A recombinant adenovirus vector containing the synNotch receptor gene for the treatment of triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) is known as the most difficult molecular subtype of breast cancer to treat. Recent studies revealed that cancer stem cells (CSCs) play a critical role in TNBC recurrence and metastasis. In this study, we developed a recombinant replication-deficient adenoviral vector (Ad-CD44-N-HIF-3α4), which contains a gene encoding a synthetic Notch (synNotch) receptor composed of the extracellular domain of CD44 (CD44-ECD) and the hypoxia-inducible factor (HIF)-3α4 connected by the Notch core regulatory region. CD44 is a transmembrane glycoprotein and known as a CSC marker in breast cancer and other malignancies. HIF-3α4 is a dominant-negative regulator of HIF-1α and HIF-2α and inhibits hypoxia-inducing effect. Both CD44 and HIF signals contribute cancer stemness and maintaining CSCs in breast cancer. The CD44-ECD in the synNotch receptor acts as the CD44 decoy receptor, and after a ligand such as a hyaluronic acid binds to the CD44-ECD, HIF-3α4 is released from the Notch core domain. We performed an in vivo study using a mouse xenograft model of MDA-MB-231, a highly invasive TNBC cell, and confirmed the significant antitumor activity of the intratumoral injections of Ad-CD44-N-HIF3α4. Our findings in this study warrant the further development of Ad-CD44-N-HIF3α4 for the treatment of patients with TNBC.

Keywords: HIF; adenovirus; breast cancer; cancer stem cells; gene therapy.

Figure 1

Mechanism of synthetic Notch (synNotch) receptor (CD44-N-HIF3α4) and construction of Ad-CD44-N-HIF3α4 (A) CD44 protein: Hyaluronic acid (HA), a ligand of CD44-extracellular domain (CD44-ECD), initiates the CD44 signaling cascade and then induces the cleavage of the intracellular domain of CD44 (CD44-ICD). CD44-ICD moves into the cell nucleus to promote tumor cell proliferation, migration, angiogenesis, and metastasis. (B) synNotch receptor (CD44-N-HIF3α4): Hypoxia-inducible factor (HIF) in hypoxic tumor microenvironments also activates the transcription of various hypoxia target genes promoting tumorigenesis and cancer stemness. Ad-CD44-N-HIF3α4 contains a gene encoding a synNotch receptor (CD44-N-HIF3α4) composed of the CD44-ECD and HIF-3α4 connected by the Notch core regulatory region. CD44-ECD in the synNotch receptor acts as a decoy receptor for the endogenous CD44 protein in the tumor microenvironment. The signal of CD44-ligands such as HA is converted via the Notch core regulatory region at the cell membrane and inhibits hypoxia-induced responses by HIF-3α4, which is released from synNotch receptor. (C) Construction of recombinant replication-deficient adenoviral vector, Ad-CD44-N-HIF3α4 A synthetic fusion gene encoding a synNotch receptor composed of the extracellular domain of CD44 (CD44-ECD) and HIF-3α4 connected by the Notch core regulatory region was introduced into the ΔE1 region of the adenovirus type 5 vector to construct a recombinant replication-deficient adenoviral vector, Ad-CD44-N-HIF3α4. HA, Hyaluronic acid; ECD, Extracellular Domain; TMD, Transmembrane Domain; ICD, Intracellular Domain; HIF, Hypoxia-inducible factor.

Figure 2

Expressions of CAR and CD44, and induction of HIF-1α mRNA by hypoxia in MDA-MB-231 and MCF-7 cells (A) The expressions of CAR and CD44 on the cell surface of MDA-MB-231 and MCF-7 cells were determined by flow cytometry. Mean fluorescence intensities (MFI) and their representative histograms are shown. Both CAR and CD44 expressions were significantly higher than their isotype controls (n=3, average ± SE bars, **p< 0.01). However, the CD44 expression in MCF-7 was relatively lower than MDA-MB-231 cells. The upper right corner of the histogram represents CD44 and CAR double positive cells. (B) The mRNA expressions of HIF-1α and VEGF in MDA-MB-231 and MCF-7 cells were measured by real-time RT-PCR under the culture conditions of O₂ concentrations of 21% and 2%. The mRNA levels of HIF-1α and VEGF in MDA-MB-231 cells were significantly increased in 2% hypoxia culture condition compared to 21% condition. In addition, mRNA level of HIF-1α in MCF-7 cells was not increased in hypoxia, while VEGF was significantly increased in hypoxia (n=3, average ± SE bars, **p< 0.01).

Figure 3

Detection of hyaluronan synthases (HAS1, HAS2, and HAS3) mRNA in MDA-MB-231 and MCF-7 cells (A) The mRNA expressions of HAS1, HAS2 and HAS3 in MDA-MB-231 and MCF-7 cells were measured by real-time RT-PCR and compared to those in non-malignant SV-HUC-1 cells. The mRNA expressions of HAS1 in MDA-MB-231 and MCF-7 cells was not increased compared to those of SV-HUC-1 cells. (B) The mRNA expression of HAS2 in MDA-MB-231 cells was significantly increased compared to that in SV-HUC-1 cells, but not in MCF-7 cells. (C) The mRNA expressions of HAS3 in MDA-MB-231 and MCF-7 cells was not increased compared to that in SV-HUC-1 cells. mRNA levels were standardized by the expression levels of control gene TATA-binding protein (TBP). (n=3, average ± SE bars, **p< 0.01).

Figure 4

Ad-CD44-N-HIF3α4 transduced CD44-ECD protein in MDA-MB231 and MCF-7 cells in dose-dependent manner but inhibits the cell growth of only MDA-MB-231 cells (A) Western blotting using anti-CD44 antibody revealed that MDA-MB-231 highly expressed endogenous CD44 protein (around 80 kDa) but MCF-7 does not express the endogenous CD44 protein. Ad-CD44-N-HIF3α4 could induce the synNotch receptor protein including CD44-ECD in dose dependent manner both in MDA-MB-231 and MCF-7. (B) Cell proliferation analysis, Ad-CD44-N-HIF3α4 significantly inhibited the cell growth of MDA-MB-231 cells in normoxia and hypoxia conditions compared to cell only (no treatment) group but did not inhibit the cell growth of MCF-7 cells (n=3, average ± SE bars, **p< 0.01).

Figure 5

Ad-CD44-N-HIF3α4 inhibited migration in MDA-MB-231 in hypoxia condition, Ad-CD44-N-HIF3α4 significantly inhibited the cell migration of MDA-MB-231 cells in hypoxia condition but not in normoxia condition. Magnification: ×100 (n=3, average ± SE bars, *p< 0.05).

Figure 6

Gene expression of HIF-3α4, SOCS3 and p53 induced by adenovirus vectors in MDA-MB-231 cells The gene expression of HIF-3α4 (A), SOCS3 (B) and p53 (C) in MDA-MB-231 cells were measured by real-time RT-PCR after Ad-CD44-N-HIF3α4, Ad-SOCS3, Ad-p53 and Ad-lacZ infections. mRNA levels were standardized by the expression levels of control gene TATA binding protein (TBP). The significantly increased levels of mRNA expressions of HIF-3α4 (A), SOCS3 (B) and p53 (C) were observed in cells infected with Ad-CD44-N-HIF3α4, Ad-SOCS3 or Ad-p53, respectively (n=3, average ± SE bars, **p< 0.01).

Figure 7

Gene expression of survivin, CCL2, VEGF and Bcl-xL in MDA-MB-231 cells infected with adenovirus vectors were measured by real-time RT-PCR. Cells were cultured under hypoxic conditions or under normoxic conditions. The significantly decreased mRNA expressions of (A) survivin and (B) CCL2 were observed in cells infected with Ad-CD44-N-HIF3α4 compared to cells infected with the other adenovirus vectors and no virus only in the culture conditions under hypoxic. The significantly decreased mRNA expressions of (C) VEGF and (D) Bcl-xL were observed in cells infected with Ad-CD44-N-HIF3α4 compared to cells infected with the other adenovirus vectors and no virus only in the culture conditions under hypoxic. mRNA levels were standardized by the expression levels of control gene TBP. (n=3, average ± SE bars, *p< 0.05,**p< 0.01).

Figure 8

Immunohistochemical staining for CD44 in MDA-MB-231 tumor injected with adenovirus vectors One million MDA-MB-231 cells were subcutaneously inoculated into nude mice for tumor formation, followed by intratumoral injections of Ad-CD44-N-HIF3α4, Ad-SOCS3, Ad-p53, Ad-LacZ or PBS. After the treatments, tumors were resected and stained for CD44 expressions. The remarkably increased expression of CD44 was detected in the cell membrane of tumor injected with Ad-CD44-N-HIF3α4 compared to tumors injected with the other adenovirus vectors or PBS. (Original magnification: x400).

Figure 9

Anti-tumor effect of Ad-CD44-N-HIF3α4 in mice with MDA-MB-231 tumors One million MDA-MB-231 cells were subcutaneously inoculated into nude mice and intratumoral injections of Ad-CD44-N-HIF3α4, Ad-SOCS3, Ad-p53, Ad-LacZ or PBS were performed every other day for 8 times. Ad-CD44-N-HIF3α4 significantly inhibited the growth of MDA-MB-231 tumors compared to Ad-p53 (p< 0.05), Ad-SOCS3, Ad-LacZ and PBS (p< 0.01). (n=5, average ± SE bars, *p< 0.05, **p< 0.01).