MYC expression correlates with PD-L1 expression in non-small cell lung cancer

doi:10.1016/j.lungcan.2017.06.006

Lung Cancer

Volume 110, August 2017, Pages 63-67

https://doi.org/10.1016/j.lungcan.2017.06.006 Get rights and content

Highlights

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MYC seems to be involved in immune evasion through inducing PD-L1 expression.
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MYC expression significantly correlated with PD-L1 expression in NSCLC tissue.
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NSCLC patients with MYC-PD-L1 double-positive samples showed poor clinical outcomes.
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MYC expression could be used for treatment response assessment for immune checkpoint inhibitor.

Abstract

Objectives Programmed death-ligand 1 (PD-L1) is a widely used biomarker for predicting immune checkpoint inhibitors, but is of limited usefulness in the prediction of drug response. MYC, a transcription factor that is overexpressed in cancers, is involved in preventing immune cells from attacking tumor cells through inducing PD-L1 expression. This study evaluated the relationship between MYC and PD-L1 expression in 84 non-small cell lung cancer (NSCLC) patients who underwent curative surgical resection.

Materials and Methods The relationship between MYC and PD-L1 was investigated by introducing pcDNA3-cMYC into A549 and H1299 cells with low PD-L1 expression and siRNA against MYC into H60 and H2009 cells with high PD-L1 expression. Expression of PD-L1 in NSCLC tissues was analyzed by immunostaining using a PD-L1 (22C3) PharmDx protocol using the Dako Automated Link 48 platform and expression of MYC was determined using anti-c-MYC (Y69) (ab320720).

Results Of 84 patients, PD-L1 was expressed in 14 (16.7%) and MYC was overexpressed in 30 (35.7%). We investigated the relationship between PD-L1 and MYC expression. There were 49 (58.3%) double-negative patients and 9 (10.7%) double-positive patients. Significant positive correlation was observed between PD-L1 and MYC expression (γ = 0.210, P = 0.029). Double-negative patients showed better disease free (31.1 vs. 7.1 months, P = 0.011) and overall survival (56.1 vs. 14.4 months, P = 0.032) than double-positive patients.

Conclusion Taken together, MYC expression significantly correlated with PD-L1 expression in NSCLC. The usefulness of MYC expression as a surrogate marker of treatment response assessment is worth evaluating for immune checkpoint inhibitor therapy and special interest are required for the subgroup of NSCLC patients, whose tumor expresses PD-L1 and MYC double positive.

Introduction

Lung cancer is a major public health issue worldwide, with a high prevalence and high mortality [1]. Throughout the era of platinum-based doublet chemotherapy, target agents have been widely used [2]. However, in cases where a treatment target cannot be identified and patients have resistance to the target agent, new breakthroughs are needed.

The tumor microenvironment interacts with cancer cells by modulating programmed death-1 receptor −1 (PD-1) and the ligand PD-L1 pathways. Expression of PD-L1 on cancer cells leads to evasion from the immune response, permitting cancer progression and metastasis [3], [4]. Immunotherapy for lung cancer had entered a golden era with the development of immune checkpoint inhibitors that interfere with cytotoxic T lymphocyte antigen-4 (CTLA-4), PD-1 and its ligand PD-L1. A number of immune checkpoint inhibitors are under development based on biological findings. The PD-1 inhibitors nivolumab and pembrolizumab have received FDA approval for lung cancer treatment [5], [7].

Demand for biomarkers for predicting therapeutic outcomes of these drugs is high. Biomarkers developed to date [5] are: (1) PD-L1 expression in cancer cells; (2) tumor microenvironment/immune effector cells (with PD-L1 expression in the tumor microenvironment, PD-L2 expression in tumor cells and infiltrating immune cells, CD8 and T-cell and effector functional markers, and gene expression of CTLA4 and CX3CL1); (3) gene alteration and phenotypic alteration of tumor cells such as tumor mutational load, oncogene mutation, and epithelial mesenchymal transition; and (4) clinicopathological biomarkers such as squamous cell histology and smoking history. In lung cancer, PD-L1 is a therapeutic target of immune checkpoint inhibitors and is used as a biomarker to predict therapeutic response. However, its usefulness as a therapeutic biomarker is challenged because of: (1) differences in antibodies and staining platforms from different companies; (2) instability of PD-L1 antigenicity; and (3) different reading criteria among researchers for predicting therapeutic responses. These issues suggest an urgent need for effective surrogate markers for predicting response to immune checkpoint inhibitors.

MYC, located on chromosome 8q24.21, is a regulator gene that regulates the expression of approximately 15% of human genes by recruiting histone acetyltransferases and binding enhancer box sequences (E-boxes) [6], [7]. Malfunction of MYC, including MYC translocation involved in the development of Burkitt lymphoma, is found in cervix, colon, breast, stomach, and lung cancer[8], [9], [10]. MYC overexpression is observed in 41% of non-small cell lung cancer (NSCLC) and associated with loss of cell differentiation [9]. In addition to its classic function, MYC seems be involved in preventing immune cells from attacking tumor cells by inducing PD-L1 and CD47 expression [11]. Using cancer cells and mouse models, Casey et al. showed that (1) suppression of MYC in mouse and human tumor cells causes a reduction in the levels of PD-L1 mRNA and protein (2) MYC directly binds to the promoters of the PD-L1 genes (3) MYC inactivation in mouse tumors down-regulates PDL1 expression and enhances the anti-tumor immune response [11]. This finding suggests that when combined with PD-L1 expression, MYC and PD-L1 double-positive lung cancer patients may exhibit unique clinical characteristics. MYC could be a surrogate biomarker for response prediction for therapy with immune checkpoint inhibitors.

To explore this possibility, we investigated the correlation of MYC and PD-L1 expression using human lung cancer tissue and evaluated the clinical implications of MYC and PD-L1 double positivity in lung cancer tissues.

Section snippets

Materials and methods

2.1 Cell lines, plasmids, and antibodies. Lung cancer cells were obtained from ATCC (Manassas, VA, USA) or the Korean Cell Line Bank (Seoul, Korea). pcDNA3-cMYC was a gift from Wafik El-Deiry (Addgene plasmid #16011) and pcDNA3 was the negative control [12]. Anti-PD-L1 (22C3) was obtained from DAKO and anti-cMYC antibody (Y69) was from Abcam^® (ab32072). Unless otherwise stated, antibodies were from Cell Signaling Technology (Danvers, MA, USA).

2.2 Study population. The study used 84 lung cancer

Results

3.1 Relationship of PD-L1 and MYC in lung cancer cells. To examine the relationship between PD-1 and MYC expression, we investigated the relationship between the expression of the two proteins in unstimulated NSCLC cell lines using immunoblots. Among the eight cell lines tested, 5 showed distinct MYC expression, 2 showed no expression and 1 showed faint expression. No significant relationship was observed between the expression of the two proteins based on estimated correlation between the

Discussion

Since the expression of PD-L1 in the tumor tissues is not only a target for immune checkpoint inhibitors but also a major biomarker for predicting therapeutic responses, there is a growing interest in the molecules that regulate the expression of PD-L1 [5]. Recent in vitro study by Casey et al. amplified interest in MYC by showing that MYC directly binds to the promoters of PD-L1 and CD47, induces their expression, and plays critical roles in immune escape [11].

The loss of control of MYC

Conclusions

In conclusion, MYC expression significantly correlated with PD-L1 expression and NSCLC patients with MYC-PD-L1 double-positive samples showed poor clinical outcomes. These findings suggested that it is worth evaluating the usefulness of MYC expression as a surrogate marker of treatment response assessment to immune checkpoint inhibitor therapy. Additional prospective clinical studies may be needed to identify NSCLC patients with PD-L1-MYC double-positive samples who may benefit from currently

Conflict of interest

The authors declare that they have no competing interests.

Acknowledgements

This study was supported by a faculty research grant of Yonsei University College of Medicine 6-2015-0152 awarded to EY KIM. The funding source has not played any role in the planning and execution of this study. There was no writing assistance for the preparation of this manuscript.

References (20)

C.G. Drake et al.
Mechanisms of immune evasion by tumors
Adv. Immunol.
(2006)
K. Shien et al.
Predictive biomarkers of response to PD-1/PD-L1 immune checkpoint inhibitors in non-small cell lung cancer
Lung Cancer
(2016)
E.Y. Kim et al.
AZD6244 inhibits cisplatin-induced ERK1/2 activation and potentiates cisplatin-associated cytotoxicity in K-ras G12D preclinical models
Cancer Lett.
(2015)
R.L. Siegel et al.
Cancer statistics, 2015
CA. Cancer J. Clin.
(2015)
J.H. Schiller et al.
Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer
N. Engl. J. Med.
(2002)
D.M. Pardoll
The blockade of immune checkpoints in cancer immunotherapy
Nat. Rev. Cancer
(2012)
J. Gearhart et al.
Pluripotency redux-advances in stem-cell research
N. Engl. J. Med.
(2007)
R. Cotterman et al.
N-Myc regulates a widespread euchromatic program in the human genome partially independent of its role as a classical transcription factor
Cancer Res.
(2008)
S.N. Finver et al.
Croce CM. Sequence analysis of the MYC oncogene involved in the t(8;14)(q24;q11) chromosome translocation in a human leukemia T-cell line indicates that putative regulatory regions are not altered
Proc. Natl. Acad. Sci. U. S. A.
(1988)
J. Lorenz et al.
Oncogene overexpression in non-small-cell lung cancer tissue prevalence and clinicopathological significance
J. Mol. Med.
(1994)

There are more references available in the full text version of this article.

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View full text

MYC expression correlates with PD-L1 expression in non-small cell lung cancer

Highlights

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Conclusions

Conflict of interest

Acknowledgements

Adv. Immunol.

Lung Cancer

Cancer Lett.

Cancer statistics, 2015

CA. Cancer J. Clin.

Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer

N. Engl. J. Med.

The blockade of immune checkpoints in cancer immunotherapy

Nat. Rev. Cancer

Pluripotency redux-advances in stem-cell research

N. Engl. J. Med.

N-Myc regulates a widespread euchromatic program in the human genome partially independent of its role as a classical transcription factor

Cancer Res.

Croce CM. Sequence analysis of the MYC oncogene involved in the t(8;14)(q24;q11) chromosome translocation in a human leukemia T-cell line indicates that putative regulatory regions are not altered

Proc. Natl. Acad. Sci. U. S. A.

Oncogene overexpression in non-small-cell lung cancer tissue prevalence and clinicopathological significance

J. Mol. Med.