In vivo silencing of Reptin blocks the progression of human hepatocellular carcinoma in xenografts and is associated with replicative senescence.

BACKGROUND & AIMS: We previously showed that Reptin is overexpressed in hepatocellular carcinoma (HCC), and that in vitro depletion of Reptin with siRNAs led to HCC cell growth arrest and apoptosis. Here, we asked whether in vivo targeting of Reptin in established tumours had a therapeutic effect. METHODS: We used lentiviral vectors to construct HuH7 and Hep3B cell lines with doxycycline (Dox)-dependent expression of Reptin (R2) or control shRNA (GL2). Cells were injected subcutaneously into immunodeficient mice, and Dox was given when tumours reached a volume of 250 mm(3). RESULTS: In vitro, the growth of GL2-Dox, GL2+Dox, and R2-Dox cells was undistinguishable whereas that of R2+Dox cells stopped 4 days after Dox treatment. The growth decrease was associated with increased apoptosis, and evidence of replicative senescence, as shown by staining for acid beta-galactosidase and the presence of senescence-associated heterochromatin foci. In xenografted mice, R2+Dox tumour growth stagnated or even regressed with prolonged treatment in contrast with the GL2-Dox, GL2+Dox, and R2-Dox tumours that progressed steadily. The blockage of tumour progression was associated with the induction of senescence and reduced cell proliferation. CONCLUSIONS: In vivo Reptin depletion leads to tumour growth arrest. Reptin may prove a valuable target in HCC.

Adenosine triphosphatase pontin is overexpressed in hepatocellular carcinoma and coregulated with reptin through a new posttranslational mechanism.

UNLABELLED: Reptin and Pontin are related ATPases associated with stoichiometric amounts in several complexes involved in chromatin remodeling, transcriptional regulation, and telomerase activity. We found that Reptin was up-regulated in hepatocellular carcinoma (HCC) and that down-regulation of Reptin led to growth arrest. We show here that Pontin messenger RNA (mRNA) is also up-regulated in human HCC 3.9-fold as compared to nontumor liver (P = 0.0004). Pontin expression was a strong independent factor of poor prognosis in a multivariate analysis. As for Reptin, depletion of Pontin in HuH7 cells with small interfering RNAs (siRNAs) led to growth arrest. Remarkably, Pontin depletion led to down-regulation of Reptin as shown with western blot, and vice versa. Whereas siRNAs induced a decrease of their cognate mRNA targets, they did not affect the transcripts of the partner protein. Translation of Pontin or Reptin was not altered when the partner protein was silenced. However, pulse-chase experiments demonstrated that newly synthesized Pontin or Reptin stability was reduced in Reptin- or Pontin-depleted cells, respectively. This phenomenon was reversed upon inhibition of proteasome or ubiquitin-activating enzyme (E1). In addition, proteasome inhibition could partly restore Pontin steady-state levels in Reptin-depleted cells, as shown by western blot. This restoration was not observed when cells were also treated with cycloheximide, thus confirming that proteasomal degradation in this setting was restricted to newly synthesized Pontin. CONCLUSION: Reptin and Pontin protein levels are strictly controlled by a posttranslational mechanism involving proteasomal degradation of newly synthesized proteins. These data demonstrate a tight regulatory and reciprocal interaction between Reptin and Pontin, which may in turn lead to the maintenance of their 1:1 stoichiometry.

A PI3K- and GTPase-independent Rac1-mTOR mechanism mediates MET-driven anchorage-independent cell growth but not migration.

Receptor tyrosine kinases (RTKs) are often overexpressed or mutated in cancers and drive tumor growth and metastasis. In the current model of RTK signaling, including that of MET, downstream phosphatidylinositol 3-kinase (PI3K) mediates both cell proliferation and cell migration, whereas the small guanosine triphosphatase (GTPase) Rac1 mediates cell migration. However, in cultured NIH3T3 and glioblastoma cells, we found that class I PI3K mediated oncogenic MET-induced cell migration but not anchorage-independent growth. In contrast, Rac1 regulated both processes in distinct ways. Downstream of PI3K, Rac1 mediated cell migration through its GTPase activity, whereas independently of PI3K, Rac1 mediated anchorage-independent growth in a GTPase-independent manner through an adaptor function. Through its RKR motif, Rac1 formed a complex with the kinase mTOR to promote its translocation to the plasma membrane, where its activity promoted anchorage-independent growth of the cell cultures. Inhibiting mTOR with rapamycin suppressed the growth of subcutaneous MET-mutant cell grafts in mice, including that of MET inhibitor-resistant cells. These findings reveal a GTPase-independent role for Rac1 in mediating a PI3K-independent MET-to-mTOR pathway and suggest alternative or combined strategies that might overcome resistance to RTK inhibitors in patients with cancer.

Reactivation of Mutant-EGFR Degradation through Clathrin Inhibition Overcomes Resistance to EGFR Tyrosine Kinase Inhibitors.

Tyrosine kinase inhibitors (TKI) targeting mutant EGFR in non-small cell lung cancer (NSCLC) have been successful to control cancer growth, but acquired resistance inevitably occurs, including mutations directly on EGFR, for example, T790M and C797S. Strategies to prevent such acquired mutations by reducing mutant-EGFR expression have met limited success. Here, we propose a new model of mutant-EGFR trafficking and demonstrate that clathrin inhibition induces rapid degradation across a large panel of endogenous mutant-EGFR (Ex19del, L858R, and Ex20Ins). This panel included mutant-EGFR (T790M) resistant to the first- and second-generation EGFR inhibitors and to the third-generation TKI osimertinib and occurs through both mutational (C797S) and nonmutational EGFR mechanisms. Clathrin-mediated endocytosis inhibition of mutant EGFR induced a macropinocytosis-dependent lysosomal pathway associated with a loss of mutant-EGFR-dependent signaling (pAKT, pERK). Moreover, induction of this macropinocytic pathway led to robust apoptosis-dependent death across all mutant-EGFR cell lines tested, including those resistant to TKIs. We, therefore, propose a novel strategy to target mutant-EGFR refractory to approved existing TKI treatments in NSCLC and where new treatment strategies remain a key area of unmet need.Significance: These findings extend our mechanistic understanding of NSCLC mutant EGFR trafficking biology, the role that trafficking may play in resistance of mutant EGFR to tyrosine kinase inhibitors, and provide new therapeutic and biological insights to tackle this fundamental issue and improve benefit to patients. Cancer Res; 78(12); 3267-79. ©2018 AACR.

Driving Cytotoxic Natural Killer Cells into Melanoma: If CCL5 Plays the Music, Autophagy Calls the Shots.

Autophagy is a quality control process executed at the basal level in almost all cell types. However, in cancer cells, autophagy is activated by several stimuli, including hypoxia. Depending on tumor type, stage, and genetic context, autophagy is a double-edged sword. Autophagy promotes regression in newly established tumors; however, it supports tumor progression in well-established tumors by maintaining cancer cell survival under stress conditions. These data, in addition to the emerging role of autophagy in impairing antitumor immunity, have attracted significant interest in developing autophagy inhibitors as a new approach to cancer treatment. The enthusiasm for developing selective drugs inhibiting autophagy has been seriously challenged by the discovery that most autophagy-related proteins display nonautophagic functions. Autophagy inhibitors chloroquine and hydroxychloroquine are currently being investigated in several clinical trials in combination with standard anticancer therapies. Here, we provide a brief overview on the nonautophagic function of autophagy-related proteins and summarize the major mechanisms whereby autophagy modulation could positively or negatively impact cancer therapies. We also focus on the emerging role of targeting autophagy in the improvement of NK-mediated antitumor immunity through the regulation of CCL5 and its receptors' expression in melanoma, and we provide some clues revealing how autophagy modulators could be exploited to improve cancer immunotherapies.

Antitumor Activity of Osimertinib, an Irreversible Mutant-Selective EGFR Tyrosine Kinase Inhibitor, in NSCLC Harboring EGFR Exon 20 Insertions.

EGFR exon 20 insertions (Ex20Ins) account for 4% to 10% of EGFR activating mutations in non-small cell lung cancer (NSCLC). EGFR Ex20Ins tumors are generally unresponsive to first- and second-generation EGFR inhibitors, and current standard of care for NSCLC patients with EGFR Ex20Ins is conventional cytotoxic chemotherapy. Therefore, the development of an EGFR TKI that can more effectively target NSCLC with EGFR Ex20Ins mutations represents a major advance for this patient subset. Osimertinib is a third-generation EGFR TKI approved for the treatment of advanced NSCLC harboring EGFR T790M; however, the activity of osimertinib in EGFR Ex20Ins NSCLC has yet to be fully assessed. Using CRISPR-Cas 9 engineered cell lines carrying the most prevalent Ex20Ins mutations, namely Ex20Ins D770_N771InsSVD (22%) or Ex20Ins V769_D770InsASV (17%), and a series of patient-derived xenografts, we have characterized osimertinib and AZ5104 (a circulating metabolite of osimertinib) activities against NSCLC harboring Ex20Ins. We report that osimertinib and AZ5104 inhibit signaling pathways and cellular growth in Ex20Ins mutant cell lines in vitro and demonstrate sustained tumor growth inhibition of EGFR-mutant tumor xenograft harboring the most prevalent Ex20Ins in vivo The antitumor activity of osimertinib and AZ5104 in NSCLC harboring EGFR Ex20Ins is further described herein using a series of patient-derived xenograft models. Together these data support clinical testing of osimertinib in patients with EGFR Ex20Ins NSCLC. Mol Cancer Ther; 17(5); 885-96. ©2018 AACR.

Beta 1-integrin-c-Met cooperation reveals an inside-in survival signalling on autophagy-related endomembranes.

Receptor tyrosine kinases (RTKs) and integrins cooperate to stimulate cell migration and tumour metastasis. Here we report that an integrin influences signalling of an RTK, c-Met, from inside the cell, to promote anchorage-independent cell survival. Thus, c-Met and ß1-integrin co-internalize and become progressively recruited on LC3B-positive 'autophagy-related endomembranes' (ARE). In cells growing in suspension, ß1-integrin promotes sustained c-Met-dependent ERK1/2 phosphorylation on ARE. This signalling is dependent on ATG5 and Beclin1 but not on ATG13, suggesting ARE belong to a non-canonical autophagy pathway. This ß1-integrin-dependent c-Met-sustained signalling on ARE supports anchorage-independent cell survival and growth, tumorigenesis, invasion and lung colonization in vivo. RTK-integrin cooperation has been assumed to occur at the plasma membrane requiring integrin 'inside-out' or 'outside-in' signalling. Our results report a novel mode of integrin-RTK cooperation, which we term 'inside-in signalling'. Targeting integrin signalling in addition to adhesion may have relevance for cancer therapy.

Measuring the role for Met endosomal signaling in tumorigenesis.

Met is a receptor tyrosine kinase, often overexpressed or mutated in human cancer. Upon activation by its ligand, the hepatocyte growth factor, Met controls several cell functions such as proliferation, migration, and survival through the activation of multiple pathways. Upon ligand binding, Met rapidly internalizes and continues to signal from endosomal compartments prior to its degradation. Importantly, this "endosomal signaling" has recently been shown to be involved in tumorigenesis and experimental metastasis. Consequently, interfering with Met endosomal signaling may provide a novel therapeutic approach in cancer treatment. However, there is a need for additional studies in various experimental models to confirm this and find the most specific ways of achieving it. Thus, outlined in this review are the techniques and tools we have been using to study Met endocytosis and Met endosomal signaling.

Receptor tyrosine kinase c-Met controls the cytoskeleton from different endosomes via different pathways.

Receptor tyrosine kinases (RTKs) are increasingly recognized as having the capacity to signal post-internalization. Signalling outputs and/or duration, and subsequent cellular outcome, are thought to be distinct when emanating from endosomes compared with those from the plasma membrane. Here we show, in invasive, basal-like human breast cell models, that different mechanisms are engaged by the RTK c-Met in two different endosomes to control the actin cytoskeleton via the key migratory signal output Rac1. Despite an acute activation of Rac1 from peripheral endosomes (PEs), c-Met needs to traffic to a perinuclear endosome (PNE) to sustain Rac1 signalling, trigger optimal membrane ruffling, cell migration and invasion. Unexpectedly, in the PNE but not in the PE, PI3K and the Rac-GEF Vav2 are required. Thus we describe a novel endosomal signalling mechanism whereby one signal output, Rac1, is stimulated through distinct pathways by the same RTK depending on which endosome it is localized to in the cell.

A direct role for Met endocytosis in tumorigenesis.

Compartmentalization of signals generated by receptor tyrosine kinase (RTK) endocytosis has emerged as a major determinant of various cell functions. Here, using tumour-associated Met-activating mutations, we demonstrate a direct link between endocytosis and tumorigenicity. Met mutants exhibit increased endocytosis/recycling activity and decreased levels of degradation, leading to accumulation on endosomes, activation of the GTPase Rac1, loss of actin stress fibres and increased levels of cell migration. Blocking endocytosis inhibited mutants' anchorage-independent growth, in vivo tumorigenesis and metastasis while maintaining their activation. One mutant resistant to inhibition by a Met-specific tyrosine kinase inhibitor was sensitive to endocytosis inhibition. Thus, oncogenicity of Met mutants results not only from activation but also from their altered endocytic trafficking, indicating that endosomal signalling may be a crucial mechanism regulating RTK-dependent tumorigenesis.

Pontin and reptin, two related ATPases with multiple roles in cancer.

Studies in model organisms or cultured human cells suggest potential implications in carcinogenesis for the AAA+ ATPases Pontin and Reptin. Both proteins are associated with several chromatin-remodeling complexes and have many functions including transcriptional regulation, DNA damage repair, and telomerase activity. They also interact with major oncogenic actors such as beta-catenin and c-myc and regulate their oncogenic function. We only now begin to get insight into the role of Pontin and Reptin in human cancers.

Overexpression and role of the ATPase and putative DNA helicase RuvB-like 2 in human hepatocellular carcinoma.

UNLABELLED: Using a proteomic analysis of human hepatocellular carcinoma (HCC), we identified the overexpression in 4 tumors of RuvB-like 2 (RUVBL2), an ATPase and putative DNA helicase known to interact with beta-catenin and cellular v-myc myelocytomatosis viral oncogene homolog (c-myc). RUVBL2 expression was further analyzed in tumors with quantitative reverse-transcription polymerase chain reaction analysis and immunohistochemistry; in addition, RUVBL2 expression in a HuH7 cell line was silenced by small interfering RNA or increased with a lentiviral vector. RUVBL2 messenger RNA overexpression was confirmed in 72 of 96 HCC cases, and it was associated with poorly differentiated tumors (P = 0.02) and a poor prognosis (P = 0.02) but not with beta-catenin mutations or c-myc levels. Although RUVBL2 was strictly nuclear in normal hepatocytes, tumoral hepatocytes exhibited additional cytoplasmic staining. There was no mutation in the coding sequence of RUVBL2 in 10 sequenced cases. Silencing RUVBL2 in HuH7 HCC cells reduced cell growth (P < 0.001) and increased apoptosis, as shown by DNA fragmentation (P < 0.001) and caspase 3 activity (P < 0.005). This was associated with an increased expression of several proapoptotic genes and with an increased conformational activation of Bak-1 and Bax. On the other hand, HuH7 cells with an overexpression of RUVBL2 grew better in soft agar (P < 0.03), had increased resistance to C2 ceramide-induced apoptosis (P < 0.001), and gave rise to significantly larger tumors when injected into immunodeficient Rag2/gammac mice (P = 0.016). CONCLUSION: RUVBL2 is overexpressed in a large majority of HCCs. RUVBL2 overexpression enhances tumorigenicity, and RUVBL2 is required for tumor cell viability. These results argue for a major role of RUVBL2 in liver carcinogenesis.