High expression of GLI1 is associated with better survival in advanced SCLC

Kozirovskis V.*1, Zandberga E.2, Magone M.1, Purkalne G.3, Linē A.2, Vikmanis U.3

Summary. Aim: Aberrant Sonic hedgehog (Shh) pathway signaling has been described in small cell lung cancer (SCLC), as well discrepancies, when analyzing expression of pathway components in SCLC cell lines vs tumor biopsies. Shh key component GLI1 was evaluated in advanced SCLC and data correlated with patient survival. Materials and Methods: GLI1 expression was analyzed by quantitative real-time polymerase chain reaction in pre-treatment fresh frozen tumor biopsies of 12 advanced SCLC patients and mRNA level of GLI1 was compared in short-term vs long-term survivor’s samples (stratified by median survival, independent samples t-test). Results: Expression of GLI1 mRNA was significantly higher in long-term (> 9.6 months, n = 6) survivor’s biopsies than in short-term (≤ 9.6 months, n = 6) survivors (p = 0.0196, 95% CI: 0.000016 to 0.000147, two-tailed independent samples t-test). Conclusion: High GLI1 mRNA expression in SCLC was found to be positive prognostic marker associated with longer survival. Further research is needed for validation of these results due to the small number of patients in the study.

Submitted: April 14, 2019.
*Correspondence: E-mail: viktors.kozirovskis@stradini.lv
Abbreviations used: OS — overall survival; SCLC — small cell lung cancer; Shh — Sonic hedgehog.

DOI: 10.32471/exp-oncology.2312-8852.vol-42-no-1.14266

Aberrant Sonic hedgehog (Shh) pathway signaling has been described in small cell lung cancer (SCLC), however, controversial results have been obtained, when analyzing expression of pathway components in SCLC cell lines vs tumor biopsies [1]. There is an increasing evidence that the embryonic signaling pathways are essential for the maintenance and chemoresistance of SCLC cancer stem cells [2]. These chemoresistant cells may be the cause for non-successful treatment of some SCLC patients, who do not respond well to therapy and survive much shorter than other patients.

The aim of this study was to evaluate the prognostic role of Shh pathway activity in SCLC. We evaluated mRNA expression of Shh pathway key component GLI1 in advanced stage SCLC samples and correlated data with patient survival.

MATERIALS AND METHODS

Patients and tissue samples. Study tissue samples were obtained along with a standard biopsies by fibrobronchoscopy from patients, diagnosed with advanced (stage III and IV) SCLC at Pauls Stradins Clinical University Hospital between October 2010 and January 2014. The Ethics Committee of the Institute of Experimental and Clinical Medicine of University of Latvia has approved this study. Written informed consent was obtained from the patients before inclusion in the study.

The tissue specimens were submerged in RNAlater solution (Thermo Fisher Scientific, USA) and stored at –20°C. Board-certified cytologist confirmed the presence of tumor cells in the sample by cytological evaluation of the smear, which was obtained from study specimen before placing it to the RNAlater. All diagnoses were histologically confirmed in standard biopsies as SCLC by board-certified pathologists. Pre-treatment biopsies from 20 SCLC patients were obtained and they were followed during treatment.

Clinical information, treatment and survival data were collected. 7 patients were excluded from the analysis due to the lack of cytological confirmation of tumor cell presence in a study biopsy. 1 patient died early during treatment from non-cancer related cause and therefore was also excluded from the analysis.

12 SCLC patients were stratified in a short-term and long-term survivors by median overall survival (OS) time (9.6 months) to correlate Shh related genes expression with patient survival. Demographics and clinical characteristics of 12 patients with qualitative RNA and survival data are shown in Tables 1 and 2.

Table 1. Characteristics of 12 patients qualified for gene expression and survival analysis. The comparison between groups was carried out by the Student’s t-test or the Chi Square test, according to the type of variable
Patient characteristics Long-term survivors (OS > 9.6 months, n = 6) Short-term survivors (OS ≤ 9.6 months, n = 6) P-value
Gender p = 1.0
Male 5 5
Female 1 1
AgeMedian (range) 61.5 (54–77) 51.5 (49–70) p = 0.078
ECOG p = 0.446
0 1 1
1 0 2
2 4 2
3 1 1
4 0 0
Stage p = 0.565
III A 1 0
III B 2 2
IV 3 4
Deviations of treatment dosing/timing due toxicity p = 0.079
None 2 5
Present 4 1
Best response p = 0.301
CR 0 0
PR 6 4
SD 0 1
PD 0 1
Disease progression type p = 0.601
Local 2 1
Distant 2 3
Combined 1 2
Not progressed 1 0
Note: CR — complete response; PR — partial response; SD — stable disease; PD — progressive disease.
Table 2. Survival and treatment data of 12 study patients
Patient ID Nr. Survival in months (days) Survival in months (days) Received treatment
Patient LC145 4 (140) 4 CE
Patient LC137 6 (191) 6 PE → RT (57.6/50.4/45 Gy)
Patient LC146 8 (250) 4 PE → 3 CAV
Patient LC102 8 (252) 5 PE
Patient LC171 8 (267) 1 PE → 5 CAV
Patient LC135 8 (269) 4 PE → RT (54/50.4 Gy) → 37.5 Gy WBRT
Patient LC130 10 (305) 5 PE → 3 CAV
Patient LC127 10 (318) 5 PE → 30 Gy WBRT
Patient LC134 12 (383) 5 PE → RT (54/54/45 Gy) → 36 Gy WBRT
Patient LC139 13 (396) 6 PE → RT (54/50.4 Gy) → PCI (30 Gy)
Patient LC121 15 (462) 6 CE → RT (54/50.4/50.4 Gy) → PCI (36 Gy) → 1 CAV
Patient LC126 82 (2499) 5 PE → RT (56/50.4 Gy) → PCI (36 Gy)
Note: CE — carboplatin/etoposide; PE — cisplatin/etoposide; RT — radiation therapy; CAV — cyclophosphamide/adriamycin/vincristine; WBRT — whole brain radiation therapy; PCI — prophylactic cranial irradiation.

Preparation of tissue mRNA. Biopsy homo­genization was done by FastPrep®-24 Instrument and Lysing Matrix D (MP Biomedicals, USA) at 0.4 m/s for 40 s. Total RNA was isolated using MirVana total RNA Isolation Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. RNA was treated with DNAse I (Thermo Fisher Scientific, USA) and RNA concentration and purity was quantified by Nanodrop ND-100 spectrophotometer. cDNA was synthesized by random hexamer priming from 1 µg of total RNA by using Revert Aid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions.

Quantitative real-time polymerase chain reaction. Quantitative real-time polymerase chain reaction was performed using 2 μl 1:10 diluted cDNA reaction mixtures, ABSolute Blue ™ SYBR green Low ROX (Thermo Fisher Scientific, USA) and ViiA 7 real — time polymerase chain reaction system (Applied Biosystems, Life Technologies, USA). Sequences of primers used in this study are available on request. To normalize the expression data, normalization factor was calculated for each cDNA from the expression values of three most stable references genes (ACTB, LRP10, YWHAZ) selected among seven frequently used housekeeping genes by using geNorm software. All quantitative real-time polymerase chain reaction experiments were performed in duplicates and the data were presented in graphs as means ± standard deviation.

Statistical analysis. GLI1 mRNA level was compared by calculating two-tailed independent samples t-test in short-term vs long-term survivor’s samples, stratified by median survival. Data were statistically interpreted using SPSS Statistics 21. P-values of < 0.05 were considered significant.

RESULTS AND DISCUSSION

A small cohort of 12 patients with qualitative RNA and survival data was analysed:

  • 6 SCLC patients in long-term survivors group (OS > 9.6 months) had mean GLI1 mRNA level of 0.000141757 ± 0.000038223 in their pre-treatment samples.
  • 6 SCLC patients in short-term survivors group (OS ≤ 9.6 months) accordingly had mean GLI1 mRNA level of 0.000060198 ± 0.000016324.

Significantly higher GLI1 mRNA levels were found by two-tailed independent samples t-test in pre-treatment biopsies of long-term SCLC survivors when comparing with short-term survivors (p = 0.0196, 95% CI: 0.000016 to 0.000147) (Figure).

 High expression of <i>GLI1</i> is associated with better survival in advanced SCLC
Figure. Dot plots showing normalized GLI1 gene expression levels in pre-treatment SCLC biopsies of long- vs short-term survivors cohorts. The median gene expression level for each group is indicated by a line inside the box. Data presented in graphs are means obtained from experiments in duplicates ± standard deviation

Negative prognostic role of GLI1 has been drawn from numerous studies and meta-analyses for breast [3] and gastric cancer [4], lung adenocarcinoma and squamous cell carcinoma [5, 6], gallbladder and liver cancer, cervical cancer, rhabdomyosarcoma, colon cancer, ovarian cancer, bladder cancer, esophageal cancer, head and neck squamous cell carcinoma, pancreatic cancer [7]. Over-expression of GLI1 tends to progressive stages and is related to unfavourable prognosis. Only in intracranial tumors GLI1 positivity was not correlated to poorer survival. Further study has confirmed the adverse effect of low nuclear GLI1 expression in glioblastomas, which is in contrast with the negative prognostic effect of high GLI1 expression reported in non-cranial malignancies [8]. Concerning GLI1 as a prognostic factor in lung adenocarcinoma there is also study that has reported longer survival in GLI1 positive tumors [9].

SCLC is a specific malignancy with a few common critical genetic and epigenetic alterations, leading to frequent GLI1 overexpression and ligand-independent GLI1 activation. GLI1 activation could be caused for example by bi-allelic inactivation of TP53 genes and TP53 deficiency observed nearly in all SCLC cases [10, 11]. Another frequently observed GLI1 activating alteration in SCLC is downregulation of Notch pathway and downregulation of PI3K-AKT oncogenic signaling. Notch pathway signaling was found to be downregulated in 77% of 110 SCLC clinical tumor specimens, while Notch family genomic alterations were found here in 25% of cases [11]. Loss of function of tumor suppressor PTEN, an endogenous inhibitor of AKT, which could be found in 10–18% of SCLC tumors [12], will also lead to increased transcriptional activity of GLI1.

Our finding with higher GLI1 expression in long-term SCLC survivors might reflect in this case lower mutational burden and only few epigenetic changes with retained GLI1 upregulation characteristic for SCLC. GLI1 downregulation and increased chemoresistance in short-term SCLC survivors may be explained by the higher number of genetic and epigenetic changes in tumor cells.

The main objects for SCLC molecular research still are cell lines and xenografts due to the clinical features of disease. Aberrant Shh pathway signaling has been described in SCLC, as well as discrepancies, when analyzing expression of pathway components in SCLC cell lines/xenografts vs tumor biopsies. Only small percentage of SCLC cell lines express transcriptional regulator GLI1, which can be used as an indicator of Shh signalling activity [13]. Contrary, when GLI1 immunohistochemical staining was performed on SCLC biopsies to investigate human tumors for GLI1 expression, 34 (85%) out of 40 SCLC tumors demonstrated GLI1 expression and 26 of these displayed medium or strong GLI1 reactivity [14]. Thus, caution should be taken when working with tumor derived cell lines, as the expression and signaling may not reflect the in vivo situation. These findings were supported also by Affymetrix oligonucleotide microarray analysis of human SCLC tumors, where significantly higher mean GLI1 expression in SCLC tissues was found when compared to that of the 20 SCLC cell lines investigated [15].

So advantage of this study was that mRNA expression of key Shh pathway member GLI1 was evaluated in SCLC fresh biopsies and survival data of patients were available. Unfortunately, small size of biopsy samples was an issue, which lead to exclusion of notable part of study patients from analysis due to the lack of cancer cells in a study specimen.

In conclusion, at our best knowledge this represents the first study investigating the prognostic role of GLI1 in SCLC patients. Our findings suggest that the Shh pathway activation might be a positive prognostic marker in SCLC contrary to its negative role observed in other malignancies. Further research is needed for validation of these results due to the small number of patients in the study.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Romalda Grigalinoviča for her extra work while being study dedicated cytologist in this project.

FUNDING

This work was supported by the Latvian National Research Programme BIOMEDICINE 2014–2017.

DISCLOSURE OF INTEREST

The authors declare no conflict of interest.

REFERENCES

  • 1. Daniel VC, Marchionni L, Hierman JS, et al. A primary xenograft model of small cell lung cancer reveals irreversible changes in gene expression imposed by culture in-vitro. Cancer Res 2009; 69: 3364–73.
  • 2. Watkins DN, Berman DM, Burkholder SG, et al. Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer. Nature 2003; 422: 313–7.
  • 3. Wang B, Yu T, Hu Y, et al. Prognostic role of Gli1 expression in breast cancer: a meta-analysis. Oncotarget 2017; 8: 81088–97.
  • 4. Jian-Hui C, Er-Tao Z, Si-Le C, et al. CD44, Sonic hedgehog, and Gli1 expression are prognostic biomarkers in gastric cancer patients after radical resection. Gastroenterol Res Pract 2016; Article ID 1013045.
  • 5. Berardi R, Santinelli A, Onofri A, et al. Hedgehog (Hh) signaling is a predictor of clinical outcome for advanced non-small cell lung cancer (NSCLC). J Carcinog Mutagen 2014; 5: 175.
  • 6. Cui Y, Cui CA, Yang ZT, et al. Gli1 expression in cancer stem-like cells predicts poor prognosis in patients with lung squamous cell carcinoma. Exp Mol Pathol 2017; 102: 347–53.
  • 7. Cheng J, Gao J, Tao K, et al. Prognostic role of Gli1 expression in solid malignancies: a meta-analysis. Sci Rep 2016; 6: Article ID 22184.
  • 8. Kim Y, Do IG, Hong M, et al. Negative prognostic effect of low nuclear GLI1 expression in glioblastomas. J Neurooncol 2017; 133: 69–76.
  • 9. Kim JE, Kim H, Choe JY, et al. High expression of Sonic hedgehog signaling proteins is related to the favorable outcome, EGFR mutation, and lepidic predominant subtype in primary lung adenocarcinoma. Ann Surg Oncol 2013; Suppl 3: 570–6.
  • 10. Mazzà D, Infante P, Colicchia V, et al. PCAF ubiquitin ligase activity inhibits Hedgehog/Gli1 signaling in p53-dependent response to genotoxic stress. Cell Death Differ 2013; 20: 1688–97.
  • 11. George J, Lim JS, Jang SJ, et al. Comprehensive genomic profiles of small cell lung cancer. Nature 2015; 524: 47–53.
  • 12. Yokomizo A, Tindall DJ, Drabkin H, et al. PTEN/MMAC1 mutations identified in small cell, but not in non-small cell lung cancers. Oncogene 1998; 17: 475–9.
  • 13. Ruiz I, Altaba A, Stecca B, et al. Hedgehog-Gli signaling in brain tumors: stem cells and paradevelopmental programs in cancer. Cancer Lett 2004; 204: 145–57.
  • 14. Vestergaard J, Pedersen MW, Pedersen N, et al. Hedgehog signaling in small-cell lung cancer: Frequent in vivo but a rare event in vitro. Lung Cancer 2006 52: 281–90.
  • 15. Pedersen N, Mortensen S, Sorensen SB, et al. Transcriptional gene expression profiling of small cell lung cancer cells. Cancer Res 2003; 63: 1943–53.
No Comments » Add comments
Leave a comment

ERROR: si-captcha.php plugin says GD image support not detected in PHP!

Contact your web host and ask them why GD image support is not enabled for PHP.

ERROR: si-captcha.php plugin says imagepng function not detected in PHP!

Contact your web host and ask them why imagepng function is not enabled for PHP.