Caloric restriction leads to druggable LSD1-dependent cancer stem cells expansion.

Caloric Restriction (CR) has established anti-cancer effects, but its clinical relevance and molecular mechanism remain largely undefined. Here, we investigate CR's impact on several mouse models of Acute Myeloid Leukemias, including Acute Promyelocytic Leukemia, a subtype strongly affected by obesity. After an initial marked anti-tumor effect, lethal disease invariably re-emerges. Initially, CR leads to cell-cycle restriction, apoptosis, and inhibition of TOR and insulin/IGF1 signaling. The relapse, instead, is associated with the non-genetic selection of Leukemia Initiating Cells and the downregulation of double-stranded RNA (dsRNA) sensing and Interferon (IFN) signaling genes. The CR-induced adaptive phenotype is highly sensitive to pharmacological or genetic ablation of LSD1, a lysine demethylase regulating both stem cells and dsRNA/ IFN signaling. CR + LSD1 inhibition leads to the re-activation of dsRNA/IFN signaling, massive RNASEL-dependent apoptosis, and complete leukemia eradication in ~90% of mice. Importantly, CR-LSD1 interaction can be modeled in vivo and in vitro by combining LSD1 ablation with pharmacological inhibitors of insulin/IGF1 or dual PI3K/MEK blockade. Mechanistically, insulin/IGF1 inhibition sensitizes blasts to LSD1-induced death by inhibiting the anti-apoptotic factor CFLAR. CR and LSD1 inhibition also synergize in patient-derived AML and triple-negative breast cancer xenografts. Our data provide a rationale for epi-metabolic pharmacologic combinations across multiple tumors.

Targeting insulin-like growth factor I with 10-23 DNAzymes: 2'-O-methyl modifications in the catalytic core enhance mRNA cleavage.

Insulin-like growth factor I (IGF-I) and its cognate receptor (IGF-1R) contribute to normal cell function and to tumorigenesis. The role of IGF-I signaling in tumor growth has been demonstrated in vivo using nucleic acid-based strategies. Here, we designed the first 10-23 DNAzymes directed against IGF-I mRNA. Unlike antisense approaches and RNA interference that require protein catalysis, DNAzymes catalyze protein-free RNA cleavage. We identified target sequences and measured catalytic properties of differently designed DNAzymes on short synthetic RNA targets and on in vitro transcribed IGF-I mRNA. The most efficient cleavers were then transfected into cells, and their inhibitory effect was analyzed using reporter gene assays. We found that increasing the size of DNAzyme flanking sequences and modifications of the termini with 2'-O-methyl residues improved cleavage rates of target RNAs. Modification of the catalytic loop with six 2'-O-methyl ribonucleotides at nonessential positions increased or decreased catalytic efficiency depending on the mRNA target site. In cells, DNAzymes with 2'-O-methyl-modified catalytic cores and flanking sequences were able to inhibit reporter gene activity because of specific recognition and cleavage of IGF-I mRNA sequences. Mutant DNAzymes with inactive catalytic cores were unable to block reporter gene expression, demonstrating that the RNA cleaving ability of 10-23 DNAzymes contributed to inhibitory mechanisms. Our results show that nuclease-resistant 2'-O-methyl-modified DNAzymes with high catalytic efficiencies are useful for inhibiting IGF-I gene function in cells.

Small interfering RNA targeted to IGF-IR delays tumor growth and induces proinflammatory cytokines in a mouse breast cancer model.

Insulin-like growth factor I (IGF-I) and its type I receptor (IGF-IR) play significant roles in tumorigenesis and in immune response. Here, we wanted to know whether an RNA interference approach targeted to IGF-IR could be used for specific antitumor immunostimulation in a breast cancer model. For that, we evaluated short interfering RNA (siRNAs) for inhibition of in vivo tumor growth and immunological stimulation in immunocompetent mice. We designed 2'-O-methyl-modified siRNAs to inhibit expression of IGF-IR in two murine breast cancer cell lines (EMT6, C4HD). Cell transfection of IGF-IR siRNAs decreased proliferation, diminished phosphorylation of downstream signaling pathway proteins, AKT and ERK, and caused a G0/G1 cell cycle block. The IGF-IR silencing also induced secretion of two proinflammatory cytokines, TNF- α and IFN-γ. When we transfected C4HD cells with siRNAs targeting IGF-IR, mammary tumor growth was strongly delayed in syngenic mice. Histology of developing tumors in mice grafted with IGF-IR siRNA treated C4HD cells revealed a low mitotic index, and infiltration of lymphocytes and polymorphonuclear neutrophils, suggesting activation of an antitumor immune response. When we used C4HD cells treated with siRNA as an immunogen, we observed an increase in delayed-type hypersensitivity and the presence of cytotoxic splenocytes against wild-type C4HD cells, indicative of evolving immune response. Our findings show that silencing IGF-IR using synthetic siRNA bearing 2'-O-methyl nucleotides may offer a new clinical approach for treatment of mammary tumors expressing IGF-IR. Interestingly, our work also suggests that crosstalk between IGF-I axis and antitumor immune response can mobilize proinflammatory cytokines.

Chronodisruption in lung cancer and possible therapeutic approaches.

A customary temporal organization of physiological functions and biological processes is necessary to maintain body homeostasis and an altered body time structure may favour carcinogenesis. There is growing evidence that GH stimulates cancer growth, IGF1 may have a role in carcinogenesis and cancer promotion, GH-IGF1 axis, TRH, TSH, thyroxine, melatonin and cortisol modulate immune cell function and the immune system is often dysfunctional in patients with malignancies. The aim of our study was to evaluate GH-IGF1 axis, hypothalamus-pituitary-thyroid axis, melatonin, cortisol, lymphocyte subsets and IL2 in lung cancer patients. Peripheral blood samples were collected at 4-hour intervals in a 24-hour period from eleven healthy male subjects (age range 35-53 years) and nine male patients suffering from non-small cell lung cancer (age range 43-63 years). In each blood sample, lymphocyte subpopulations (CD3+, CD4+, CD8+, CD16+, CD20+, CD25+, HLA-DR+, γδTcR bearing cells) were analyzed and GH, IGF1, TRH, TSH, FT4, melatonin, cortisol and IL2 were measured. Circadian rhythmicity was evaluated and MESOR, amplitude and acrophase values were compared. In healthy subjects a significant circadian rhythm could be demonstrated with midday peaks for CD8+, CD16+, γδTCR expressing cells and cortisol, and peaks during the night for CD3+, CD4+, GH, TSH and melatonin. A borderline significant rhythm was also observed for CD20+, with a peak late in the evening. IGF1, TRH, FT4 and IL2 values did not show rhythmic variation. In cancer patients a significant circadian rhythm could be demonstrated with diurnal peak for CD16+ and peaks during the night for CD4+ and melatonin. GH, IGF1, TRH, TSH, FT4, cortisol and IL2 values did not show rhythmic variation. MESOR of CD8+ (P<0.0001), CD20+ (P=0.05), γδTCR expressing cells (P=0.01), IGF1 (P<0.001) and TSH (P=0.032) was higher in healthy subjects, whereas MESOR of CD16+ (P<0.0001), CD25+ (P=0.001), GH (P<0.001), TRH (P=0.002), FT4 (P=0.030), cortisol (P=0.01) and IL2 (P=0.02) was higher in cancer patients. Amplitude of circadian variation of γδTCR expressing cells (P=0.01), TSH (P<0.001) and cortisol (P=0.01) was higher in healthy subjects, whereas amplitude of circadian variation of CD4+ was higher in cancer patients (P=0.02). In conclusion, non-small cell lung cancer patients show severe alterations of periodic and quantitative characteristics of neuroendocrine and immune parameters with loss of circadian rhythmicity and internal desynchronization, leading to chronodisruption.