Lysyl oxidase activity regulates oncogenic stress response and tumorigenesis

Abstract

Cellular senescence, a stable proliferation arrest, is induced in response to various stresses. Oncogenic stress-induced senescence (OIS) results in blocked proliferation and constitutes a fail-safe program counteracting tumorigenesis. The events that enable a tumor in a benign senescent state to escape from OIS and become malignant are largely unknown. We show that lysyl oxidase activity contributes to the decision to maintain senescence. Indeed, in human epithelial cell the constitutive expression of the LOX or LOXL2 protein favored OIS escape, whereas inhibition of lysyl oxidase activity was found to stabilize OIS. The relevance of these in vitro observations is supported by in vivo findings: in a transgenic mouse model of aggressive pancreatic ductal adenocarcinoma (PDAC), increasing lysyl oxidase activity accelerates senescence escape, whereas inhibition of lysyl oxidase activity was found to stabilize senescence, delay tumorigenesis, and increase survival. Mechanistically, we show that lysyl oxidase activity favors the escape of senescence by regulating the focal-adhesion kinase. Altogether, our results demonstrate that lysyl oxidase activity participates in primary tumor growth by directly impacting the senescence stability.

Figure 1

HEC-TM cells enter senescence after MEK activation. (a) HEC-TM cells were treated for 3 days with 4-OHT to activate MEK and were monitored the following days. (b) Cell extracts were prepared and analyzed by immunoblotting with the indicated antibodies at d0 or at d5. Tubulin was used as a loading control. (c) Cells were PFA-fixed and crystal violet-stained. (d) Cells were fixed and assayed for SA-β-Gal activity. (e) RNAs were prepared and RT-qPCRs were performed against the indicated genes. Expression was normalized against the level of actin mRNA. The experiments shown in this figure are representative of at least 3 independent experiments

Figure 2

LOX expression and LOXL2 expression favor escape from OIS. HEC-TM cells were infected with an empty control or a LOX- or LOXL2-encoding vector and puromycin selected. (a) Cell lysates were prepared and analyzed by immunobloting with an anti-flag antibody. (b) HEC-TM cells were treated or not for 3 days with 4-OHT (d0), PFA-fixed and crystal violet-stained to measure cell growth at the indicated time points. (c) Cells were treated as in (b), fixed, and assayed for SA-β-Gal activity. (d) RNAs were prepared at d3 and RT-qPCRs were performed against the indicated senescence markers. Expression was normalized against the level of actin mRNA. (e) HEC-TM cells expressing a LOX-encoding, a LOXL-encoding or an empty control vector were counted after each passage and seeded at the same density. Population doublings are presented. The experiments shown in this figure are representative of at least three independent experiments

Figure 3

Activity of secreted LOX or LOXL2 favors escape from OIS. (a) Supernatants of LOX- or LOXL2-expressing HEC-TM cells or of ones containing the empty control vector were concentrated and analyzed by immunoblotting with an anti-flag antibody. (b) LOX activity was measured in concentrated supernatants and normalized to 100% for the ctrl cells (±S.E.M.). (c) HEC-TM cells were treated for 3 days with 4-OHT +/− BAPN together with the indicated supernatant, PFA-fixed and crystal violet-stained to measure cell growth on d3, and (d) assayed for SA-β-Gal activity on d3. The experiments shown in this figure are representative of at least three independent experiments

Figure 4

LOX activity inhibition blocks spontaneous escape from OIS. (a) Protocol of treatment to induce senescence and inhibition of its reversal in HEC-TM cells. (b) HEC-TM cells were treated for 3 days with 4-OHT, with or without BAPN. Cells were PFA-fixed and crystal violet-stained to measure cell growth at various time points. (c) Cells were treated as in (b), fixed and assayed for SA-β-Gal activity or (d) RNA were prepared and analyzed by RT-qPCR against the indicated genes and normalized with respect to actin. (e) HEC-TM cells were treated or not with BAPN every day. After each passage, cells were counted and seeded at the same density. Population doublings are presented. The experiments shown in this figure are representative of at least three independent experiments

Figure 5

LOX activity favors escape from senescence in vivo in a model of PDAC. Pdx1-Cre;LSL-Kras^G12D/+; Ink4a/Arf^lox/lox (KIA) mice were injected with concentrated LOX or control supernatant every day from day 23 after birth. Mice were killed 39 days after birth and their pancreases fixed before analysis. (a) A Sirius red staining of collagen was performed at the indicated times. Collagen fiber organization is illustrated by pictures made by polarized light microscopy (scale bar: 200 μm). (b) SA-β-Gal assays were performed on pancreas samples at the indicated times. (c) IHC performed against the Dec1 senescence marker. (d) IHC against the Wnt16B senescence marker. (e) IHC against the Ki67 proliferation marker. For (c–e) Scale bar: 50 μm

Figure 6

LOX inhibition stabilizes senescence and delays carcinogenesis in vivo. Pdx1-Cre;LSL-Kras^G12D/+;Ink4a/Arf^lox/lox (KIA) mice were injected or not with BAPN, three times a week from day 25 after birth. Mice were killed 45 days after birth and their pancreases fixed before analysis. (a) Sirius red staining of collagen was performed and representative photographs obtained by polarized light microscopy are displayed (scale bar: 200 μm). (b) SA-β-Gal assays were performed on pancreas samples at the indicated times. (c) IHC against the Dec1 senescence marker. (d) IHC against the Wnt16B senescence marker. (e) IHC against the Ki67 proliferation marker. For (c–e) scale bar: 50 μm. (f) KIA mice were injected (n=12) or not (n=75) with the LOX inhibitor BAPN. Kaplan–Meier analyses of the probability to survive were performed. P-values were calculated using a log rank test

Figure 7

FAK is modulated by LOX activity. (a) HEC-TM wells were treated for 3 days with 4-OHT. Cell extracts were prepared and analyzed by immunoblotting with antibodies directed against P-ERK, P-FAK, FAK, and tubulin. (b) HEC-TM cells were treated for 3 days with 4-OHT, with or without BAPN. BAPN treatment was carried out for 4 days. Cell extracts were prepared and analyzed by immunoblotting with antibodies directed against P-FAK, FAK, and tubulin. (c) Pancreases of mice, 39 days after birth, treated or not with LOX supernatants, were analyzed by IHC with an antibody targeting P-FAK. (d) Pancreases of mice, 45 days after birth, treated or not with BAPN, were analyzed by IHC with an antibody targeting P-FAK

Figure 8

FAK activity participates in senescence modulation by LOX activity. (a) HEC-TM cells were treated with 4-OHT, with or without FAK inhibitors, for 3 days and then cultured for an additional 4 days with or without the indicated FAK inhibitors (d4), fixed, and crystal violet-stained. (b) Cells were fixed and their SA-β-Gal activity assayed on d0 and d4. (c) RNA was prepared and RT-qPCR was performed against senescence markers. (d) HEC-TR cells were infected with a FAK-encoding or an empty control retroviral vector and neomycin-selected. Immunofluorescence against the Flag tag was performed. (e) Selected cells were treated with 4-OHT+BAPN for 3 days (d0) and with BAPN for 4 additional days (d4). Cells were next fixed, crystal violet-stained, and (f) assayed for SA-β-Gal activity, or (g) RNAs were prepared and RT-qPCR performed on a senescence marker