Loss of Pkd1 limits susceptibility to colitis and colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers, with an annual incidence of ~135,000 in the US, associated with ~50,000 deaths. Autosomal dominant polycystic kidney disease (ADPKD), associated with mutations disabling the PKD1 gene, affects as many as 1 in 1000. Intriguingly, some studies have suggested that individuals with germline mutations in PKD1 have reduced incidence of CRC, suggesting a genetic modifier function. Using mouse models, we here establish that loss of Pkd1 greatly reduces CRC incidence and tumor growth induced by loss of the tumor suppressor Apc. Growth of Pkd1-/-;Apc-/- organoids was reduced relative to Apc-/- organoids, indicating a cancer cell-intrinsic activity, even though Pkd1 loss enhanced activity of pro-oncogenic signaling pathways. Notably, Pkd1 loss increased colon barrier function, with Pkd1-deficient animals resistant to DSS-induced colitis, associated with upregulation of claudins that decrease permeability, and reduced T cell infiltration. Notably, Pkd1 loss caused greater sensitivity to activation of CFTR, a tumor suppressor in CRC, paralleling signaling relations in ADPKD. Overall, these data and other data suggest germline and somatic mutations in PKD1 may influence incidence, presentation, and treatment response in human CRC and other pathologies involving the colon.

Patterns of Ciliation and Ciliary Signaling in Cancer.

Among the factors that have been strongly implicated in regulating cancerous transformation, the primary monocilium (cilium) has gained increasing attention. The cilium is a small organelle extending from the plasma membrane, which provides a localized hub for concentration of transmembrane receptors. These receptors transmit signals from soluble factors (including Sonic hedgehog (SHH), platelet-derived growth factor (PDGF-AA), WNT, TGFß, NOTCH, and others) that regulate cell growth, as well as mechanosensory cues provided by flow or extracellular matrix. Ciliation is regulated by cell cycle, with most cells that are in G0 (quiescent) or early G1 ciliation and cilia typically absent in G2/M cells. Notably, while most cells organized in solid tissues are ciliated, cancerous transformation induces significant changes in ciliation. Most cancer cells lose cilia; medulloblastomas and basal cell carcinomas, dependent on an active SHH pathway, rely on ciliary maintenance. Changes in cancer cell ciliation are driven by core oncogenic pathways (EGFR, KRAS, AURKA, PI3K), and importantly ciliation status regulates functionality of those pathways. Ciliation is both influenced by targeted cancer therapies and linked to therapeutic resistance; recent studies suggest ciliation may also influence cancer cell metabolism and stem cell identity. We review recent studies defining the relationship between cilia and cancer.

Nedd9 Restrains Autophagy to Limit Growth of Early Stage Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most common cancer worldwide. With overall 5-year survival estimated at <17%, it is critical to identify factors that regulate NSCLC disease prognosis. NSCLC is commonly driven by mutations in KRAS and TP53, with activation of additional kinases such as SRC promoting tumor invasion. In this study, we investigated the role of NEDD9, a SRC activator and scaffolding protein, in NSCLC tumorigenesis. In an inducible model of NSCLC dependent on Kras mutation and Trp53 loss (KP mice), deletion of Nedd9 (KPN mice) led to the emergence of larger tumors characterized by accelerated rates of tumor growth and elevated proliferation. Orthotopic injection of KP and KPN tumors into the lungs of Nedd9-wild-type and -null mice indicated the effect of Nedd9 loss was cell-autonomous. Tumors in KPN mice displayed reduced activation of SRC and AKT, indicating that activation of these pathways did not mediate enhanced growth of KPN tumors. NSCLC tumor growth has been shown to require active autophagy, a process dependent on activation of the kinases LKB1 and AMPK. KPN tumors contained high levels of active LKB1 and AMPK and increased autophagy compared with KP tumors. Treatment with the autophagy inhibitor chloroquine completely eliminated the growth advantage of KPN tumors. These data for the first time identify NEDD9 as a negative regulator of LKB1/AMPK-dependent autophagy during early NSCLC tumor growth. SIGNIFICANCE: This study demonstrates a novel role for the scaffolding protein NEDD9 in regulating LKB1-AMPK signaling in early stage non-small cell lung cancer, suppressing autophagy and tumor growth.

Unexpected Activities in Regulating Ciliation Contribute to Off-target Effects of Targeted Drugs.

PURPOSE: For many tumors, signaling exchanges between cancer cells and other cells in their microenvironment influence overall tumor signaling. Some of these exchanges depend on expression of the primary cilium on nontransformed cell populations, as extracellular ligands including Sonic Hedgehog (SHH), PDGFRα, and others function through receptors spatially localized to cilia. Cell ciliation is regulated by proteins that are themselves therapeutic targets. We investigated whether kinase inhibitors of clinical interest influence ciliation and signaling by proteins with ciliary receptors in cancer and other cilia-relevant disorders, such as polycystic kidney disease (PKD). EXPERIMENTAL DESIGN: We screened a library of clinical and preclinical kinase inhibitors, identifying drugs that either prevented or induced ciliary disassembly. Specific bioactive protein targets of the drugs were identified by mRNA depletion. Mechanism of action was defined, and activity of select compounds investigated. RESULTS: We identified multiple kinase inhibitors not previously linked to control of ciliation, including sunitinib, erlotinib, and an inhibitor of the innate immune pathway kinase, IRAK4. For all compounds, activity was mediated through regulation of Aurora-A (AURKA) activity. Drugs targeting cilia influenced proximal cellular responses to SHH and PDGFRα. In vivo, sunitinib durably limited ciliation and cilia-related biological activities in renal cells, renal carcinoma cells, and PKD cysts. Extended analysis of IRAK4 defined a subset of innate immune signaling effectors potently affecting ciliation. CONCLUSIONS: These results suggest a paradigm by which targeted drugs may have unexpected off-target effects in heterogeneous cell populations in vivo via control of a physical platform for receipt of extracellular ligands.

Microscopy-Based Automated Live Cell Screening for Small Molecules That Affect Ciliation.

The primary monocilium, or cilium, is a single antenna-like organelle that protrudes from the surface of most mammalian cell types, and serves as a signaling hub. Mutations of cilia-associated genes result in severe genetic disorders termed ciliopathies. Among these, the most common is autosomal dominant polycystic kidney disease (ADPKD); less common genetic diseases include Bardet-Biedl syndrome, Joubert syndrome, nephronophthisis, and others. Important signaling cascades with receptor systems localized exclusively or in part at cilia include Sonic Hedgehog (SHH), platelet derived growth factor alpha (PDGFRα), WNTs, polycystins, and others. Changes in ciliation during development or in pathological conditions such as cancer impacts signaling by these proteins. Notably, ciliation status of cells is coupled closely to the cell cycle, with cilia protruding in quiescent (G0) or early G1 cells, declining in S/G2, and absent in M phase, and has been proposed to contribute to cell cycle regulation. Because of this complex biology, the elaborate machinery regulating ciliary assembly and disassembly receives input from many cellular proteins relevant to cell cycle control, development, and oncogenic transformation, making study of genetic factors and drugs influencing ciliation of high interest. One of the most effective tools to investigate the dynamics of the cilia under different conditions is the imaging of live cells. However, developing assays to observe the primary cilium in real time can be challenging, and requires a consideration of multiple details related to the cilia biology. With the dual goals of identifying small molecules that may have beneficial activity through action on human diseases, and of identifying ciliary activities of existing agents that are in common use or development, we here describe creation and evaluation of three autofluorescent cell lines derived from the immortalized retinal pigmented epithelium parental cell line hTERT-RPE1. These cell lines stably express the ciliary-targeted fluorescent proteins L13-Arl13bGFP, pEGFP-mSmo, and tdTomato-MCHR1-N-10. We then describe methods for use of these cell lines in high throughput screening of libraries of small molecule compounds to identify positive and negative regulators of ciliary disassembly.

Ciliary signalling in cancer.

Although tumours initiate from oncogenic changes in a cancer cell, subsequent tumour progression and therapeutic response depend on interactions between the cancer cells and the tumour microenvironment (TME). The primary monocilium, or cilium, provides a spatially localized platform for signalling by Hedgehog, Notch, WNT and some receptor tyrosine kinase pathways and mechanosensation. Changes in ciliation of cancer cells and/or cells of the TME during tumour development enforce asymmetric intercellular signalling in the TME. Growing evidence indicates that some oncogenic signalling pathways as well as some targeted anticancer therapies induce ciliation, while others repress it. The links between the genomic profile of cancer cells, drug treatment and ciliary signalling in the TME likely affect tumour growth and therapeutic response.

Ganetespib limits ciliation and cystogenesis in autosomal-dominant polycystic kidney disease (ADPKD).

Autosomal-dominant polycystic kidney disease (ADPKD) is associated with progressive formation of renal cysts, kidney enlargement, hypertension, and typically end-stage renal disease. In ADPKD, inherited mutations disrupt function of the polycystins (encoded by PKD1 and PKD2), thus causing loss of a cyst-repressive signal emanating from the renal cilium. Genetic studies have suggested ciliary maintenance is essential for ADPKD pathogenesis. Heat shock protein 90 (HSP90) clients include multiple proteins linked to ciliary maintenance. We determined that ganetespib, a clinical HSP90 inhibitor, inhibited proteasomal repression of NEK8 and the Aurora-A activator trichoplein, rapidly activating Aurora-A kinase and causing ciliary loss in vitro. Using conditional mouse models for ADPKD, we performed long-term (10 or 50 wk) dosing experiments that demonstrated HSP90 inhibition caused durable in vivo loss of cilia, controlled cystic growth, and ameliorated symptoms induced by loss of Pkd1 or Pkd2. Ganetespib efficacy was not increased by combination with 2-deoxy-d-glucose, a glycolysis inhibitor showing some promise for ADPKD. These studies identify a new biologic activity for HSP90 and support a cilia-based mechanism for cyst repression.-Nikonova, A. S., Deneka, A. Y., Kiseleva, A. A., Korobeynikov, V., Gaponova, A., Serebriiskii, I. G., Kopp, M. C., Hensley, H. H., Seeger-Nukpezah, T. N., Somlo, S., Proia, D. A., Golemis, E. A. Ganetespib limits ciliation and cystogenesis in autosomal-dominant polycystic kidney disease (ADPKD).

Combination of Eribulin and Aurora A Inhibitor MLN8237 Prevents Metastatic Colonization and Induces Cytotoxic Autophagy in Breast Cancer.

Recent findings suggest that the inhibition of Aurora A (AURKA) kinase may offer a novel treatment strategy against metastatic cancers. In the current study, we determined the effects of AURKA inhibition by the small molecule inhibitor MLN8237 both as a monotherapy and in combination with the microtubule-targeting drug eribulin on different stages of metastasis in triple-negative breast cancer (TNBC) and defined the potential mechanism of its action. MLN8237 as a single agent and in combination with eribulin affected multiple steps in the metastatic process, including migration, attachment, and proliferation in distant organs, resulting in suppression of metastatic colonization and recurrence of cancer. Eribulin application induces accumulation of active AURKA in TNBC cells, providing foundation for the combination therapy. Mechanistically, AURKA inhibition induces cytotoxic autophagy via activation of the LC3B/p62 axis and inhibition of pAKT, leading to eradication of metastases, but has no effect on growth of mammary tumor. Combination of MLN8237 with eribulin leads to a synergistic increase in apoptosis in mammary tumors, as well as cytotoxic autophagy in metastases. These preclinical data provide a new understanding of the mechanisms by which MLN8237 mediates its antimetastatic effects and advocates for its combination with eribulin in future clinical trials for metastatic breast cancer and early-stage solid tumors. Mol Cancer Ther; 15(8); 1809-22. ©2016 AACR.