Correction: Mitochondrial clearance by the STK38 kinase supports oncogenic Ras-induced cell transformation.

[This corrects the article DOI: 10.18632/oncotarget.9875.].

Characterization of Breast Cancer Preclinical Models Reveals a Specific Pattern of Macrophage Polarization.

Drug discovery efforts have focused on the tumor microenvironment in recent years. However, few studies have characterized the stroma component in patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMs). In this study, we characterized the stroma in various models of breast cancer tumors in mice. We performed transcriptomic and flow cytometry analyses on murine populations for a series of 25 PDXs and the two most commonly used GEMs (MMTV-PyMT and MMTV-erBb2). We sorted macrophages from five models. We then profiled gene expression in these cells, which were also subjected to flow cytometry for phenotypic characterization. Hematopoietic cell composition, mostly macrophages and granulocytes, differed between tumors. Macrophages had a specific polarization phenotype related to their M1/M2 classification and associated with the expression of genes involved in the recruitment, invasion and metastasis processes. The heterogeneity of the stroma component of the models studied suggests that tumor cells modify their microenvironment to satisfy their needs. Our observations suggest that such models are of relevance for preclinical studies.

Mitochondrial clearance by the STK38 kinase supports oncogenic Ras-induced cell transformation.

Oncogenic Ras signalling occurs frequently in many human cancers. However, no effective targeted therapies are currently available to treat patients suffering from Ras-driven tumours. Therefore, it is imperative to identify downstream effectors of Ras signalling that potentially represent promising new therapeutic options. Particularly, considering that autophagy inhibition can impair the survival of Ras-transformed cells in tissue culture and mouse models, an understanding of factors regulating the balance between autophagy and apoptosis in Ras-transformed human cells is needed. Here, we report critical roles of the STK38 protein kinase in oncogenic Ras transformation. STK38 knockdown impaired anoikis resistance, anchorage-independent soft agar growth, and in vivo xenograft growth of Ras-transformed human cells. Mechanistically, STK38 supports Ras-driven transformation through promoting detachment-induced autophagy. Even more importantly, upon cell detachment STK38 is required to sustain the removal of damaged mitochondria by mitophagy, a selective autophagic process, to prevent excessive mitochondrial reactive oxygen species production that can negatively affect cancer cell survival. Significantly, knockdown of PINK1 or Parkin, two positive regulators of mitophagy, also impaired anoikis resistance and anchorage-independent growth of Ras-transformed human cells, while knockdown of USP30, a negative regulator of PINK1/Parkin-mediated mitophagy, restored anchorage-independent growth of STK38-depleted Ras-transformed human cells. Therefore, our findings collectively reveal novel molecular players that determine whether Ras-transformed human cells die or survive upon cell detachment, which potentially could be exploited for the development of novel strategies to target Ras-transformed cells.

AMOTL1 Promotes Breast Cancer Progression and Is Antagonized by Merlin.

The Hippo signaling network is a key regulator of cell fate. In the recent years, it was shown that its implication in cancer goes well beyond the sole role of YAP transcriptional activity and its regulation by the canonical MST/LATS kinase cascade. Here we show that the motin family member AMOTL1 is an important effector of Hippo signaling in breast cancer. AMOTL1 connects Hippo signaling to tumor cell aggressiveness. We show that both canonical and noncanonical Hippo signaling modulates AMOTL1 levels. The tumor suppressor Merlin triggers AMOTL1 proteasomal degradation mediated by the NEDD family of ubiquitin ligases through direct interaction. In parallel, YAP stimulates AMOTL1 expression. The loss of Merlin expression and the induction of Yap activity that are frequently observed in breast cancers thus result in elevated AMOTL1 levels. AMOTL1 expression is sufficient to trigger tumor cell migration and stimulates proliferation by activating c-Src. In a large cohort of human breast tumors, we show that AMOTL1 protein levels are upregulated during cancer progression and that, importantly, the expression of AMOTL1 in lymph node metastasis appears predictive of the risk of relapse. Hence we uncover an important mechanism by which Hippo signaling promotes breast cancer progression by modulating the expression of AMOTL1.

Innate immune cell-produced IL-17 sustains inflammation in bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune skin disease characterized by the binding of autoantibodies to components of the hemidesmosome structure, resulting in an inflammatory response and subepidermal blister formation. To investigate the role of immune orientation in the inflammatory processes associated with disease progression, blister fluid, serum, and biopsy specimens were collected from 31 consecutive BP patients. Blister fluids displayed high levels of IL-6, IL-17, IL-22, and IL-23, whereas transforming growth factor-ß was increased in BP sera. However, neither immunocytochemistry on a trans-differentiation model of IL-17-producing peripheral blood mononuclear cells nor immunohistochemistry on BP biopsy specimens could demonstrate the presence of T helper type 17 lymphocytes. Instead, innate immune cells, especially neutrophils, produced IL-17 at the skin lesional site. Of note, superpotent topical corticosteroid application quickly and markedly reduced both IL-17 expression and clinical signs of BP. Consistently, IL-17 upregulated matrix-metalloprotease-9 and neutrophil elastase expression, two proteases involved in blister formation, thereof further demonstrating its role in the progress of BP. Finally, IL-17-induced matrix degradation, originated from neutrophil activation, initiated the formation of an amplification loop of the inflammatory response that could represent the underlying phenomenon leading to the maintenance and even disease extent. Thus, our results could open new therapeutic strategies for BP patients.

Vasculature analysis of patient derived tumor xenografts using species-specific PCR assays: evidence of tumor endothelial cells and atypical VEGFA-VEGFR1/2 signalings.

BACKGROUND: Tumor endothelial transdifferentiation and VEGFR1/2 expression by cancer cells have been reported in glioblastoma but remain poorly documented for many other cancer types. METHODS: To characterize vasculature of patient-derived tumor xenografts (PDXs), largely used in preclinical anti-angiogenic assays, we designed here species-specific real-time quantitative RT-PCR assays. Human and mouse PECAM1/CD31, ENG/CD105, FLT1/VEGFR1, KDR/VEGFR2 and VEGFA transcripts were analyzed in a large series of 150 PDXs established from 8 different tumor types (53 colorectal, 14 ovarian, 39 breast and 15 renal cell cancers, 6 small cell and 5 non small cell lung carcinomas, 13 cutaneous melanomas and 5 glioblastomas) and in two bevacizumab-treated non small cell lung carcinomas xenografts. RESULTS: As expected, mouse cell proportion in PDXs -evaluated by quantifying expression of the housekeeping gene TBP- correlated with all mouse endothelial markers and human VEGFA RNA levels. More interestingly, we observed human PECAM1/CD31 and ENG/CD105 expression in all tumor types, with higher rate in glioblastoma and renal cancer xenografts. Human VEGFR expression profile varied widely depending on tumor types with particularly high levels of human FLT1/VEGFR1 transcripts in colon cancers and non small cell lung carcinomas, and upper levels of human KDR/VEGFR2 transcripts in non small cell lung carcinomas. Bevacizumab treatment induced significant low expression of mouse Pecam1/Cd31, Eng/Cd105, Flt1/Vegfr1 and Kdr/Vefr2 while the human PECAM1/CD31 and VEGFA were upregulated. CONCLUSIONS: Taken together, our results strongly suggest existence of human tumor endothelial cells in all tumor types tested and of both stromal and tumoral autocrine VEGFA-VEGFR1/2 signalings. These findings should be considered when evaluating molecular mechanisms of preclinical response and resistance to tumor anti-angiogenic strategies.

Targeting Bcl-2/Bcl-XL induces antitumor activity in uveal melanoma patient-derived xenografts.

PURPOSE: Uveal melanoma (UM) is associated with a high risk of metastases and lack of efficient therapies. Reduced capacity for apoptosis induction by chemotherapies is one obstacle to efficient treatments. Human UM is characterized by high expression of the anti-apoptotic protein Bcl-2. Consequently, regulators of apoptosis such as Bcl-2 family inhibitors may constitute an attractive approach to UM therapeutics. In this aim, we have investigated the efficacy of the Bcl-2/Bcl-XL inhibitor S44563 on 4 UM Patient-Derived Xenografts (PDXs) and derived-cell lines. EXPERIMENTAL DESIGN: Four well characterized UM PDXs were used for in vivo experiments. S44563 was administered alone or combined with fotemustine either concomitantly or after the alkylating agent. Bcl-2, Bcl-XL, and Mcl-1 expressions after S44563 administration were evaluated by immunohistochemistry (IHC). RESULTS: S44563 administered alone by at 50 and 100 mg/kg i.p. induced a significant tumour growth inhibition in only one xenograft model with a clear dose effect. However, when S44563 was concomitantly administered with fotemustine, we observed a synergistic activity in 3 out of the 4 tested models. In addition, S44563 administered after fotemustine induced a tumour growth delay in 2 out of 3 tested xenografts. Finally, IHC analyses showed that Bcl-2, Bcl-XL, and Mcl-1 expression were not modified after S44563 administration. CONCLUSION: The novel anti-apoptotic experimental compound S44563, despite a relative low efficacy when administered alone, increased the efficacy of fotemustine in either concomitant or sequential combinations or indeed subsequent to fotemustine. These data support further exploration of potential therapeutic effect of Bcl-2/Bcl-xl inhibition in human UM.

Specific targeting of caspase-9/PP2A interaction as potential new anti-cancer therapy.

PURPOSE: PP2A is a serine/threonine phosphatase critical to physiological processes, including apoptosis. Cell penetrating peptides are molecules that can translocate into cells without causing membrane damage. Our goal was to develop cell-penetrating fusion peptides specifically designed to disrupt the caspase-9/PP2A interaction and evaluate their therapeutic potential in vitro and in vivo. EXPERIMENTAL DESIGN: We generated a peptide containing a penetrating sequence associated to the interaction motif between human caspase-9 and PP2A (DPT-C9h), in order to target their association. Using tumour cell lines, primary human cells and primary human breast cancer (BC) xenografts, we investigated the capacity of DPT-C9h to provoke apoptosis in vitro and inhibition of tumour growth (TGI) in vivo. DPT-C9h was intraperitoneally administered at doses from 1 to 25 mg/kg/day for 5 weeks. Relative Tumour Volume (RTV) was calculated. RESULTS: We demonstrated that DPT-C9h specifically target caspase-9/PP2A interaction in vitro and in vivo and induced caspase-9-dependent apoptosis in cancer cell lines. DPT-C9h also induced significant TGI in BC xenografts models. The mouse-specific peptide DPT-C9 also induced TGI in lung (K-Ras model) and breast cancer (PyMT) models. DPT-C9h has a specific effect on transformed B cells isolated from chronic lymphocytic leukemia patients without any effect on primary healthy cells. Finally, neither toxicity nor immunogenic responses were observed. CONCLUSION: Using the cell-penetrating peptides blocking caspase-9/PP2A interactions, we have demonstrated that DPT-C9h had a strong therapeutic effect in vitro and in vivo in mouse models of tumour progression.

Optimization of tumor xenograft dissociation for the profiling of cell surface markers and nutrient transporters.

Metabolic adaptations and changes in the expression of nutrient transporters are known to accompany tumorigenic processes. Nevertheless, in the context of solid tumors, studies of metabolism are hindered by a paucity of tools allowing the identification of cell surface transporters on individual cells. Here, we developed a method for the dissociation of human breast cancer tumor xenografts combined with quantification of cell surface markers, including metabolite transporters. The expression profiles of four relevant nutrient transporters for cancer cells' metabolism, Glut1, ASCT2, PiT1 and PiT2 (participating to glucose, glutamine and inorganic phosphate, respectively), as detected by new retroviral envelope glycoprotein-derived ligands, were distinctive of each tumor, unveiling underlying differences in metabolic pathways. Our tumor dissociation procedure and nutrient transporter profiling technology provides opportunities for future basic research, clinical diagnosis, prognosis and evaluation of therapeutic responses, as well as for drug discovery and development.