Immunoglobulin G expression in lung cancer and its effects on metastasis

Abstract

Lung cancer is one of the leading malignancies worldwide, but the regulatory mechanism of its growth and metastasis is still poorly understood. We investigated the possible expression of immunoglobulin G (IgG) genes in squamous cell carcinomas and adenocarcinomas of the lung and related cancer cell lines. Abundant mRNA of IgG and essential enzymes for IgG synthesis, recombination activation genes 1, 2 (RAG1, 2) and activation-induced cytidine deaminase (AID) were detected in the cancer cells but not in adjacent normal lung tissue or normal lung epithelial cell line. The extents of IgG expression in 86 lung cancers were found to associate with clinical stage, pathological grade and lymph node metastasis. We found that knockdown of IgG with siRNA resulted in decreases of cellular proliferation, migration and attachment for cultured lung cancer cells. Metastasis-associated gene 1 (MTA1) appeared to be co-expressed with IgG in lung cancer cells. Statistical analysis showed that the rate of IgG expression was significantly correlated to that of MTA1 and to lymph node metastases. Inhibition of MTA1 gene expression with siRNA also led to decreases of cellular migration and attachment for cultured lung cancer cells. These evidences suggested that inhibition of cancer migration and attachment induced by IgG down-regulation might be achieved through MTA1 regulatory pathway. Our findings suggest that lung cancer-produced IgG is likely to play an important role in cancer growth and metastasis with significant clinical implications.

Figure 1. IgG expression in LSCC and LAC with IHC.

A–C, Igγ (A), Igκ (B) and Igλ (C) immunostainings are positive in the cytoplasm and cellular membrane of LSCC on serial sections. Note: The IgG positive cancer cells are distributed at the periphery of the tumor mass. D–F, positive signals for Igγ (D), Igκ (E) and Igλ (F) are shown in the cytoplasm and cellular membrane of a LAC. G–I, Igγ (G), Igκ (H) and Igλ (I) expressions are detected in lymphocytes of tonsil tissues as a positive control. J–L, Igγ (J), pan CK (K) and CD20 (L) are expressed in LSCC on serial sections. M–O, Igγ (M), pan CK (N) and CD20 (O) are expressed in serial sections of LAC. Igγ(P), Igκ (Q) and Igλ (R) are not expressed in normal epithelial cells adjacent to tumor mass on serial sections. Black arrowheads point to the same cancer cell on serial sections. Black arrows point to positive lymphocytes. With AEC colorization, the positive immunostainings with AEC are red in color. Note: IgG is positive in both cancer cells and infiltrating lymphocytes with the latter stronger than the former. Scale bar: 20 µm.

Figure 2. Igγ Antibody preabsorption tests in LSCC and LAC.

A–C, the mouse anti-human Igγ monoclonal antibody: pure human IgG  = 1∶1, 1∶5, and 1∶10 in LSCC sections. D–F, the similar gradient ratio of Igγ: pure human IgG in LAC sections. With the increase of antigen concentration (pure human IgG), the positive IgG signal was weakened. Bar: 20 µm.

Figure 3. Receptors for Igγ were not expressed on the cellular membrane of lung cancer cells.

CD16 (Fcγ receptor III) is shown in LSCC (A) and LAC (E) tissues. CD32 (Fcγ receptor II) is displayed in LSCC (B) and LAC (F) tissues. CD64 (Fcγ receptor I) is shown in LSCC (C) and LAC (G) tissues. FcRn (neonatal Fcγ receptor) is shown in LSCC (D) and LAC (H) tissues. In those sections, positive signals are only expressed in the cytoplasm and membrane of lymphocytes, while no positive signals are found in cancer cells. CD16 (I), CD32 (J), CD64 (K) and FcRn (L) are expressed in biopsied human tonsil tissues as positive controls. Bar: 20 µm.

Figure 4. IgG expression in lung cancer tissue with IHC and ISH.

Colocalization of the IgG mRNA and protein in LSCC and LAC tissue samples demonstrated with ISH and IHC. A–C, Igγ immunostaining (A, red) and mRNA signals (B, purple, with antisense probe) are positive on serial sections of a LSCC. C is a negative control with sense probe. D–F, Igγ immunostaining (D, red) and mRNA signals (E, purple, with antisense probe) are positive on serial sections of a LAC. F is negative with sense probe. G–I, Tonsil tissues are used as a positive control. G is for Igγ with IHC showing positive lymphocytes. H is for the antisense probe with ISH showing positive lymphocytes. I is with sense probe in ISH showing no positive signal. J–L, Igγ protein (J) and mRNA (K, the antisense probe) are not expressed in normal epithelial cells adjacent to tumor mass. L is the sense probe. Black arrowheads point to the same cancer cells on serial sections. Black arrows point to positive lymphocytes.

Figure 5. IgG expression in lung cancer tissue with LMD coupled with RT-PCR.

A is the representative photos of LMD in LSCC, LAC and normal lung adjacent to tumor mass. B, the bands represented that IgG synthesis associated genes were detected in LSCC, LAC and normal lung with LMD coupled with RT-PCR. No CD19 was detected, thereby excluding possible B lymphocyte contamination. Raji cell line was used as a positive control. Water was used as a negative control in place of the primers. RNA treated with DNase (abbreviated as R) was used as a template to exclude possible false positivity in 1R-6R. NL is the abbreviation of normal lung.

Figure 6. The sequencing results of IgG mRNA extracted from LSCC and LAC.

A is for the sequences of Vγ, B is for Vκ, and C is for Vλ.

Figure 7. IgG expression in lung cancer cell lines with ISH and immunofluorescence.

A–C, Igγ (A), Igκ (B) and Igλ (C) are expressed in A549 cells with immunofluorescence. D–F, Igγ (D), Igκ (E) and Igλ (F) are expressed in SK-MES-1 cells. G–I, Igγ (G), Igκ (H) and Igλ (I) are in Raji cells as positive control. J–L, Igγ (J), Igκ (K) and Igλ (L) are not expressed in Beas2B cells. Positive signals (purple) for IGHG1 antisense probe are seen in the cytoplasm of A549 (M), SK-MES-1 (O) and Raji (Q) cells. N, P and R show negative results (sense probe) in A549, SK-MES-1 and Raji cells. No positive signals are found in Beas2B cells (S, the antisense probe; and T, the sense probe). To highlight the cells without purple signal, methyl green was use to counterstain the nuclei. Scale bars: 20 µm.

Figure 8. IgG expression in lung cancer cell lines with RT-PCR and Western blot.

A, the bands represented that IgG synthesis associated genes were detected with RT-PCR in A549 and SK-MES-1, whereas not in Beas2B. B shows that IgG whole molecule, Igγ, Igκ and Igλ are detected in A549, SK-MES-1, whereas not in Beas2B with Western blot. Human pure IgG was used as molecular weight standard to compare with that from lung cancer cells. Raji cell line was a positive control. C shows that RAG1, RAG2 and AID are expressed in lung cancer cells, whereas not in Beas2B. Raji cell line was served as a positive control.

Figure 9. IgG expression was strongly correlated to MTA1 expression and IgG down-regulation lowered MTA1 expression.

A, IgG was down-regulated with specific IGHG1 siRNA transfection with Real-time PCR. B, MTA1 expression was lowered following the decrease of IgG expression detected with Real-time PCR. C, typical bands represent the changes of IgG and MTA1 expressions after IGHG1 siRNA interference in A549 cell line. D, similar changes in SK-MES-1 cell line. E, Igγ (red, positive staining in cytoplasm) was mostly co-localized with MTA1 (purplish red, positive staining in nuclei) in lung cancers. Black arrows or arrowheads show typical cancer cells labeling with IgG and MTA1. F, lung cancers with weak IgG expression have a lower expression level of MTA1 in comparison to strong IgG expression cancers. G, lung cancers with lymph node metastasis have much higher MTA1 expression than non-metastatic cancers. The histograms represent the four independent assays. Data are presented as mean ± S.E. *, p<0.05. Scale bars: 20 µm.

Figure 10. IgG down-regulation inhibits lung cancer cell migration and attachment.

With specific IGHG1 siRNA transfection, the ability of migration and attachment of lung cancer cells was reduced markedly in A549 and SK-MES-1 cells, whereas not in Beas2B cells. A, representative photographs (200×) of transwell assays. B, representative photographs (200×) of attachment assays. The histograms represent the four independent assays. Data are presented as mean ± S.E. *, p<0.05.

Figure 11. IgG down-regulation inhibits cell migration by wound healing assay in lung cancer cells.

Forty-eight hours after scratching the cultured cells, scratched areas were photographed. The results show that for the untreated and siRNA-scrambled groups, the wound area is markedly narrowed in A549 and SK-MES-1 but not in Beas2B.