GM-1111 reduces radiation-induced oral mucositis in mice by targeting pattern recognition receptor-mediated inflammatory signaling.

PURPOSE: Oral mucositis (OM) is a common, painful side effect of radiation therapy used for the treatment of head and neck cancer (HNC). Activation of the innate immune system upon irradiation has been identified as a key precipitating event of OM. To better understand OM's pathogenesis, we studied pattern recognition receptors (PRRs) and their downstream pro-inflammatory cytokines in a mouse model of radiation-induced OM. We also tested therapeutic efficacy of GM-1111 that targets innate immune system to reduce radiation-induced OM. METHODS AND MATERIALS: The pathogenesis of OM was studied in a single X-ray induced mouse model. The severity of OM was measured by visual and microscopical examinations. The irradiation-induced changes of PRRs and their downstream effector cytokine gene expression levels were determined. The efficacy of GM-1111 to reduce OM was tested in single and fractionated irradiation mouse models. The impact of the drug on tumor response to radiation therapy was also tested in a mouse model of human HNC. RESULTS: Radiation-induced tissue ulcerations were radiation-dosage and -time dependent. The lesions showed selective increases in PRR and pro-inflammatory cytokine gene expression levels. Once daily administration of GM-1111 (≥30 mg/kg, s.c.) significantly reduced the severity and the incidence of OM. The drug had little effect on PRRs but significantly inhibited downstream pro-inflammatory cytokine genes. GM-1111 did not interfere radiation therapy to induce HNC SCC-25 tumor regression. Instead, we observed significant drug-induced tumor regression. CONCLUSIONS: Radiation induces tissue damages. The increased expression levels of PRRs and their downstream pro-inflammatory cytokine genes in the damaged tissues suggest their important contribution to the pathogenesis of OM. Drug GM-1111 that targets these innate immune molecules may be a potential drug candidate as an intervention for OM.

Microenvironmental Regulation of Long Noncoding RNA LINC01133 Promotes Cancer Stem Cell-Like Phenotypic Traits in Triple-Negative Breast Cancers.

The fibrotic tumor microenvironment is a critical player in the pathogenesis of triple-negative breast cancers (TNBCs), with the presence of fibroblastic infiltrates particularly correlating with tumors that are clinically advanced. On this front, we previously demonstrated that TNBCs are highly enriched in fibroblastic stromal progenitor cells called mesenchymal stem/stromal cells (MSCs) and that such cells play critical roles in promoting TNBC initiation and progression. How TNBC cells respond to MSC stimulation, however, is not fully understood, and stands to reveal contextual signals used by TNBC cells during tumor development and provide biomarkers and therapeutic targets of pertinence to TNBC management. Here, we report that MSCs strongly induced the long noncoding RNA (lncRNA) LINC01133 in neighboring TNBC cells. Indeed, although lncRNAs have been tightly associated with cancer development, their contributions to breast cancer in general, and to TNBC pathogenesis in particular, have not been fully elucidated, and we set out to determine if LINC01133 regulated malignant traits in TNBC cells. We establish that LINC01133 is sufficient, on its own, in promoting phenotypic and growth characteristics of cancer stem cell-like cells, and that it is a direct mediator of the MSC-triggered miR-199a-FOXP2 pathway in TNBC models. Furthermore, we show that LINC01133 is a critical regulator of the pluripotency-determining gene Kruppel-Like Factor 4 (KLF4), and that it represents a biomarker and prognosticator of disease outcome in the clinic. Collectively, our findings introduce LINC01133 as a novel functional driver of malignancy and a potential theranostic in TNBC. Stem Cells 2019;37:1281-1292.

Pentraxin-3 is a PI3K signaling target that promotes stem cell-like traits in basal-like breast cancers.

Basal-like breast cancers (BLBCs) exhibit hyperactivation of the phosphoinositide 3-kinase (PI3K) signaling pathway because of the frequent mutational activation of the PIK3CA catalytic subunit and the genetic loss of its negative regulators PTEN (phosphatase and tensin homolog) and INPP4B (inositol polyphosphate-4-phosphatase type II). However, PI3K inhibitors have had limited clinical efficacy in BLBC management because of compensatory amplification of PI3K downstream signaling loops. Therefore, identification of critical PI3K mediators is paramount to the development of effective BLBC therapeutics. Using transcriptomic analysis of activated PIK3CA-expressing BLBC cells, we identified the gene encoding the humoral pattern recognition molecule pentraxin-3 (PTX3) as a critical target of oncogenic PI3K signaling. We found that PTX3 abundance is stimulated, in part, through AKT- and nuclear factor κB (NF-κB)-dependent pathways and that presence of PTX3 is necessary for PI3K-induced stem cell-like traits. We further showed that PTX3 expression is greater in tumor samples from patients with BLBC and that it is prognostic of poor patient survival. Our results thus reveal PTX3 as a newly identified PI3K-regulated biomarker and a potential therapeutic target in BLBC.

Silencing FOXP2 in breast cancer cells promotes cancer stem cell traits and metastasis.

In a recent article in Cell Stem Cell, we showed that mesenchymal stem cells (MSCs), progenitor cells that populate the breast tumor stroma, induce microRNA-mediated FOXP2 repression in breast cancer cells (BCCs), thus promoting cancer stem cell (CSC) and metastatic traits. Here, we discuss the implications of these findings for understanding metastatic CSC genesis.

MSC-regulated microRNAs converge on the transcription factor FOXP2 and promote breast cancer metastasis.

Mesenchymal stem/stromal cells (MSCs) are progenitor cells shown to participate in breast tumor stroma formation and to promote metastasis. Despite expanding knowledge of their contributions to breast malignancy, the underlying molecular responses of breast cancer cells (BCCs) to MSC influences remain incompletely understood. Here, we show that MSCs cause aberrant expression of microRNAs, which, led by microRNA-199a, provide BCCs with enhanced cancer stem cell (CSC) properties. We demonstrate that such MSC-deregulated microRNAs constitute a network that converges on and represses the expression of FOXP2, a forkhead transcription factor tightly associated with speech and language development. FOXP2 knockdown in BCCs was sufficient in promoting CSC propagation, tumor initiation, and metastasis. Importantly, elevated microRNA-199a and depressed FOXP2 expression levels are prominent features of malignant clinical breast cancer and are associated significantly with poor survival. Our results identify molecular determinants of cancer progression of potential utility in the prognosis and therapy of breast cancer.

Mesenchymal stem cells in tumor development: emerging roles and concepts.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that participate in the structural and functional maintenance of connective tissues under normal homeostasis. They also act as trophic mediators during tissue repair, generating bioactive molecules that help in tissue regeneration following injury. MSCs serve comparable roles in cases of malignancy and are becoming increasingly appreciated as critical components of the tumor microenvironment. MSCs home to developing tumors with great affinity, where they exacerbate cancer cell proliferation, motility, invasion and metastasis, foster angiogenesis, promote tumor desmoplasia and suppress anti-tumor immune responses. These multifaceted roles emerge as a product of reciprocal interactions occurring between MSCs and cancer cells and serve to alter the tumor milieu, setting into motion a dynamic co-evolution of both tumor and stromal tissues that favors tumor progression. Here, we summarize our current knowledge about the involvement of MSCs in cancer pathogenesis and review accumulating evidence that have placed them at the center of the pro-malignant tumor stroma.

Lysosomal proteases are involved in generation of N-terminal huntingtin fragments.

N-terminal mutant huntingtin (N-mhtt) fragments form inclusions and cause cell death in vitro. Mutant htt expression stimulates autophagy and increases levels of lysosomal proteases. Here, we show that lysosomal proteases, cathepsins D, B and L, affected mhtt processing and levels of cleavage products (cp) known as A and B, which form inclusions. Adding inhibitors of cathepsin D, B and L to clonal striatal cells reduced mhtt, especially mhtt fragment cp A. Mutant htt fully degraded in cathepsin-L-treated lysates but formed stable N-mhtt fragments upon exposure to cathepsin D. Mutagenesis analysis of htt cDNA suggested that cathepsin D and the protease for cp A may cleave htt in the same region. Brain lysates from HD knock-in mice expressed N-mhtt fragments that accumulated with cathepsin D treatment and declined with aspartyl protease inhibition. Findings implicate lysosomal proteases in formation of N-mhtt fragments and clearance of mhtt.

Huntingtin is present in the nucleus, interacts with the transcriptional corepressor C-terminal binding protein, and represses transcription.

Huntingtin is a protein of unknown function that contains a polyglutamine tract, which is expanded in patients with Huntington's disease (HD). We investigated the localization and a potential function for huntingtin in the nucleus. In human fibroblasts from normal and HD patients, huntingtin localized diffusely in the nucleus and in subnuclear compartments identified as speckles, promyelocytic leukemia protein bodies, and nucleoli. Huntingtin-positive nuclear bodies redistributed after treatment with sodium butyrate. By Western blot, purified nuclei had low levels of full-length huntingtin compared with the cytoplasm but contained high levels of N- and C-terminal huntingtin fragments, which tightly bound the nuclear matrix. Full-length huntingtin co-immunoprecipitated with the transcriptional corepressor C-terminal binding protein, and polyglutamine expansion in huntingtin reduced this interaction. Full-length wild-type and mutant huntingtin repressed transcription when targeted to DNA. Truncated N-terminal mutant huntingtin repressed transcription, whereas the corresponding wild-type fragment did not repress transcription. We speculate that wild-type huntingtin may function in the nucleus in the assembly of nuclear matrix-bound protein complexes involved with transcriptional repression and RNA processing. Proteolysis of mutant huntingtin may alter nuclear functions by disrupting protein complexes and inappropriately repressing transcription in HD.