Transcriptome profiling of human col\onic cells exposed to the gut pathobiont Streptococcus gallolyticus subsp. gallolyticus.

Streptococcus gallolyticus sp. gallolyticus (SGG) is a gut pathobiont involved in the development of colorectal cancer (CRC). To decipher SGG contribution in tumor initiation and/or acceleration respectively, a global transcriptome was performed in human normal colonic cells (FHC) and in human tumoral colonic cells (HT29). To identify SGG-specific alterations, we chose the phylogenetically closest relative, Streptococcus gallolyticus subsp. macedonicus (SGM) as control bacterium. We show that SGM, a bacterium generally considered as safe, did not induce any transcriptional changes on the two human colonic cells. The transcriptional reprogramming induced by SGG in normal FHC and tumoral HT29 cells was significantly different, although most of the genes up- and down-regulated were associated with cancer disease. Top up-regulated genes related to cancer were: (i) IL-20, CLK1, SORBS2, ERG1, PIM1, SNORD3A for normal FHC cells and (ii) TSLP, BHLHA15, LAMP3, ZNF27B, KRT17, ATF3 for cancerous HT29 cells. The total number of altered genes were much higher in cancerous than in normal colonic cells (2,090 vs 128 genes being affected, respectively). Gene set enrichment analysis reveals that SGG-induced strong ER- (endoplasmic reticulum) stress and UPR- (unfolded protein response) activation in colonic epithelial cells. Our results suggest that SGG induces a pro-tumoral shift in human colonic cells particularly in transformed cells potentially accelerating tumor development in the colon.

Global proteomic identifies multiple cancer-related signaling pathways altered by a gut pathobiont associated with colorectal cancer.

In this work, we investigated the oncogenic role of Streptococcus gallolyticus subsp. gallolyticus (SGG), a gut bacterium associated with colorectal cancer (CRC). We showed that SGG UCN34 accelerates colon tumor development in a chemically induced CRC murine model. Full proteome and phosphoproteome analysis of murine colons chronically colonized by SGG UCN34 revealed that 164 proteins and 725 phosphorylation sites were differentially regulated. Ingenuity Pathway Analysis (IPA) indicates a pro-tumoral shift specifically induced by SGG UCN34, as ~ 90% of proteins and phosphoproteins identified were associated with digestive cancer. Comprehensive analysis of the altered phosphoproteins using ROMA software revealed up-regulation of several cancer hallmark pathways such as MAPK, mTOR and integrin/ILK/actin, affecting epithelial and stromal colonic cells. Importantly, an independent analysis of protein arrays of human colon tumors colonized with SGG showed up-regulation of PI3K/Akt/mTOR and MAPK pathways, providing clinical relevance to our findings. To test SGG's capacity to induce pre-cancerous transformation of the murine colonic epithelium, we grew ex vivo organoids which revealed unusual structures with compact morphology. Taken together, our results demonstrate the oncogenic role of SGG UCN34 in a murine model of CRC associated with activation of multiple cancer-related signaling pathways.

PAK1-Dependent Antitumor Effect of AAC-11‒Derived Peptides on Sézary Syndrome Malignant CD4+ T Lymphocytes.

Sézary syndrome is an aggressive form of cutaneous T-cell lymphoma characterized by the presence of a malignant CD4+ T-cell clone in both blood and skin. Its pathophysiology is still poorly understood, and the development of targeted therapies is hampered by the absence of specific target proteins. AAC-11 plays important roles in cancer cell progression and survival and thus has been considered as an anticancer therapeutic target. In this study, we show that a peptide called RT39, comprising a portion of AAC-11‒binding site to its protein partners coupled to the penetratin sequence, induces the specific elimination of the malignant T-cell clone both ex vivo on the circulating cells of patients with Sézary syndrome and in vivo in a subcutaneous xenograft mouse model. RT39 acts by direct binding to PAK1 that is overexpressed, located in the plasma membrane, and constitutively activated in Sézary cells, resulting in their selective depletion by membranolysis. Along with the absence of toxicity, our preclinical efficacy evidence suggests that RT39 might represent a promising alternative therapeutic tool for Sézary syndrome because it spares the nonmalignant immune cells and, contrary to antibody-based immunotherapies, does not require the mobilization of the cellular immunity that shows heavy deficiencies at advanced stages of the disease.

Efficient Therapeutic Delivery by a Novel Cell-Penetrating Peptide Derived from Acinus.

In this study, we have identified a novel cell-penetrating sequence, termed hAP10, from the C-terminus of the human protein Acinus. hAP10 was able to efficiently enter various normal and cancerous cells, likely through an endocytosis pathway, and to deliver an EGFP cargo to the cell interior. Cell penetration of a peptide, hAP10DR, derived from hAP10 by mutation of an aspartic acid residue to an arginine was dramatically increased. Interestingly, a peptide containing a portion of the heptad leucine repeat region domain of the survival protein AAC-11 (residues 377-399) fused to either hAP10 or hAP10DR was able to induce tumor cells, but not normal cells, death both ex vivo on Sézary patients' circulating cells and to inhibit tumor growth in vivo in a sub-cutaneous xenograft mouse model for the Sézary syndrome. Combined, our results indicate that hAP10 and hAP10DR may represent promising vehicles for the in vitro or in vivo delivery of bioactive cargos, with potential use in clinical settings.

Prophylactic and therapeutic antileukemic effects induced by the AAC-11-derived Peptide RT53.

Despite considerable progress, the treatment of acute leukemia continues to be a challenge for a significant majority of patients. Using a well-characterized preclinical mouse model of acute promyelocytic leukemia (APL), we evaluated here the antileukemic efficacy of RT53, an anticancer peptide with potential immunological properties. Our results indicate that RT53 possesses a direct antileukemic effect, even at a late stage. We also demonstrate that a single injection of a vaccine consisting of leukemic blasts exposed to RT53, which induces the hallmarks of immunogenic cell death, was highly effective in preventing leukemia development in both prophylactic and therapeutic settings. The vaccine comprising RT53-treated APL cells generated long-term antileukemic protection and depletion experiments indicated that CD4 + T cells were of crucial importance for vaccine efficacy. Combined, our results provide the rationale for the exploration of RT53-based therapies for the treatment of acute leukemia.

The anticancer peptide RT53 induces immunogenic cell death.

In recent years, immunogenic cell death (ICD) has emerged as a revolutionary concept in the development of novel anticancer therapies. This particular form of cell death is able, through the spatiotemporally defined emission of danger signals by the dying cell, to induce an effective antitumor immune response, allowing the immune system to recognize and eradicate malignant cells. To date, only a restricted number of chemotherapeutics can trigger ICD of cancer cells. We previously reported that a peptide, called RT53, spanning the heptad leucine repeat region of the survival protein AAC-11 fused to a penetrating sequence, selectively induces cancer cell death in vitro and in vivo. Interestingly, B16F10 melanoma cells treated by RT53 were able to mediate anticancer effects in a tumor vaccination model. Stimulated by this observation, we investigated whether RT53 might mediate ICD of cancer cells. Here, we report that RT53 treatment induces all the hallmarks of immunogenic cell death, as defined by the plasma membrane exposure of calreticulin, release of ATP and the exodus of high-mobility group box 1 protein (HMGB1) from dying cancer cells, through a non-regulated, membranolytic mode of action. In a prophylactic mouse model, vaccination with RT53-treated fibrosarcomas prevented tumor growth at the challenge site. Finally, local intratumoral injection of RT53 into established cancers led to tumor regression together with T-cell infiltration and the mounting of an inflammatory response in the treated animals. Collectively, our results strongly suggest that RT53 can induce bona fide ICD of cancer cells and illustrate its potential use as a novel antitumor and immunotherapeutic strategy.

Significance of Streptococcus gallolyticus subsp. gallolyticus Association With Colorectal Cancer.

Streptococcus gallolyticus subsp. gallolyticus Sgg (formerly known as S. bovis type I) is the main causative agent of septicemia and infective endocarditis (IE) in elderly and immunocompromised persons. It belongs to the few opportunistic bacteria, which have been strongly associated to colorectal cancer (CRC). A literature survey covering a period of 40 years (1970-2010) revealed that 65% of patients diagnosed with an invasive Sgg infection had a concomitant colorectal neoplasia. Sgg is associated mainly with early adenomas and may thus constitute an early marker for CRC screening. Sgg has been described as a normal inhabitant of the rumen of herbivores and in the digestive tract of birds. It is more rarely detected in human intestinal tract (2.5-15%). Recent molecular analyses indicate possible zoonotic transmission of Sgg. Thanks to the development of a genetic toolbox and to comparative genomics, a number of factors that are important for Sgg pathogenicity have been identified. This review will highlight the role of Sgg pili in host colonization and how their phase-variable expression contributes to mitigate the host immune responses and finally their use as serological diagnostic tool. We will then present experimental data addressing the core question whether Sgg is a cause or consequence of CRC. We will discuss a few recent studies examining the etiological versus non-etiological participation of Sgg in colorectal cancer with the underlying mechanisms.

Evaluation of planar bioluminescence imaging and microPET/CT for therapy monitoring in a mouse model of pigmented metastatic melanoma.

Bioluminescence imaging (BLI) is widely used for in-vivo monitoring of anti-cancer therapy in mice. [18F]MEL050 is a Positron Emission Tomography (PET) radiotracer which specifically targets melanin. We evaluated planar BLI and [18F]MEL050-PET/CT for therapy (pro-apoptotic peptide LZDP) monitoring in a mouse model of metastatic pigmented melanoma. Twelve B6-albino mice were intravenously injected with B16-F10-luc2 cells on day 0 (D0). The mice received daily from D2 to D17 either an inactive peptide (G1, n=6), or LZDP (G2, n=6). They underwent both BLI and [18F]MEL050-PET/CT imaging on D2, D8 and D17. The number of visible tumors was determined on BLI and PET/CT. [18F]MEL050 uptake in tumor sites was quantified on PET/CT. After sacrifice (D17), the number of black tumors was counted ex-vivo. On D2, BLI and PET/CT images were visually negative. On D8, BLI detected 8 tumor sites in 4/6 mice of G1 vs 5 in 3/6 mice of G2 (NS); PET/CT was visually negative. On D17, BLI detected 17 tumor sites in 5/6 mice of G1 vs 10 in 4/6 mice of G2 (NS). PET/CT detected 18 tumor sites in 4/4 mice of G1 vs 14 in 3/4 mice of G2 (NS). Mean %ID/g of [18F]MEL050 in tumor sites was lower in G2 than in G1 on D17 (P<0.001), whereas bioluminescence intensity was not different between the 2 groups. Ex-vivo examination confirmed lower number of tumors in G2 (P<0.03). In the small number of animals tested in this study, [18F]MEL050-PET/CT and ex-vivo examination could affirm anti-tumoral effect of LZDP, but not BLI.

[18F]MEL050 as a melanin-targeted PET tracer: Fully automated radiosynthesis and comparison to 18F-FDG for the detection of pigmented melanoma in mice primary subcutaneous tumors and pulmonary metastases.

INTRODUCTION: Melanoma is a highly malignant cutaneous tumor of melanin-producing cells. MEL050 is a synthetic benzamide-derived molecule that specifically binds to melanin with high affinity. Our aim was to implement a fully automated radiosynthesis of [18F]MEL050, using for the first time, the AllInOne™ synthesis module (Trasis), and to evaluate the potential of [18F]MEL050 for the detection of pigmented melanoma in mice primary subcutaneous tumors and pulmonary metastases, and to compare it with that of [18F]FDG. METHODS: Automated radiosynthesis of [18F]MEL050, including HPLC purification and formulation, were performed on an AllInOne™ synthesis module. [18F]MEL050 was synthesized using a one-step bromine-for-fluorine nucleophilic heteroaromatic substitution. Melanoma models were induced by subcutaneous (primary tumor) or intravenous (pulmonary metastases) injection of B16-F10-luc2 cells in NMRI mice. The maximum percentage of [18F]MEL050 Injected Dose per g of lung tissue (%ID/g Max) was determined on PET images, compared to [18F]FDG and correlated to in vivo bioluminescence imaging. RESULTS: The automated radiosynthesis of [18F]MEL050 required an overall radiosynthesis time of 48min, with a yield of 13-18% (not-decay corrected) and radiochemical purity higher than 99%. [18F]MEL050 PET/CT images were concordant with bioluminescence imaging, showing increased radiotracer uptake in all primary subcutaneous tumors and pulmonary metastases of mice. PET quantification of radiotracers uptake in tumors and muscles demonstrated similar tumor-to-background ratio (TBR) with [18F]MEL050 and [18F]FDG in subcutaneous tumors and higher TBR with [18F]MEL050 than with [18F]FDG in pulmonary metastases. CONCLUSION: We successfully implemented the radiosynthesis of [18F]MEL050 using the AllInOne™ module, including HPLC purification and formulation. In vivo PET/CT validation of [18F]MEL050 was obtained in mouse models of pigmented melanoma, where higher [18F]MEL050 uptake was observed in sub-millimetric pulmonary metastases, comparatively to [18F]FDG.

A Cell-Penetrating Peptide Targeting AAC-11 Specifically Induces Cancer Cells Death.

AAC-11 is an antiapoptotic protein that is upregulated in most cancer cells. Increased expression of AAC-11 confers a survival advantage when cancer cells are challenged with various stresses and contributes to tumor invasion and metastases, whereas its deregulation reduces resistance to chemotherapeutic drugs. The antiapoptotic effect of AAC-11 may be clinically relevant as its expression correlates with poor prognosis in several human cancers. Thus, inactivation of AAC-11 might constitute an attractive approach for developing cancer therapeutics. We have developed an AAC-11-derived cell-penetrating peptide, herein named RT53, mimicking in part the heptad leucine repeat region of AAC-11, which functions as a protein-protein interaction module, and that can prevent AAC-11 antiapoptotic properties. In this study, we investigated the anticancer effects of RT53. Our results indicate that RT53 selectively kills cancer cells while sparing normal cells. RT53 selectively inserts into the membranes of cancer cells, where it adopts a punctate distribution and induces membranolysis and release of danger-associated molecular pattern molecules. Systemic administration of RT53 inhibited the growth of preexisting BRAF wild-type and V600E mutant melanoma xenograft tumors through induction of apoptosis and necrosis. Toxicological studies revealed that repetitive injections of RT53 did not produce significant toxicity. Finally, RT53-killed B16F10 cells induced tumor growth inhibition in immunocompetent mice following a rechallenge with live cancer cells of the same type. Collectively, our data demonstrate that RT53 possesses tumor-inhibitory activity with no toxicity in mice, suggesting its potential as a therapeutic agent for the treatment of melanoma and probably other cancers. Cancer Res; 76(18); 5479-90. ©2016 AACR.

DNA-PKcs plays role in cancer metastasis through regulation of secreted proteins involved in migration and invasion.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a major role in DNA damage signaling and repair and is also frequently overexpressed in tumor metastasis. We used isogenic cell lines expressing different levels of DNA-PKcs to investigate the role of DNA-PKcs in metastatic development. We found that DNA-PKcs participates in melanoma primary tumor and metastasis development by stimulating angiogenesis, migration and invasion. Comparison of conditioned medium content from DNA-PKcs-proficient and deficient cells reveals that DNA-PKcs controls secretion of at least 103 proteins (including 44 metastasis-associated with FBLN1, SERPINA3, MMP-8, HSPG2 and the inhibitors of matrix metalloproteinases, such as α-2M and TIMP-2). High throughput analysis of secretomes, proteomes and transcriptomes, indicate that DNA-PKcs regulates the secretion of 85 proteins without affecting their gene expression. Our data demonstrate that DNA-PKcs has a pro-metastatic activity via the modification of the tumor microenvironment. This study shows for the first time a direct link between DNA damage repair and cancer metastasis and highlights the importance of DNA-PKcs as a potential target for anti-metastatic treatment.

A preclinical study combining the DNA repair inhibitor Dbait with radiotherapy for the treatment of melanoma.

Melanomas are highly radioresistant tumors, mainly due to efficient DNA double-strand break (DSB) repair. Dbait (which stands for DNA strand break bait) molecules mimic DSBs and trap DNA repair proteins, thereby inhibiting repair of DNA damage induced by radiation therapy (RT). First, the cytotoxic efficacy of Dbait in combination with RT was evaluated in vitro in SK28 and 501mel human melanoma cell lines. Though the extent of RT-induced damage was not increased by Dbait, it persisted for longer revealing a repair defect. Dbait enhanced RT efficacy independently of RT doses. We further assayed the capacity of DT01 (clinical form of Dbait) to enhance efficacy of "palliative" RT (10 × 3 Gy) or "radical" RT (20 × 3 Gy), in an SK28 xenografted model. Inhibition of repair of RT-induced DSB by DT01 was revealed by the significant increase of micronuclei in tumors treated with combined treatment. Mice treated with DT01 and RT combination had significantly better tumor growth control and longer survival compared to RT alone with the "palliative" protocol [tumor growth delay (TGD) by 5.7-fold; median survival: 119 vs 67 days] or the "radical" protocol (TGD by 3.2-fold; median survival: 221 vs 109 days). Only animals that received the combined treatment showed complete responses. No additional toxicity was observed in any DT01-treated groups. This preclinical study provides encouraging results for a combination of a new DNA repair inhibitor, DT01, with RT, in the absence of toxicity. A first-in-human phase I study is currently under way in the palliative management of melanoma in-transit metastases (DRIIM trial).

DNA-PK target identification reveals novel links between DNA repair signaling and cytoskeletal regulation.

The DNA-dependent protein kinase (DNA-PK) may function as a key signaling kinase in various cellular pathways other than DNA repair. Using a two-dimensional gel electrophoresis approach and stable DNA double-strand break-mimicking molecules (Dbait32Hc) to activate DNA-PK in the nucleus and cytoplasm, we identified 26 proteins that were highly phosphorylated following DNA-PK activation. Most of these proteins are involved in protein stability and degradation, cell signaling and the cytoskeleton. We investigated the relationship between DNA-PK and the cytoskeleton and found that the intermediate filament (IF) vimentin was a target of DNA-PK in vitro and in cells. Vimentin was phosphorylated at Ser459, by DNA-PK, in cells transfected with Dbait32Hc. We produced specific antibodies and showed that Ser459-P-vimentin was mostly located at cell protrusions. In migratory cells, the vimentin phosphorylation induced by Dbait32Hc was associated with a lower cellular adhesion and migration capacity. Thus, this approach led to the identification of downstream cytoplasmic targets of DNA-PK and revealed a connection between DNA damage signaling and the cytoskeleton.