Identification of a HLA-A*0201-restricted immunogenic epitope from the universal tumor antigen DEPDC1

Abstract

The identification of universal tumor-specific antigens shared between multiple patients and/or multiple tumors is of great importance to overcome the practical limitations of personalized cancer immunotherapy. Recent studies support the involvement of DEPDC1 in many aspects of cancer traits, such as cell proliferation, resistance to induction of apoptosis and cell invasion, suggesting that it may play key roles in the oncogenic process. In this study, we report that DEPDC1 expression is upregulated in most types of human tumors, and closely linked to a poorer prognosis; therefore, it might be regarded as a novel universal oncoantigen potentially suitable for targeting many different cancers. In this regard, we report the identification of a HLA-A*0201 allele-restricted immunogenic DEPDC1-derived epitope, which is able to induce cytotoxic T lymphocytes (CTL) exerting a strong and specific functional response in vitro toward not only peptide-loaded cells but also triple negative breast cancer (TNBC) cells endogenously expressing the DEPDC1 protein. Such CTL are also therapeutically active against human TNBC xenografts in vivo upon adoptive transfer in immunodeficient mice. Overall, these data provide evidence that this DEPDC1-derived antigenic epitope can be exploited as a new tool for developing immunotherapeutic strategies for HLA-A*0201 patients with TNBC, and potentially many other cancers.

Keywords: Cancer immunotherapy; DEPDC1; cytotoxic T lymphocytes; triple negative breast cancer; tumor antigen.

Figure 1.

DEPDC1 is upregulated in different human cancers. (A) DEPDC1 mRNA expression in normal (white) and tumor tissues (gray) as reported from microarray studies in the Oncomine database. Breast: (t-test = 10.953; p-value = 1.181E−14); bladder (t-test = 6.217; p-value = 8.82E−9); brain (t-test = 9.929; p-value = 7.56E−12); cervix (t-test = 7.688; p-value = 9.13E−9); colon (t-test = 3.978; p-value = 9.98E−5); esophagus (t-test = 10.994; p-value = 8.17E−19); stomach (t-test = 7.378; p-value = 4.54E−10); nasopharynx (t-test = 5.832; p-value = 1.71E−6); leukemia (t-test = 4.400; p-value = 8.57E−4); liver (t-test = 6.180; p-value = 2.18E−7); lung (t-test = 16.310; p-value = 7.19E−18); skin (t-test = 5.874; p-value = 3.54E−5); ovary (t-test = 14.643; p-value = 2.12E−7) (TCGA Ovarian, No Associated Paper, 2013); pancreas (t-test = 4.794; p-value = 8.57E−6). Numbers above each box plot refer to the number of samples reported. (B) Endogenous expression of DEPDC1 protein in several human tumor cell lines (left panels) and in different normal human tissues (right panels), as assessed by western blot analysis. MDA-MB-231shDEPDC1 refers to cells with DEPDC1 silenced by shRNA, and served as an internal negative control.

Figure 2.

Identification of DEPDC1-derived immunogenic epitopes. The IFNγ production from CD8⁺ T cells stimulated for three times with autologous DCs pulsed with each DEPDC1-derived peptide was measured in response to unpulsed or pulsed LCL cells, by cytokine intracellular staining. LCL cells loaded with Gag-17_77–85 peptide served as a negative control. Data are presented as the percentage of CD8⁺ T cells positive for IFNγ staining (**p < 0.01; not statistically significant (p > 0.05) if not indicated).

Figure 3.

Characterization of DEPDC1#5 peptide-stimulated T cell cultures. (A) Flow cytometry analysis of T cells subsets in cell cultures after three stimulations with autologous DC pulsed with DEPDC1#5 peptide. The mean percentage and standard deviation are shown (n = 24 healthy donors). (B) Percentages of CD8⁺ T cells within subsets defined by the expression of CD45RA and CCR7 differentiation markers (CD45RA⁺CCR7⁺ naive, CD45RA⁻CCR7⁺ central memory (CM), CD45RA⁻CCR7⁻ effector memory (EM) and CD45RA⁺CCR7⁻ terminally differentiated effector memory (Temra) cells; n = 11 healthy donors)). (C) Tetramer staining of CD8⁺ T cells after sequential rounds of stimulation. The mean percentage ± SD of CD8⁺/tetramer⁺ lymphocytes in cultures is shown for each stimulation.

Figure 4.

Functional characterization of DEPDC1#5 peptide-stimulated CTLs. (A) Western blot analysis of DEPDC1 protein expression in MDA-MB-231 and 293 cell lines. MDA-MB-231shDEPDC1 are silenced for DEPDC1, while shCTRL refer to cells silenced with a scrambled control siRNA. (B) Intracellular IFNγ staining of CD8⁺ T cells stimulated for three times with autologous DEPDC1#5 peptide-pulsed DC. Data are presented as the mean percentage ± SD of CD8⁺ T cells positive for intracellular IFNγ staining (*p< 0.05; n = 3 healthy donors). (C, D) Lytic activity and specificity of DEPDC1#5 peptide-stimulated CTL. Cytotoxicity of DEPDC1#5 peptide-stimulated CD8⁺ T cells generated as in (B) was analyzed by a 6-h chromium release assays against the reported targets. Results are expressed as LU₃₀ × 10⁶ effectors (*p < 0.05; ***p < 0.001; ****p < 0.0001; not statistically significant (p > 0.05) if not indicated).

Figure 5.

In vitro tumor growth restraining activity by DEPDC1#5 peptide-stimulated CTLs. The figure reports the inhibition of MDA-MB-231 outgrowth by DEPDC1#5 peptide-stimulated T cells after 4 weeks of culture. MDA-MB-231 cultured alone or in presence of control CTL (Ctrl-CTL), and NIH 3T3 cells alone or co-cultured with DEPDC1#5 peptide-stimulated CTL were used as negative controls. Results are expressed as percentage of target cell growth inhibition and the numbers above each bar refer to the target:CTL ratio (n = 3 healthy donors).

Figure 6.

Assessment of therapeutic efficacy in vivo of DEPDC1#5 peptide-stimulated CTL. (A) On day 0, NSG mice were injected into the mammary fat pad with 1 × 10⁶ luc-transduced MDA-MB-231 cells, and were treated intra-tumorally at days 3, 6 and 10 with 1 × 10⁷ DEPDC1#5 peptide-stimulated CTLs (n = 9) or Melan-A peptide-stimulated CTL (n = 8). The untreated group of mice received three injections of PBS1X (n = 18). (B) Tumor growth was monitored by BLI measurement as photon flux. Left panels show the primary tumor BLI of three representative mice for each group after 29 d from tumor injection, while right panel reports tumor growth of all animals at different time points (mean of photons/second ± SD; ***p < 0.001; the ANOVA was used for statistical analysis). (C) Distant metastatic colonization was evaluated by BLI when a primary tumor size of about 700 mm³ was reached in each group. The left panels show the BLI of distant metastases in three representative mice for each group, while right panel reports the photons/second detected in each individual mouse belonging to the different groups (*p < 0.05; **p < 0.01).