain. The NBDs and R-domain are cytosolic and how they are assembled with the MSDs to achieve the native CFTR structure is not clear. Human DnaJ 2 (Hdj-2) is a co-chaperone of heat shock cognate 70 (Hsc70) which is localized to the cytosolic face of the ER. Whether Hdj-2 directs Hsc70 to facilitate the assembly of cytosolic regions on CFTR was investigated. We report that immature ER forms of CFTR and ΔF508 CFTR can be isolated in complexes with Hdj-2 and Hsc70. The ΔF508 mutation is localized in NBD1 and causes the CFTR to misfold. Levels of complex formation between ΔF508 CFTR and Hdj-2/Hsp70 were~2-fold higher than those with CFTR. The earliest stage at which Hdj-2/Hsc70 could bind CFTR translation intermediates coincided with the expression of NBD1 in the cytosol. Interestingly, complex formation between Hdj-2 and nascent CFTR was greatly reduced after expression of the R-domain. In experiments with purified components, Hdj-2 and Hsc70 acted synergistically to suppress NBD1 aggregation. Collectively, these data suggest that Hdj-2 and Hsc70 facilitate early steps in CFTR assembly. A putative step in the CFTR folding pathway catalyzed by Hdj-2/Hsc70 is the formation of an intramolecular NBD1-R-domain complex. Whether this step is defective in the biogenesis of ΔF508 CFTR will be discussed.

A variety of biomarkers have been developed to monitor growth of ovarian cancer and to detect disease at an early interval. CA125 (MUC16) has provided a useful serum tumor marker for monitoring response to chemotherapy, detecting disease recurrence, distinguishing malignant from benign pelvic masses, and potentially improving clinical trial design. A rapid fall in CA125 during chemotherapy predicts a favorable prognosis and could be used to redistribute patients on multiarmed randomized clinical trials. Several studies now document that CA125 can serve as a surrogate marker for response in phase II trials. Serial measurement of CA125 might also provide a useful marker for monitoring stabilization of disease with cytostatic targeted therapeutic agents. The greatest potential for serum markers may be in detecting ovarian cancer at an early stage. A rising CA125 can be used to trigger transvaginal sonography (TVS) in a small fraction of patients. An algorithm has been developed that calculates risk of ovarian cancer based on serial CA125 values and refers patients at highest risk for TVS. Use of the algorithm is currently being evaluated in a trial with 200,000 women in the UK that will test critically the ability of a two-stage screening strategy to improve survival in ovarian cancer. Whatever the outcome, as 20% of ovarian cancers have little or no expression of CA125, additional serum markers will be required to detect all patients in an initial phase of screening. More than 30 serum markers have been evaluated alone and in combination with CA125 by different investigators. Some of the most promising include: HE4, mesothelin, M-CSF, osteopontin, kallikrein(s), and soluble EGF receptor. Two proteomic approaches have been used: one examines the pattern of peaks on mass spectroscopy and the other uses proteomic analysis to identify a limited number of critical markers that can be assayed by more conventional methods. Both approaches are promising and require further development. Several groups are placing markers on multiplex platforms to permit simultaneous assay of multiple markers with very small volumes of serum. Mathematical techniques are being developed to analyze combinations of marker levels to improve sensitivity and specificity. In the future, serum markers should improve the sensitivity of detecting recurrent disease as well as facilitate earlier detection of ovarian cancer.

Specification of Hsp70 action in cellular protein metabolism may occur through the formation of specialized Hsp70:Hsp40 pairs. To test this model, we compared the ability of purified Sis1 and Ydj1 to regulate the ATPase and protein-folding activity of Hsp70 Ssa1 and Ssb1/2 proteins. Ydj1 and Sis1 could both functionally interact with Ssa1, but not the Ssb1/2 proteins, to refold luciferase. Interestingly, Ydj1:Ssa1 could promote up to four times more luciferase folding than Sis1:Ssa1. This functional difference was explored and could not be accounted for by differences in the ability of Sis1 and Ydj1 to regulate Ssa1 ATPase activity. Instead, differences in the chaperone function of Ydj1 and Sis1 were observed. Ydj1 was dramatically more effective than Sis1 at suppressing the thermally induced aggregation of luciferase. Paradoxically, Sis1 and Ydj1 could bind similar quantities of chemically denatured luciferase. The polypeptide binding domain of Sis1 was found to lie between residues 171-352 and correspond to its conserved carboxyl terminus. The conserved carboxyl terminus of Ydj1 is also known to participate in the binding of nonnative polypeptides. Thus, Ydj1 appears more efficient at assisting Ssa1 in folding luciferase because its contains a zinc finger-like region that is absent from Sis1. Ydj1 and Sis1 are structurally and functionally distinct Hsp40 proteins that can specify Ssa1 action by generating Hsp70:Hsp40 pairs that exhibit different chaperone activities.

Ydj1 is a member of the Hsp40 (DnaJ-related) chaperone family that facilitates cellular protein folding by regulating Hsp70 ATPase activity and binding unfolded polypeptides. Ydj1 contains four conserved subdomains that appear to represent functional units. To define the action of these regions, protease-resistant Ydj1 fragments and Ydj1 mutants were analyzed for activities exhibited by the unmodified protein. The Ydj1 mutant proteins analyzed were unable to support growth of yeast at elevated temperatures and were found to have alterations in the J-domain (Ydj1 H34Q), zinc finger-like region (Ydj1 C159T), and conserved carboxyl terminus (Ydj1 G315D). Fragment Ydj1 (1-90) contains the J-domain and a small portion of the G/F-rich region and could regulate Hsp70 ATPase activity but could not suppress the aggregation of the model protein rhodanese. Ydj1 H34Q could not regulate the ATPase activity of Hsp70 but could bind unfolded polypeptides. The J-domain functions independently and was sufficient to regulate Hsp70 ATPase activity. Fragment Ydj1 (179 -384) could suppress rhodanese aggregation but was unable to regulate Hsp70. Ydj1 (179 -384) contains the conserved carboxyl terminus of DnaJ but is missing the J-domain, G/F-rich region, and a major portion of the zinc finger-like region. Ydj1 G315D exhibited severe defects in its ability to suppress rhodanese aggregation and form complexes with unfolded luciferase. The conserved carboxyl terminus of Ydj1 appeared to participate in the binding of unfolded polypeptides. Ydj1 C159T could form stable complexes with unfolded proteins and suppress protein aggregation but was inefficient at refolding denatured luciferase. The zinc finger-like region of Ydj1 appeared to function in conjunction with the conserved carboxyl terminus to fold proteins. However, Ydj1 does not require an intact zinc finger-like region to bind unfolded polypeptides. These data suggest that the combined functions of the J-domain, zinc finger-like region, and the conserved carboxyl terminus are required for Ydj1 to cooperate with Hsp70 and facilitate protein folding in the cell.Hsp40 proteins make up an essential family of molecular chaperones that function to specify the cellular processes facilitated by Hsp70 (1-5). Prokaryotic and eukaryotic cells contain multiple Hsp40 proteins that act in specific pairs with Hsp70 at different cellular locations via conserved mechanisms (6 -9). Escherichia coli DnaJ, the progenitor of the Hsp40 family, functions to promote protein folding, degradation of misfolded proteins, and bacteriophage DNA replication (3, 10, 11). The mammalian Hsp40 protein Auxillin directs Hsp70 to uncoat clathrin-coated vesicles (12, 13). The cysteine string protein of Drosophilia is palmitolyated and localized to neurosecretory vesicles, where it is proposed to direct Hsp70 to catalyze putative protein assembly/disassembly events required for neurotransmitter release (14, 15). The yeast protein Tim44 is a component of the import machinery that directs the mitochondrial cognate of Hsp70 ...

Summary Regulators of mitosis have been successfully targeted to enhance response to taxane chemotherapy. Here, we show that the Salt Inducible Kinase 2 (SIK2) localizes at the centrosome, plays a key role in the initiation of mitosis and regulates the localization of the centrosome linker protein, C-Nap1, through S2392 phosphorylation. Interference with the known SIK2 inhibitor PKA induced SIK2-dependent centrosome splitting in interphase while SIK2 depletion blocked centrosome separation in mitosis, sensitizing ovarian cancers to paclitaxel in culture and in xenografts. Depletion of SIK2 also delayed G1/S transition and reduced AKT phosphorylation. Higher expression of SIK2 significantly correlated with poor survival in patients with high-grade serous ovarian cancers. These data identify SIK2 as a plausible target for therapy in ovarian cancers.

Elevated preoperative NLR is an independent predictor of worse RFS for patients with stage IIA colon cancer and a potential biomarker to identify candidates for adjuvant chemotherapy.