Roles of exosomes in metastatic colorectal cancer.

Metastases remain a major cause of cancer morbidity and mortality. This is a multistep process that involves aberrant cell communication, leading to tumor cell dissemination from the primary tumor and colonization of distinct organs for secondary tumor formation. The mechanisms promoting this pathological process are not fully understood, although they may be of obvious therapeutic interest. Exosomes are small cell-secreted vesicles that contain a large variety of proteins, lipids, and nucleic acids with important signaling activities, and that represent an evolutionarily conserved mechanism for cell-to-cell communication. Not surprisingly, exosome activities have gained strong interest in cancer biology and might play essential roles in metastasis development. Here, we will describe recent findings on the role of exosomes in cancer metastasis formation, particularly in colorectal cancer (CRC). We will also discuss the potential therapeutic value of these vesicles in metastatic cancer.

Design of a small molecule against an oncogenic noncoding RNA.

The design of precision, preclinical therapeutics from sequence is difficult, but advances in this area, particularly those focused on rational design, could quickly transform the sequence of disease-causing gene products into lead modalities. Herein, we describe the use of Inforna, a computational approach that enables the rational design of small molecules targeting RNA to quickly provide a potent modulator of oncogenic microRNA-96 (miR-96). We mined the secondary structure of primary microRNA-96 (pri-miR-96) hairpin precursor against a database of RNA motif-small molecule interactions, which identified modules that bound RNA motifs nearby and in the Drosha processing site. Precise linking of these modules together provided Targaprimir-96 (3), which selectively modulates miR-96 production in cancer cells and triggers apoptosis. Importantly, the compound is ineffective on healthy breast cells, and exogenous overexpression of pri-miR-96 reduced compound potency in breast cancer cells. Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), a small-molecule RNA target validation approach, shows that 3 directly engages pri-miR-96 in breast cancer cells. In vivo, 3 has a favorable pharmacokinetic profile and decreases tumor burden in a mouse model of triple-negative breast cancer. Thus, rational design can quickly produce precision, in vivo bioactive lead small molecules against hard-to-treat cancers by targeting oncogenic noncoding RNAs, advancing a disease-to-gene-to-drug paradigm.

Identification of an EGFRvIII-JNK2-HGF/c-Met-Signaling Axis Required for Intercellular Crosstalk and Glioblastoma Multiforme Cell Invasion.

Glioblastoma multiforme (GBM) is the most aggressive and common form of adult brain cancer. Current therapeutic strategies include surgical resection, followed by radiotherapy and chemotherapy. Despite such aggressive multimodal therapy, prognosis remains poor, with a median patient survival of 14 months. A proper understanding of the molecular drivers responsible for GBM progression are therefore necessary to instruct the development of novel targeted agents and enable the design of effective treatment strategies. Activation of the c-Jun N-terminal kinase isoform 2 (JNK2) is reported in primary brain cancers, where it associates with the histologic grade and amplification of the epidermal growth factor receptor (EGFR). In this manuscript, we demonstrate an important role for JNK2 in the tumor promoting an invasive capacity of EGFR variant III, a constitutively active mutant form of the receptor commonly found in GBM. Expression of EGFR variant III induces transactivation of JNK2 in GBM cells, which is required for a tumorigenic phenotype in vivo. Furthermore, JNK2 expression and activity is required to promote increased cellular invasion through stimulation of a hepatocyte growth factor-c-Met signaling circuit, whereby secretion of this extracellular ligand activates the receptor tyrosine kinase in both a cell autonomous and nonautonomous manner. Collectively, these findings demonstrate the cooperative and parallel activation of multiple RTKs in GBM and suggest that the development of selective JNK2 inhibitors could be therapeutically beneficial either as single agents or in combination with inhibitors of EGFR and/or c-Met.

FGFR3 has tumor suppressor properties in cells with epithelial phenotype.

BACKGROUND: Due to frequent mutations in certain cancers, FGFR3 gene is considered as an oncogene. However, in some normal tissues, FGFR3 can limit cell growth and promote cell differentiation. Thus, FGFR3 action appears paradoxical. RESULTS: FGFR3 expression was forced in pancreatic cell lines. The receptor exerted dual effects: it suppressed tumor growth in pancreatic epithelial-like cells and had oncogenic properties in pancreatic mesenchymal-like cells. Distinct exclusive pathways were activated, STATs in epithelial-like cells and MAP Kinases in mesenchymal-like cells. Both FGFR3 splice variants had similar effects and used the same intracellular signaling. In human pancreatic carcinoma tissues, levels of FGFR3 dropped in tumors. CONCLUSION: In tumors from epithelial origin, FGFR3 signal can limit tumor growth, explaining why the 4p16.3 locus bearing FGFR3 is frequently lost and why activating mutations of FGFR3 in benign or low grade tumors of epithelial origin are associated with good prognosis. The new hypothesis that FGFR3 can harbor both tumor suppressive and oncogenic properties is crucial in the context of targeted therapies involving specific tyrosine kinase inhibitors (TKIs). TKIs against FGFR3 might result in adverse effects if used in the wrong cell context.

Comprehensive analysis of PTEN status in breast carcinomas.

PTEN plays a well-established role in the negative regulation of the PI3K pathway, which is frequently activated in several cancer types, including breast cancer. A nuclear function in the maintenance of chromosomal stability has been proposed for PTEN but is yet to be clearly defined. In order to improve understanding of the role of PTEN in mammary tumorigenesis in terms of a possible gene dosage effect, its PI3K pathway function and its association with p53, we undertook comprehensive analysis of PTEN status in 135 sporadic invasive ductal carcinomas. Four PTEN status groups were defined; complete loss (19/135, 14%), reduced copy number (19/135, 14%), normal (86/135, 64%) and complex (11/135, 8%). Whereas the PTEN complete loss status was significantly associated with estrogen receptor (ER) negativity (p=0.006) and in particular the basal-like phenotype (p<0.0001), a reduced PTEN copy number was not associated with hormone receptor status or a particular breast cancer subtype. Overall, PI3K pathway alteration was suggested to be involved in 59% (79/134) of tumors as assessed by human epidermal growth factor receptor 2 overexpression, PIK3CA mutation or a complete loss of PTEN. A complex PTEN status was identified in a tumor subgroup which displayed a specific, complex DNA profile at the PTEN locus with a strikingly similar highly rearranged pan-genomic profile. All of these tumors had relapsed and were associated with a poorer prognosis in the context of node negative disease (p=1.4 × 10(-13) ) thus may represent a tumor subgroup with a common molecular alteration which could be targeted to improve clinical outcome.

In vivo gene transfer targeting in pancreatic adenocarcinoma with cell surface antigens.

BACKGROUND: Pancreatic ductal adenocarcinoma is a deadly malignancy resistant to current therapies. It is critical to test new strategies, including tumor-targeted delivery of therapeutic agents. This study tested the possibility to target the transfer of a suicide gene in tumor cells using an oncotropic lentiviral vector. RESULTS: Three cell surface markers were evaluated to target the transduction of cells by lentiviruses pseudotyped with a modified glycoprotein from Sindbis virus. Only Mucin-4 and the Claudin-18 proteins were found efficient for targeted lentivirus transductions in vitro. In subcutaneous xenografts of human pancreatic cancer cells models, Claudin-18 failed to achieve efficient gene transfer but Mucin-4 was found very potent. Human pancreatic tumor cells were modified to express a fluorescent protein detectable in live animals by bioimaging, to perform a direct non invasive and costless follow up of the tumor growth. Targeted gene transfer of a bicistronic transgene bearing a luciferase gene and the herpes simplex virus thymidine kinase gene into orthotopic grafts was carried out with Mucin-4 oncotropic lentiviruses. By contrast to the broad tropism VSV-G carrying lentivirus, this oncotropic lentivirus was found to transduce specifically tumor cells, sparing normal pancreatic cells in vivo. Transduced cells disappeared after ganciclovir treatment while the orthotopic tumor growth was slowed down. CONCLUSION: This work considered for the first time three aspect of pancreatic adenocarcinoma targeted therapy. First, lentiviral transduction of human pancreatic tumor cells was possible when cells were grafted orthotopically. Second, we used a system targeting the tumor cells with cell surface antigens and sparing the normal cells. Finally, the TK/GCV anticancer system showed promising results in vivo. Importantly, the approach presented here appeared to be a safer, much more specific and an as efficient way to perform gene delivery in pancreatic tumors, in comparison with a broad tropism lentivirus. This study will be useful in future designing of targeted therapies for pancreatic cancer.

Regulation of Src tumor activity by its N-terminal intrinsically disordered region.

The membrane-anchored Src tyrosine kinase is involved in numerous pathways and its deregulation is involved in human cancer. Our knowledge on Src regulation relies on crystallography, which revealed intramolecular interactions to control active Src conformations. However, Src contains a N-terminal intrinsically disordered unique domain (UD) whose function remains unclear. Using NMR, we reported that UD forms an intramolecular fuzzy complex involving a conserved region with lipid-binding capacity named Unique Lipid-Binding Region (ULBR), which could modulate Src membrane anchoring. Here we show that the ULBR is essential for Src's oncogenic capacity. ULBR inactive mutations inhibited Src transforming activity in NIH3T3 cells and in human colon cancer cells. It also reduced Src-induced tumor development in nude mice. An intact ULBR was required for MAPK signaling without affecting Src kinase activity nor sub-cellular localization. Phospho-proteomic analyses revealed that, while not impacting on the global tyrosine phospho-proteome in colon cancer cells, this region modulates phosphorylation of specific membrane-localized tyrosine kinases needed for Src oncogenic signaling, including EPHA2 and Fyn. Collectively, this study reveals an important role of this intrinsically disordered region in malignant cell transformation and suggests a novel layer of Src regulation by this unique region via membrane substrate phosphorylation.

IR spectral imaging of secreted mucus: a promising new tool for the histopathological recognition of human colonic adenocarcinomas.

AIMS: During colonic carcinogenesis, mucin-type glycoproteins are known to undergo quantitative and qualitative alterations. The aim of this study was to determine the value of infrared (IR) spectral histology for the histopathological recognition of colonic adenocarcinomas based on mucin-associated IR spectral markers. METHODS AND RESULTS: Paraffin-embedded tissue sections of normal human colon and adenocarcinomas were analysed directly by IR-microspectroscopy (IR-MSP), without prior chemical dewaxing. IR-MSP imaging combined with multivariate analysis permitted the construction of IR colour-coded images of the tissue sections providing spatially resolved biochemical information. This allowed localization of mucin-rich areas and provided label-free spectral-based staining of secreted mucus related to the biochemical heterogeneity of its mucin content. IR images of secreted mucus display the same spectral clusters in both normal and adenocarcinomatous colonic tissues, but with significant differences in surface percentages. Such differences allow a distinction between these two tissue types. Spectral variations associated with changes of mucin secondary structure were the most accurate mucus spectral marker for discriminating between normal colon and adenocarcinomas in the sample set. CONCLUSIONS: IR-MSP imaging provides a new type of histology, independent of visual morphology, presenting tremendous possibilities for discovery and clinical monitoring of cancer markers.

Collagen Kinase Receptors as Potential Therapeutic Targets in Metastatic Colon Cancer.

Colorectal cancer (CRC) is one of the leading causes of tumor-related death worldwide. While surgery can cure patients with early stage CRC, the 5-year survival rate is only 10% for patients with metastatic disease. Therefore, new anti-metastatic therapies are needed for this cancer. Metastatic spread defines the dissemination of cancer cells with tumor-initiating capacities from the primary tumor and their colonization of distinct organs, mainly the liver, for secondary tumor formation. Although the underlying mechanisms are not fully understood, components of the tumor microenvironment have gained strong interest. Among the known metastatic-promoting factors, collagens are extracellular matrix components that are deposited within the tumor, the tumor microenvironment, and at metastatic site(s), and are recognized to play essential roles during metastasis development. Here, we review recent findings on the metastatic role of the collagen receptors Discoidin Domain Receptors 1 and 2 (DDR1 and DDR2) in CRC and discuss the therapeutic value of targeting these receptor tyrosine kinases in this cancer.

Src Family Tyrosine Kinases in Intestinal Homeostasis, Regeneration and Tumorigenesis.

Src, originally identified as an oncogene, is a membrane-anchored tyrosine kinase and the Src family kinase (SFK) prototype. SFKs regulate the signalling induced by a wide range of cell surface receptors leading to epithelial cell growth and adhesion. In the intestine, the SFK members Src, Fyn and Yes regulate epithelial cell proliferation and migration during tissue regeneration and transformation, thus implicating conserved and specific functions. In patients with colon cancer, SFK activity is a marker of poor clinical prognosis and a potent driver of metastasis formation. These tumorigenic activities are linked to SFK capacity to promote the dissemination and tumour-initiating capacities of epithelial tumour cells. However, it is unclear how SFKs promote colon tumour formation and metastatic progression because SFK-encoding genes are unfrequently mutated in human cancer. Here, we review recent findings on SFK signalling during intestinal homeostasis, regeneration and tumorigenesis. We also describe the key nongenetic mechanisms underlying SFK tumour activities in colorectal cancer, and discuss how these mechanisms could be exploited in therapeutic strategies to target SFK signalling in metastatic colon cancer.

Mesenchymal Stem Cell Administration Attenuates Colon Cancer Progression by Modulating the Immune Component within the Colorectal Tumor Microenvironment.

We here determine the influence of mesenchymal stem cell (MSC) therapy on the progression of solid tumors. The influence of MSCs was investigated in human colorectal cancer cells as well as in an immunocompetent rat model of colorectal carcinogenesis representative of the human pathology. Treatment with bone marrow (BM)-derived MSCs significantly reduced both cancer initiation and cancer progression by increasing the number of tumor-free animals as well as decreasing the number and the size of the tumors by half, thereby extending their lifespan. The attenuation of cancer progression was mediated by the capacity of the MSCs to modulate the immune component. Specifically, in the adenocarcinomas (ADKs) of MSC-treated rats, the infiltration of CD68+ monocytes/macrophages was 50% less while the presence of CD3+ lymphocytes increased almost twofold. The MSCs reprogrammed the macrophages to become regulatory cells involved in phagocytosis thereby inhibiting the production of proinflammatory cytokines. Furthermore, the MSCs decreased NK (Natural Killer) and rTh17 cell activities, Treg recruitment, the presence of CD8+ lymphocytes and endothelial cells while restoring Th17 cell activity. The expression of miR-150 and miR-7 increased up to fivefold indicating a likely role for these miRNAs in the modulation of tumor growth. Importantly, MSC administration limited the damage of healthy tissues and attenuated tumor growth following radiotherapy. Taken together, we here show that that MSCs have durable action on colon cancer development by modulating the immune component of the tumor microenvironment. In addition, we identify two miRNAs associated with the capacity of MSCs to attenuate cancer growth. Stem Cells Translational Medicine 2019;8:285&300.

DDR1 inhibition as a new therapeutic strategy for colorectal cancer.

The clinical management of metastatic colorectal cancer (mCRC) is still a major challenge. Recently, we discovered that nilotinib, an approved treatment for chronic myeloid leukaemia, inhibits invasive and metastatic properties of CRC cells by targeting the kinase activity of receptor for collagens DDR1 (Discoïdin Domain Receptor tyrosine kinase 1), suggesting that nilotinib could be an effective strategy to treat mCRC.

Inhibition of DDR1-BCR signalling by nilotinib as a new therapeutic strategy for metastatic colorectal cancer.

The clinical management of metastatic colorectal cancer (mCRC) faces major challenges. Here, we show that nilotinib, a clinically approved drug for chronic myeloid leukaemia, strongly inhibits human CRC cell invasion in vitro and reduces their metastatic potential in intrasplenic tumour mouse models. Nilotinib acts by inhibiting the kinase activity of DDR1, a receptor tyrosine kinase for collagens, which we identified as a RAS-independent inducer of CRC metastasis. Using quantitative phosphoproteomics, we identified BCR as a new DDR1 substrate and demonstrated that nilotinib prevents DDR1-mediated BCR phosphorylation on Tyr177, which is important for maintaining ß-catenin transcriptional activity necessary for tumour cell invasion. DDR1 kinase inhibition also reduced the invasion of patient-derived metastatic and circulating CRC cell lines. Collectively, our results indicate that the targeting DDR1 kinase activity with nilotinib may be beneficial for patients with mCRC.

PAR-2 activation increases human intestinal mucin secretion through EGFR transactivation.

PAR-2 (protease-activated receptors-2) are G protein-coupled receptors whose action on mucin secretion by intestinal epithelial cells is still unknown. The aim of this study was to examine the effect of PAR-2 activation on mucin secretion in the human colonic goblet cell line HT29-Cl.16E and the intracellular pathways involved. We found that PAR-2 mRNA was constitutively expressed by HT29-Cl.16E cells as well as by isolated human normal colonocytes. The PAR-2-activating peptide SLIGKV-NH(2) elicited rapid mucin secretion in HT29-Cl.16E, which was partially inhibited by calcium chelator BAPTA. Inhibitors of MAPK activation (PD98059) and EGFR tyrosine kinase activity (AG1478) abrogated PAR-2-induced ERK1/2 and EGFR tyrosine phosphorylation, respectively, and subsequent mucin secretion. Finally, PAR-2-induced EGFR transactivation was involved upstream of ERK1/2 activation. Our results show that the activation of PAR-2 expressed by human intestinal epithelial cells enhances mucin secretion, a component of the intestinal innate defence, via a pathway involving EGFR transactivation.

Characterization of the glycosidic linkage of underivatized disaccharides by interaction with Pb(2+) ions.

Electrospray ionization in combination with tandem mass spectrometry and lead cationization is used to characterize the linkage position of underivatized disaccharides. Lead(II) ions react mainly with disaccharides by proton abstraction to generate [Pb(disaccharide)(m)-H](+) ions (m = 1-2). At low cone voltages, an intense series of doubly charged ions of general formula [Pb(disaccharide)(n)](2+) are also observed. Our study shows that MS/MS experiments have to be performed to differentiate Pb(2+)-coordinated disaccharides. Upon collision, [Pb(disaccharide)-H](+) species mainly dissociate according to glycosidic bond cleavage and cross-ring cleavages, leading to the elimination of C(n)H(2n)O(n) neutrals (n = 2-4). The various fragmentation processes allow the position of the glycosidic bond to be unambiguously located. Distinction between glc-glc and glc-fru disaccharides also appears straightforward. Furthermore, for homodimers of D-glucose our data demonstrate that the anomericity of the glycosidic bond can be characterized for the 1 --> n linkages (n = 2, 4, 6). Consequently, Pb(2+) cationization combined with tandem mass spectrometry appears particularly useful to identify underivatized disaccharides.

Development of an HTS-Compatible Assay for the Discovery of Ulk1 Inhibitors.

A rapidly accumulating body of work suggests the autophagy pathway is an attractive therapeutic target for neurodegenerative diseases and cancer. To validate autophagy as an anticancer strategy and to assess if systemic inhibition of the pathway will have deleterious effects on normal tissues and physiology, highly selective autophagy inhibitors are needed. While several inducers and inhibitors of autophagy are known, all are nonspecific and none target the enzymes that execute the pathway. A central upstream regulator of the autophagy pathway is the serine/threonine kinase Ulk1 (UNC-51-like kinase-1). Selective molecular probes that function as Ulk1-specific inhibitors are needed to improve our understanding of the autophagy pathway. To identify inhibitors of Ulk1 kinase activity, we developed an HTS-compatible, homogeneous biochemical assay using AlphaScreen technology. This novel assay design uses purified stress-activated Ulk1 and monitors phosphorylation of its full-length native substrate, Atg13. This assay was optimized and validated in a 384-well format by screening the Sigma LOPAC library. Here we report that the Ulk1 AlphaScreen assay is robust and reproducible, with a Z' factor value of 0.83 ± 0.02 and a signal to background ratio of 20 ± 1.2. Thus, this assay can be used to screen large chemical libraries to discover novel inhibitors of Ulk1.

Therapeutic targeting of casein kinase 1δ in breast cancer.

Identification of specific drivers of human cancer is required to instruct the development of targeted therapeutics. We demonstrate that CSNK1D is amplified and/or overexpressed in human breast tumors and that casein kinase 1δ (CK1δ) is a vulnerability of human breast cancer subtypes overexpressing this kinase. Specifically, selective knockdown of CK1δ, or treatment with a highly selective and potent CK1δ inhibitor, triggers apoptosis of CK1δ-expressing breast tumor cells ex vivo, tumor regression in orthotopic models of triple-negative breast cancer, including patient-derived xenografts, and tumor growth inhibition in human epidermal growth factor receptor 2-positive (HER2(+)) breast cancer models. We also show that Wnt/ß-catenin signaling is a hallmark of human tumors overexpressing CK1δ, that disabling CK1δ blocks nuclear accumulation of ß-catenin and T cell factor transcriptional activity, and that constitutively active ß-catenin overrides the effects of inhibition or silencing of CK1δ. Thus, CK1δ inhibition represents a promising strategy for targeted treatment in human breast cancer with Wnt/ß-catenin involvement.

Proteases present in some pancreatic cyst fluids may affect mucin immunoassay by degrading antibodies and antigens.

OBJECTIVES: Biomarker detection in pancreatic cyst fluids is of importance to improve the diagnosis of mucinous cystadenoma, a precancerous lesion. However, assay protocols are generally established for serum testing. METHODS: Immunoradiometric assay of gastric M1/MUC5AC mucin was performed on pancreatic cyst fluids with well-characterized monoclonal antibodies. RESULTS: Among 1466 pancreatic cyst fluids tested, about 10% to 15% of samples presented abnormal behaviors: (i) radioactivity measured after immunoradiometric assay much lower than the blank of the assay and (ii) increasing dilution of the fluids leading to apparent increase of M1/MUC5AC concentration. In contrast, none of the 109 hepatic cyst fluids tested presented interference.We demonstrate that some (n = 54) interfering fluids cause mucin degradation as well as antibody degradation. Western blot analysis showed that the C-terminal part of the M1/MUC5AC apomucin is most sensitive to degradation. CONCLUSIONS: The presence of proteases that degrade antibodies as well as mucin may explain the pitfalls observed in 3.6% of the samples. To detect this interference, each fluid has to be systematically tested at 1:100 dilution in the presence of a saturating concentration of M1/MUC5AC mucin standard and in the absence of antiprotease reagents. Detection of interference could prevent false results caused by mucin degradation in situ.