MVP expression in the prediction of clinical outcome of locally advanced oral squamous cell carcinoma patients treated with radiotherapy
© Henriquez-Hernandez et al.; licensee BioMed Central Ltd. 2012
Received: 11 April 2012
Accepted: 13 August 2012
Published: 29 August 2012
To explore the role of Major Vault Protein (MVP) in oral cavity squamous cell carcinoma patients.
Subjects and Methods
131 consecutive patients suffering from oral cavity squamous cell carcinoma were included in the study. In the whole series, the mean follow-up for survivors was 123.11 ± 40.36 months. Patients in tumour stages I and II were referred to surgery; patients in stage III-IV to postoperative radiotherapy (mean dose = 62.13 ± 7.74 Gy in 1.8–2 Gy/fraction). MVP expression was studied by immunohistochemistry in paraffin-embedded tumour tissue.
MVP expression was positive in 112 patients (85.5%) and no relation was found with clinic pathological variables. MVP overexpression (those tumours with moderate or strong expression of the protein) was related to insulin-like growth factor receptor-1 (IGF-1R) expression (P = 0.014). Tumour stage of the disease was the most important prognostic factor related to survival. Tumours overexpressing MVP and IGF-1R were strongly related to poor disease-free survival (P = 0.008, Exp(B) = 2.730, CI95% (1.302-5.724)) and cause-specific survival (P = 0.014, Exp(B) = 2.570, CI95% (1.215-5.437)) in patients achieving tumour stages III-IV, in multivariate analysis.
MVP and IGF-1R expression were related in oral squamous cell carcinoma and conferred reduced long-term survival in patients suffering from advanced stages of the disease.
KeywordsMVP IGF-1R Oral carcinoma Radiotherapy Predictive factor
Ataxia Telangiectasia Mutated
Distant-Metastasis Free Survival
Insulin-like growth factor-1 receptor
Local Disease Free Survival
Major Vault Protein
Oral cavity squamous cell carcinoma
Regional Disease Free Survival.
Oral carcinoma is commonly treated by surgery or radiotherapy as local treatments. Although long-term survival is improving with advances in therapy, outcomes in locally advanced cases remain suboptimal. There is a clear need for new prognostic indicators, which could be used in diagnostics and, consequently, in the selection of the most effective treatment method.
Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins (the 110 kDa major vault protein (MVP), the two minor vault poly(ADP-ribose) polymerase (VPARP), and the 240 kDa telomerase-associated protein-1 (TEP1)) and small untranslated RNA (vRNA) . Vaults have been classically associated to multidrug resistance . However, it has been pointed that MVP interacts with several proteins involved in relevant cellular mechanisms as PTEN, Erk, or EGF. Furthermore, the expression of MVP was associated to a malignant phenotype in some cancers, indicating a direct involvement in tumour development and progression . MVP has been associated to resistance to radiotherapy , probably due to its role in preventing apoptosis by inhibiting the COP-1/p53 axis . Various DNA damaging agents, including ultraviolet irradiation, induce increased MVP transcription and protein levels . This suggests that vaults may have a role in facilitating DNA repair processes, which is consistent with previous work showing that VPARP- and TEP1-deficient mice have an increased incidence of carcinogen-induced colon tumours . At the clinical level, the role of MVP in predicting response to radiotherapy was recently first addressed . In that study, MVP was related to poor outcome after radiotherapy in 78 patients suffering from oropharyngeal carcinoma. The estimated effects of MVP overexpression appeared somewhat larger in the tongue cancer patients compared with the tonsil cancer patients for loco-regional failure and cancer-specific death . However, the underlying mechanisms behind this observation are not understood and the role of MVP in oral cavity squamous cell carcinoma has not been deeply studied, especially in combination with other biological markers.
Insulin-like growth factor-1 receptor (IGF-1R) is a transmembrane tyrosine kinase receptor commonly overexpressed in many cancers. Activation of IGF-1R leads to activation of the ras, raf and MAPK pathways, resulting in increased proliferation; and of the PI3K pathway which in turn results in the prevention of apoptosis. IGF-1R activation has been associated with increased radioresistance by increasing cell proliferation and prevention of apoptosis [9, 10]. The expression of IGF-1R directly influences radioresistance . In that sense, we have previously reported that IGF-1R overexpression is associated with reduced long-term local control in cervical  and oral cancer patients . An association between MVP and IGF-1R expression has been reported in cervical patients . Thus, the combined overexpression of MVP and IGF-1R conferred reduced long-term survival in patients suffering from cervical cancer who achieved clinical complete response to radiochemotherapy .
The aim of the present study was to assess the expression of MVP in oral cavity squamous cell carcinoma patients, its relation with clinical and pathologic prognostic factors and its role in predicting clinical outcome. In addition, we explored the relation to IGF-1R expression in this cohort of patients.
Materials and Methods
Characteristics of patients in study (n = 131)
Surgery + Radiotherapy
All patients were referred to surgery. Additionally, 14 patients were referred to functional cervical dissection, 102 patients were referred to radical cervical dissection and 1 patient was referred to bilateral cervical dissection. Fourteen patients did not suffer neck dissection. Those patients in stages III-IV were referred to postoperative radiotherapy (RT). Patients in stages I and II were referred to RT if tumour positive margins were found at surgery. Thus, 84 patients received postoperative RT up to a mean dose of 62.13 ± 7.74 Gy (median 65 Gy, range 30–70.20 Gy) in 1.8–2 Gy fractions. No patients were treated with adjuvant or neoadjuvant chemotherapy.
MVP expression was studied by immunohistochemistry in all patients as previously described [14, 15]. In short, sections from paraffin-embedded tissue tumour biopsies were incubated with mouse anti-MVP monoclonal antibody (LRP/MVP Ab-2, Clon 1032, Neomarkers, CA) applied at a 1:100 dilution and incubated for 1 hour at room temperature in a moist chamber. A secondary biotinylated antibody (DAKO Detection Kit, LSBA, Carpintería, CA) and a prepared complex peroxidase–streptavidin biotinylated (DAKO, LSBA, Carpintería, CA) were used afterwards. Staining was revealed (DAKO, DAB Chromogen, Carpintería, CA) and counterstained with Harris hematoxylin. The primary antibody was omitted in one section as a negative control and a strongly positive tumour for MVP was used as a positive control.
Outcome after treatment was assessed as Local Disease Free Survival (LDFS), Regional Disease Free Survival (RDFS), Distant-Metastasis Free Survival (DMFS), Disease-Free Survival (DFS), and Cause-Specific Survival (CSS). Correlations between previously described variables and outcome during follow-up were analyzed by the Kaplan–Meier survival test and the differences were calculated by the log-rank test. Cox regression test was used for multivariate analysis. Statistical analysis was performed by SPSS 15.0 software.
Relation of MVP and IGF-1R expression in oral tumours
IGF-1R Low expression
Actuarial survival ratios after 15 years of follow-up in the whole series were 65.6, 70.2, 90.6, 49.6, and 57.1% for LDFS, RDFS, DMFS, DFS, and CSS, respectively. Tumour grade (I vs. II-III) and stage (I-II vs. III-IV) were the most important prognostic factors for CSS in multivariate analysis (Exp(B) = 3.009, confidence interval [CI] 95% (1.255-7.214), P = 0.014; Exp(B) = 3.085, CI95% (1.565-6.083), P = 0.001, respectively). MVP and IGF-1R expression were not differentially expressed according to tumour location and were not predictive factors in the whole series (Data not shown).
Relation of prognostic variables and clinical outcome in locally advanced oral carcinoma patients (stages III-IV), n = 66
P = 0.683
P = 0.744
P = 0.524
P = 0.707
P = 0.485
P = 0.882
P = 0.537
P = 0.387
P = 0.574
P = 0.866
P = 0.330
P = 0.053
P = 0.047
P = 0.024
P = 0.008
P = 0.001
P = 0.077
P = 0.388
P = 0.003
P = 0.001
P = 0.214
P = 0.362
P = 0.030
P = 0.020
P = 0.010
P = 0.016
P = 0.153
P = 0.898
P = 0.029
P = 0.009
This study was carried out to explore the clinical association between MVP expression and prognosis on a series of OCSCC patients treated by surgery and radiotherapy. MVP was overexpressed in 67 (51.1%) of our oral cavity carcinoma patients. Only one study has reported data of MVP expression in head and neck cancer . Silva et al. detected positive expression of MVP in 49 out of 78 (62.8%) patients suffering from carcinoma in the posterior third of tongue and the tonsil.
Tumour stage was the most important prognostic factor for survival in our series. The rates of local recurrence for clinical stages III or IV tumours may be as high as 40%. In our whole series, MVP was not a predictive factor for survival. Nonetheless, when patients were segregated according to low (I-II) or high (III-IV) tumour stage, MVP expression was predictive for DMFS, DFS, and CSS. The fact that patients with advanced stages of the disease suffer more amount of adverse events related to the tumour, together with the radioresistance role of MVP previously reported in tongue and cervical carcinoma, helps to explain the predictive role of this oncoprotein only in patients suffering OCSCC in stage III-IV, all treated with surgery and RT. MVP may have a role in favouring increased genetic instability by reducing DNA damage repair by means of non-homologous end joining (NHEJ) and downregulating Ku70/80 expression . The most important radiation-induced DNA damage is the double-strand breaks (DSB), mainly repaired by NHEJ. Ku70/80 are key genes in the NHEJ repair pathway for radiation-induced DNA DSB. If vaults are overexpressed, NHEJ repair may be suppressed by several mechanisms; thus, genomic instability could arise because either repair of DSB damage by homologous repair is prevented or NHEJ repair is defective. The PARP-1, part of Vault complex, promotes homologous repair over NHEJ, suppressing Ku70/80 function. These events are associated with the decision of damaged cells to survive and proliferate, favouring tumour progression and reducing tumour response to oncologic treatment through the development of resistant cell phenotypes. Our results are in agreement with the published study of Silva et al.  where MVP has strongly predicted the clinical outcome in 78 oropharyngeal cancer patients who received primary radiotherapy with a curative intent. Because OCSCC in advanced stages (III-IV) were all referred to postoperative radiotherapy, the role of MVP was only observed in this subset of patients and not in the whole series.
We reported here the first clinical evidence of a direct relation between IGF-1R and MVP expression in OCSCC. The association between both oncoproteins has been previously reported in cervical carcinoma patients . It seems that both proteins must be overexpressed to confer radioresistance and subsequent poor clinical outcome in cervical cancer. It would be suggested that both MVP and IGF-1R increase proliferation by activation of Src, PTEN, ERK and inhibit apoptosis through the ATM COP1/P53 axis, resulting in tumour resistance to DNA damage agents. It has been reported an association between MVP expression and cell proliferation, as well as other molecules involved in apoptosis . Thus, resistance to radiotherapy in the clinical setting as a result of MVP [8, 14] may be causes by the enhanced of proliferation and inhibition of apoptosis, as well as by the regulation of DSB repair by suppressing NHEJ repair through the inhibition of Ku70/80 . The IGF-1R cooperates with MVP in preventing apoptosis by upregulating BCL-2 and, to a lesser extent, downregulating BAX , and it may also affect NHEJ repair through the AKT/p38MAPK pathways, mainly by modifying Ku expression . Activation of IGF-1R triggers a cascade of reactions involving signal transduction pathways: Ras, Raf, mitogen-activated protein kinase and phosphoinositol-3-kinase (PI3K)/AKT/BAD (Bcl-xL/Bcl2-associated death promoter). IGF-1R overexpression has been linked to disease progression, increased resistance to radiotherapy, and poor prognosis [9, 12, 18]. IGF-1R gene seems to be a novel downstream target in an Ataxia Telangiectasia Mutated (ATM)-mediated DNA damage response pathway. The potential role of the IGF-1R gene as a target ing an ATM-dependent pathway, involved in regulating the radiation response, was recently inferred . Thus, deregulated expression of the IGF-1R gene after ionizing radiation may be linked to genomic instability and enhanced transforming capacity .
The expression of MVP has a complex regulation not fully understood, influenced by the expression of other oncoproteins and other important clinical factors as hypoxia. In that sense, high MVP expression is related to severe hypoxia in clinical cervical tumours . Interestingly, hypoxia inhibits the NHEJ DNA repair through down-regulating Ku70/80 expression, combined with increased angiogenesis and altered p53 , and an inverse association between MVP and KU70/80 exists . Although the prognosis role of MVP is still contradictory, at a clinical level, vaults have been proposed as a useful predictive marker associated with radiotherapy resistance , and the interaction between IGF-1R and MVP can help to explain the predictive role of these oncoproteins in response to radiotherapy .
MVP and IGF-1R expression were related in OCSCC tumours and conferred reduced long-term survival in patients suffering advanced stages of the disease. The present results could help to understand the biological behavior of this tumor type according to the expression not only of IGF-1R, but also of MVP. In our opinion, combined MVP/IGF-1R expression could be of relevance in predicting the clinical outcome in this tumour type. However, new studies including PTEN, P53, BCL-2, cell proliferation, hypoxia or PI3K markers would be necessary to precisely define the role of these proteins on common molecular pathways. These preliminary results need to be confirmed by a larger clinical trial.
This work was subsidized by grants: Fondo de Investigación Sanitaria, FIS 1261/02.
- Kickhoefer VA, Siva AC, Kedersha NL, Inman EM, Ruland C, Streuli M, Rome LH: The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase. J Cell Biol 1999, 146:917–928.PubMedView Article
- Mossink MH, van Zon A, Scheper RJ, Sonneveld P, Wiemer EA: Vaults: a ribonucleoprotein particle involved in drug resistance? Oncogene 2003, 22:7458–7467.PubMedView Article
- Steiner E, Holzmann K, Elbling L, Micksche M, Berger W: Cellular functions of vaults and their involvement in multidrug resistance. Curr Drug Targets 2006, 7:923–934.PubMedView Article
- Fichtner I, Paal K, Borgmann A, Badiali L, Wurm R, Henze G: Chemo- and radiation sensitivity of xenografted acute lymphoblastic leukemias–correlation to the expression of multidrug resistance proteins. Anticancer Res 2003, 23:2657–2664.PubMed
- Yi C, Li S, Chen X, Wiemer EA, Wang J, Wei N, Deng XW: Major vault protein, in concert with constitutively photomorphogenic 1, negatively regulates c-Jun-mediated activator protein 1 transcription in mammalian cells. Cancer Res 2005, 65:5835–5840.PubMedView Article
- Shimamoto Y, Sumizawa T, Haraguchi M, Gotanda T, Jueng HC, Furukawa T, Sakata R, Akiyama S: Direct activation of the human major vault protein gene by DNA-damaging agents. Oncol Rep 2006, 15:645–652.PubMed
- Raval-Fernandes S, Kickhoefer VA, Kitchen C, Rome LH: Increased susceptibility of vault poly(ADP-ribose) polymerase-deficient mice to carcinogen-induced tumorigenesis. Cancer Res 2005, 65:8846–8852.PubMedView Article
- Silva P, West CM, Slevin N, Valentine H, Ryder WD, Hampson L, Bibi R, Sloan P, Thakker N, Homer J, Hampson I: Tumor expression of major vault protein is an adverse prognostic factor for radiotherapy outcome in oropharyngeal carcinoma. Int J Radiat Oncol Biol Phys 2007, 69:133–140.PubMedView Article
- Turner BC, Haffty BG, Narayanan L, Yuan J, Havre PA, Gumbs AA, Kaplan L, Burgaud JL, Carter D, Baserga R, Glazer PM: Insulin-like growth factor-I receptor overexpression mediates cellular radioresistance and local breast cancer recurrence after lumpectomy and radiation. Cancer Res 1997, 57:3079–3083.PubMed
- Yu D, Watanabe H, Shibuya H, Miura M: Redundancy of radioresistant signaling pathways originating from insulin-like growth factor I receptor. J Biol Chem 2003, 278:6702–6709.PubMedView Article
- Chinnaiyan P, Allen GW, Harari PM: Radiation and new molecular agents, part II: targeting HDAC, HSP90, IGF-1R, PI3K, and Ras. Semin Radiat Oncol 2006, 16:59–64.PubMedView Article
- Lloret M, Lara PC, Bordon E, Pinar B, Rey A, Falcon O, Molano F, Hernandez MA: IGF-1R expression in localized cervical carcinoma patients treated by radiochemotherapy. Gynecol Oncol 2007, 106:8–11.PubMedView Article
- Lara PC, Bordon E, Rey A, Moreno M, Lloret M, Henriquez-Hernandez LA: IGF-1R expression predicts clinical outcome in patients with locally advanced oral squamous cell carcinoma. Oral Oncol 2011, 47:615–619.PubMedView Article
- Lloret M, Lara PC, Bordon E, Rey A, Falcon O, Apolinario RM, Clavo B, Ruiz A: MVP expression is related to IGF1-R in cervical carcinoma patients treated by radiochemotherapy. Gynecol Oncol 2008, 110:304–307.PubMedView Article
- Henriquez-Hernandez LA, Lloret M, Pinar B, Bordon E, Rey A, Lubrano A, Lara PC: BCL-2, in combination with MVP and IGF-1R expression, improves prediction of clinical outcome in complete response cervical carcinoma patients treated by radiochemotherapy. Gynecol Oncol 2011, 122:585–589.PubMedView Article
- Lloret M, Lara PC, Bordon E, Fontes F, Rey A, Pinar B, Falcon O: Major vault protein may affect nonhomologous end-joining repair and apoptosis through Ku70/80 and bax downregulation in cervical carcinoma tumors. Int J Radiat Oncol Biol Phys 2009, 73:976–979.PubMedView Article
- Cosaceanu D, Budiu RA, Carapancea M, Castro J, Lewensohn R, Dricu A: Ionizing radiation activates IGF-1R triggering a cytoprotective signaling by interfering with Ku-DNA binding and by modulating Ku86 expression via a p38 kinase-dependent mechanism. Oncogene 2007, 26:2423–2434.PubMedView Article
- Rocha RL, Hilsenbeck SG, Jackson JG, VanDenBerg CL, Weng C, Lee AV, Yee D: Insulin-like growth factor binding protein-3 and insulin receptor substrate-1 in breast cancer: correlation with clinical parameters and disease-free survival. Clin Cancer Res 1997, 3:103–109.PubMed
- Peretz S, Jensen R, Baserga R, Glazer PM: ATM-dependent expression of the insulin-like growth factor-I receptor in a pathway regulating radiation response. Proc Natl Acad Sci U S A 2001, 98:1676–1681.PubMedView Article
- Shahrabani-Gargir L, Pandita TK, Werner H: Ataxia-telangiectasia mutated gene controls insulin-like growth factor I receptor gene expression in a deoxyribonucleic acid damage response pathway via mechanisms involving zinc-finger transcription factors Sp1 and WT1. Endocrinology 2004, 145:5679–5687.PubMedView Article
- Lara PC, Lloret M, Clavo B, Apolinario RM, Henriquez-Hernandez LA, Bordon E, Fontes F, Rey A: Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression. Radiat Oncol 2009, 4:29.PubMedView Article
- Lara PC, Lloret M, Clavo B, Apolinario RM, Bordon E, Rey A, Falcon O, Alonso AR, Belka C: Hypoxia downregulates Ku70/80 expression in cervical carcinoma tumors. Radiother Oncol 2008, 89:222–226.PubMedView Article
- Lara PC, Pruschy M, Zimmermann M, Henriquez-Hernandez LA: MVP and vaults: a role in the radiation response. Radiat Oncol 2011, 6:148.PubMedView Article
- Valenciano A, Henriquez-Hernandez LA, Moreno M, Lloret M, Lara PC: Role of IGF-1 Receptor in Radiation Response. Transl Oncol 2012, 5:1–9.PubMedView Article
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