Esquema de la tesis
46 1.2.1Fibras naturales
1.2.2 Tipos de fibras Naturales
Exposure of UT-SCC cells to 0.1% O2 for up to 72 hours led to diverse effects on the mRNA
expression of PERK, ATF4 and LAMP3 (see Figure 5A, B and C). mRNA expression of both
PERK and ATF4 was not universally induced after hypoxic exposure (see Figure 5A and B). A
significant induction of expression by hypoxia was found in five and two cell lines, respectively. The other cell lines showed a significant inhibition of expression (five and two cell lines, respectively) or effects were not significant (two and eight cell lines, respectively). Figure
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103
5C shows that LAMP3 mRNA expression is significantly downregulated in seven of the twelve
UT-SCC cell lines tested. In contrast, two cell lines showed a significant induction after hypoxic exposure and in three cell lines effects were not significant.
Next, three UT-SCC cell lines were selected for analysis of protein expression after hypoxia.
UT-SCC5 (PERK, ATF4 and LAMP3 mRNA induction after hypoxia), UT-SCC24A
(induction of PERK mRNA, no effect on ATF4 mRNA and inhibition of LAMP3 mRNA
after hypoxia) and UT-SCC45 (PERK, ATF4 and LAMP3 mRNA inhibition after hypoxia)
were chosen to obtain a diverse panel with regard to the effect of hypoxia on the UPR. Results on the protein level were not always identical to the effects found on the mRNA level (see Figure 5D). Intriguingly, expression of PERK, ATF4 and LAMP3 was reduced in both UT- SCC5 and UT-SCC24A cells. UT-SCC45 cells showed an induction at 24 hours of hypoxic exposure for PERK and LAMP3, whereas ATF4 expression was inhibited at all time points.
Data on expression of phospho-eIF2α are in conflict with PERK, ATF4 and LAMP3 as a
substantial induction in all three cell lines after hypoxic incubation was found (see Figure 5D).
FIGURE 5 Effect of hypoxic exposure (0.1% O2) on (A) PERK, (B) ATF4 and (C) LAMP3 mRNA expression in
twelve UT-SCC cell lines. Data are presented as mean + SD. Results are from two representative experiments with three replicates each. D. Protein expression of PERK, p-eIF2α, ATF4 and LAMP3 after hypoxic exposure (0.1% O2)
of three UT-SCC cell lines. α-tubulin represents the loading control. Numbers below the bands indicate densitometric analysis corrected for the corresponding α-tubulin.
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CA9 expression is induced in UT-SCC5, -24A and -45 cells upon hypoxic exposure
Next, we wanted to verify that hypoxic culture actually induces a transcriptional response in UT-SCC cells. Therefore, the mRNA expression of the endogenous hypoxia marker carbonic
anhydrase 9 (CA9) was evaluated in UT-SCC5, -24A and -45 cells. CA9 is a known
downstream target of hypoxia-inducible factor 1 (HIF1) the master regulator of the hypoxic
response. All cell lines displayed a massive induction of CA9 expression, ranging from a 50-
fold induction in UT-SCC45 at 24 hours to a 25,000-fold induction in UT-SCC5 after 72 hours (see Figure 6A). This indicates that these cells have a functional HIF-pathway and can respond to hypoxic conditions.
ER-stress induces PERK, ATF4 and LAMP3 mRNA expression in UT-SCC5, -24A
and -45 cells
Next, the effect of ER-stress on mRNA expression of PERK, ATF4 and LAMP3 was
examined in three UT-SCC cell lines. Therefore, cells were exposed to tunicamycin, an inhibitor of N-linked glycosylation, which induces ER-stress and consequentially the UPR. A
24-hour tunicamycin treatment was sufficient to induce a vast increase in PERK, ATF4 and
LAMP3 mRNA expression in all three cell lines (see Figure 6 B, C and D). This indicates that the PERK/ATF4/LAMP3-arm is not defective in these cell lines.
FIGURE 6 A. Effect of hypoxic exposure (0.1% O2) on CA9 mRNA expression in three UT-SCC cell lines. Effect of
treatment with 1 µg/ml tunicamycin for 24 hours in three UT-SCC cell lines on mRNA expression of (B) PERK, (C)
ATF4, and (D) LAMP3. Data are presented as mean + SD. Results are from two representative experiments with three replicates each.
LAMP3INHNSCC
105 DISCUSSION
In this study, LAMP3 expression was assessed in a cohort of head and neck squamous cell carcinomas. Patients with positive lymph nodes and patients developing metastases showed higher LAMP3 expression levels in their primary tumour. In addition, patients with high LAMP3 expression had a significantly worse metastasis-free survival than patients with low LAMP3 expression in their tumours. These data indicate that also in head and neck squamous cell carcinomas LAMP3 is of importance for tumour progression. Further examination of the
association of LAMP3 with hypoxia revealed that in contrast to other tumour types24,26,
LAMP3 was not linked with hypoxia in HNSCC. Instead, expression was located exclusively in normoxic areas of both primary tumours and xenografts. Culturing HNSCC cells under
hypoxic conditions revealed that in most cell lines hypoxia did not induce expression of PERK,
ATF4 or LAMP3. Instead, in most cell lines, expression levels of these factors remained
unaltered or were even inhibited after exposure to hypoxia. This is distinctly different from
tumour cells of other origins, in which the UPR was vastly induced by hypoxia24,26. The
observation that phosphorylated eIF2α accumulates during hypoxia, whereas its downstream
targets ATF4 and LAMP3 do not, underlines the unusual manner in which the UPR appears to
be regulated in these cells. However, phosphorylation of eIF2α does not rely exclusively on
PERK. eIF2α can be phosphorylated by other factors, but this is in response to stresses other
than hypoxia35,36. Whether eIF2α is phosphorylated by a PERK-independent mechanism in
HNSCC cells and what effect this has on ATF4 activity remains to be elucidated.
The data presented in the current study show that expression of PERK and ATF4 is still present in HNSCC cells, but is not always induced by hypoxia. Several studies have shown that
PERK and ATF4 are induced during conditions of (severe) hypoxia20-23,37 and that these
factors are essential for tumour survival18,37,38. In xenografts of transformed mouse embryonic
fibroblasts ATF4 colocalised with hypoxia as indicated by the marker EF5 (ref. 37). In addition,
tumours with defective PERK-signalling had a higher number of apoptotic cells in hypoxic areas compared to tumours with intact PERK-signalling. Hypoxia induces LAMP3 expression via PERK and ATF4 in tumour cell lines of multiple origins (i.e. colon, breast, prostate, lung
and cervical cancer cells)24. However, none of the studies above examined HNSCC. We found
that ER-stress provoked by tunicamycin in HNSCC cells did induce a response of the UPR. This demonstrates that the PERK-arm of the UPR is not defective in HNSCC. Previously,
PERK-/- tumours were found to exhibit less and smaller hypoxic areas compared to tumours
where PERK is still intact37. Extensive hypoxic regions were still present in HNSCC
xenografts, which is different from the effects of defective PERK-signalling. The expression of other hypoxia-regulated genes was conventionally induced under hypoxic conditions. Together, these results clearly show that HNSCC represent a group of tumours that respond atypically to hypoxia with regard to the PERK/ATF4/LAMP3-arm of the UPR. We hypothesise that there are two possibilities as to what is happening in these cells. Either 0.1% O2 for up to 72 hours is not sufficient to induce ER-stress in these cells or pathways other than the PERK/ATF4-arm
are active to deal with hypoxia-induced stress. Our current in vitro data are limited to
examination of effects after hypoxia at 0.1% O2. The UPR is maximally induced under
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106
different, slower kinetics26. Nevertheless, it would be interesting to validate these results in a
broader range of oxygen concentrations, as this may provide more information on the oxygen dependency of the observed effects. However, if hypoxia does not induce stress in HNSCC cells, then apparently these cells are very well adapted to hypoxic conditions. Previously,
survival of UT-SCC cells was found to be relatively insensitive to 0.5% O2 (ref. 32), indicating
that hypoxia is well tolerated in these cell lines. The absence of a hypoxia-induced response on the PERK/ATF4-arm in HNSCCs may have important implications for how these tumours deal with conditions of hypoxia. Perhaps HNSCC cells behave differently under hypoxic conditions in terms of their metabolism. As the UPR is mainly activated to deal with an overload of misfolded proteins in the ER, absence of a response during hypoxic conditions suggests that these cells have a low requirement for protein folding. In addition, it would be interesting to see how hypoxic exposure affects the other two pathways of the UPR besides the PERK-arm. So far, it is unclear whether there is crosstalk between the different arms of the
UPR, although redundancy has been suggested16,40.
In conclusion, this study examined the prognostic value of LAMP3 in HNSCC. Patients with high LAMP3 levels had a worse survival, which despite the small size of the cohort, was highly statistically significant for metastasis-free survival. Intriguingly, LAMP3 expression was localised exclusively in normoxic areas of tumours and xenografts. Analysis of mRNA and protein expression of PERK, ATF4 and LAMP3 in HNSCC cell lines revealed that in a number of cell lines expression was not induced, but unaltered or even inhibited after exposure to 0.1% O2. These data suggest that HNSCC represent a subset of tumours that deal with cellular stress induced by hypoxia in an atypical manner.
ACKNOWLEDGEMENTS
We are grateful to JASPER LOK for technical assistance.
REFERENCES
1. BROWN,J.M. Evidence for acutely hypoxic cells in mouse tumours, and a possible mechanism of reoxygenation. Br J Radiol 1979; 52: 650-656.
2. THOMLINSON,R.H., GRAY,L.H. The histological structure of some human lung cancers and the possible implications for radiotherapy. Br J Cancer 1955; 9: 539-549.
3. RADEMAKERS,S.E., SPAN,P.N., KAANDERS,J.H. et al. Molecular aspects of tumour hypoxia. Mol Oncol 2008;
2: 41-53.
4. BRIZEL,D.M., SCULLY,S.P., HARRELSON,J.M. et al. Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma. Cancer Res 1996; 56: 941-943.
5. BRIZEL,D.M., SIBLEY,G.S., PROSNITZ,L.R. et al. Tumor hypoxia adversely affects the prognosis of carcinoma of the head and neck. Int J Radiat Oncol Biol Phys 1997; 38: 285-289.
6. FYLES,A.W., MILOSEVIC,M., WONG,R. et al. Oxygenation predicts radiation response and survival in patients with cervix cancer. Radiother Oncol 1998; 48: 149-156.
7. HOCKEL,M., SCHLENGER,K., ARAL,B. et al. Association between tumor hypoxia and malignant progression in
advanced cancer of the uterine cervix. Cancer Res 1996; 56: 4509-4515.
8. NORDSMARK,M., OVERGAARD,M., OVERGAARD,J. Pretreatment oxygenation predicts radiation response in
advanced squamous cell carcinoma of the head and neck. Radiother Oncol 1996; 41: 31-39.
9. SUNDFOR,K., LYNG,H., ROFSTAD,E.K. Tumour hypoxia and vascular density as predictors of metastasis in
squamous cell carcinoma of the uterine cervix. Br J Cancer 1998; 78: 822-827.
10. JANSSENS,G.O., RADEMAKERS,S.E., TERHAARD,C.H. et al. Accelerated radiotherapy with carbogen and nicotinamide for laryngeal cancer: results of a phase III randomized trial. J Clin Oncol 2012; 30: 1777-1783. 11. KAANDERS,J.H., WIJFFELS,K.I., MARRES,H.A. et al. Pimonidazole binding and tumor vascularity predict for
LAMP3INHNSCC
107
12. VAUPEL,P., THEWS,O., HOECKEL,M. Treatment resistance of solid tumors: role of hypoxia and anemia. Med
Oncol 2001; 18: 243-259.
13. HOCKEL,M., VAUPEL,P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl
Cancer Inst 2001; 93: 266-276.
14. SUTHERLAND,R.M. Tumor hypoxia and gene expression--implications for malignant progression and therapy.
Acta Oncol 1998; 37: 567-574.
15. HARRIS,A.L. Hypoxia--a key regulatory factor in tumour growth. Nat Rev Cancer 2002; 2: 38-47.
16. FELDMAN,D.E., CHAUHAN,V., KOONG,A.C. The unfolded protein response: a novel component of the
hypoxic stress response in tumors. Mol Cancer Res 2005; 3: 597-605.
17. KOUMENIS,C., WOUTERS,B.G. "Translating" tumor hypoxia: unfolded protein response (UPR)-dependent and
UPR-independent pathways. Mol Cancer Res 2006; 4: 423-436.
18. ROUSCHOP,K.M., VAN DEN BEUCKEN,T., DUBOIS,L. et al. The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP1LC3B and ATG5. J Clin Invest 2010; 120: 127-141.
19. WOUTERS,B.G., KORITZINSKY,M. Hypoxia signalling through mTOR and the unfolded protein response in
cancer. Nat Rev Cancer 2008; 8: 851-864.
20. KOUMENIS,C., NACZKI,C., KORITZINSKY,M. et al. Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor eIF2alpha. Mol Cell Biol 2002; 22: 7405-7416.
21. BLAIS,J.D., FILIPENKO,V., BI,M. et al. Activating transcription factor 4 is translationally regulated by hypoxic stress. Mol Cell Biol 2004; 24: 7469-7482.
22. AMERI,K., LEWIS,C.E., RAIDA,M. et al. Anoxic induction of ATF-4 through HIF-1-independent pathways of protein stabilization in human cancer cells. Blood 2004; 103: 1876-1882.
23. RZYMSKI,T., MILANI,M., PIKE,L. et al. Regulation of autophagy by ATF4 in response to severe hypoxia. Oncogene 2010; 29: 4424-4435.
24. MUJCIC,H., RZYMSKI,T., ROUSCHOP,K.M. et al. Hypoxic activation of the unfolded protein response (UPR)
induces expression of the metastasis-associated gene LAMP3. Radiother Oncol 2009; 92: 450-459.
25. KANAO, H., ENOMOTO,T., KIMURA,T. et al. Overexpression of LAMP3/TSC403/DC-LAMP promotes metastasis in uterine cervical cancer. Cancer Res 2005; 65: 8640-8645.
26. NAGELKERKE,A., MUJCIC,H., BUSSINK,J. et al. Hypoxic regulation and prognostic value of LAMP3 expression
in breast cancer. Cancer 2011; 117: 3670-3681.
27. NAGELKERKE,A., BUSSINK,J., MUJCIC,H. et al. Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response. Breast Cancer Res 2013; 15: R2.
28. NAGELKERKE,A., BUSSINK,J., VAN DER KOGEL,A.J. et al. The PERK/ATF4/LAMP3-arm of the unfolded
protein response affects radioresistance by interfering with the DNA damage response. Radiother Oncol 2013. 29. HOOGSTEEN,I.J., LOK,J., MARRES,H.A. et al. Hypoxia in larynx carcinomas assessed by pimonidazole binding
and the value of CA-IX and vascularity as surrogate markers of hypoxia. Eur J Cancer 2009; 45: 2906-2914. 30. NIJKAMP,M.M., HOOGSTEEN,I.J., SPAN,P.N. et al. Spatial relationship of phosphorylated epidermal growth
factor receptor and activated AKT in head and neck squamous cell carcinoma. Radiother Oncol 2011; 101: 165- 170.
31. TROOST,E.G., LAVERMAN,P., PHILIPPENS,M.E. et al. Correlation of [18F]FMISO autoradiography and pimonidazole [corrected] immunohistochemistry in human head and neck carcinoma xenografts. Eur J Nucl Med Mol Imaging 2008; 35: 1803-1811.
32. STEGEMAN,H., KAANDERS,J.H., WHEELER,D.L. et al. Activation of AKT by hypoxia: a potential target for
hypoxic tumors of the head and neck. BMC Cancer 2012; 12: 463.
33. DE KOK,J.B., ROELOFS,R.W., GIESENDORF,B.A. et al. Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab Invest 2005; 85: 154-159.
34. NAGELKERKE,A., MUJCIC,H., WOUTERS,B. et al. 18S is an appropriate housekeeping gene for in vitro hypoxia
experiments. Br J Cancer 2010; 103: 590; author reply 591-592.
35. DE HARO,C., MENDEZ,R., SANTOYO,J. The eIF-2alpha kinases and the control of protein synthesis. FASEB J 1996; 10: 1378-1387.
36. KAUFMAN, R. J. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene
transcriptional and translational controls. Genes Dev 1999; 13: 1211-1233.
37. BI,M., NACZKI,C., KORITZINSKY,M. et al. ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth. EMBO J 2005; 24: 3470-3481.
38. BLAIS,J.D., ADDISON,C. L., EDGE,R. et al. Perk-dependent translational regulation promotes tumor cell adaptation and angiogenesis in response to hypoxic stress. Mol Cell Biol 2006; 26: 9517-9532.
39. MAZURE,N.M., POUYSSEGUR,J. Hypoxia-induced autophagy: cell death or cell survival? Curr Opin Cell Biol
2010; 22: 177-180.
40. LEE,A.H., IWAKOSHI,N.N., GLIMCHER,L.H. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol Cell Biol 2003; 23: 7448-7459.