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. 2014;13(18):2913-30.
doi: 10.4161/15384101.2014.946858.

A knockdown with smoke model reveals FHIT as a repressor of Heme oxygenase 1

Jennifer A Boylston  1 , Charles Brenner
Affiliations

A knockdown with smoke model reveals FHIT as a repressor of Heme oxygenase 1

Jennifer A Boylston et al. Cell Cycle. 2014.

Abstract

Fragile histidine triad (FHIT) gene deletions are among the earliest and most frequent events in carcinogenesis, particularly in carcinogen-exposed tissues. Though FHIT has been established as an authentic tumor suppressor, the mechanism underlying tumor suppression remains opaque. Most experiments designed to clarify FHIT function have analyzed the consequence of re-expressing FHIT in FHIT-negative cells. However, carcinogenesis occurs in cells that transition from FHIT-positive to FHIT-negative. To better understand cancer development, we induced FHIT loss in human bronchial epithelial cells with RNA interference. Because FHIT is a demonstrated target of carcinogens in cigarette smoke, we combined FHIT silencing with cigarette smoke extract (CSE) exposure and measured gene expression consequences by RNA microarray. The data indicate that FHIT loss enhances the expression of a set of oxidative stress response genes after exposure to CSE, including the cytoprotective enzyme heme oxygenase 1 (HMOX1) at the RNA and protein levels. Data are consistent with a mechanism in which Fhit protein is required for accumulation of the transcriptional repressor of HMOX1, Bach1 protein. We posit that by allowing superinduction of oxidative stress response genes, loss of FHIT creates a survival advantage that promotes carcinogenesis.

Keywords: ARE, antioxidant response element; ApppA, diadenosine triphosphate; BACH1; BACH1, BTB and CNC homology 1 gene; BMC, bone marrow cell; CPT, camptothecin; CSE, cigarette smoke extract; Cigarette smoke; FHIT; FHIT, fragile histidine triad gene; HMOX1; HMOX1, heme oxygenase 1 gene; MMC, mitomycin C; NRF2; Nrf2, nuclear factor erythroid derived 2-like 2 protein; Oxidative Stress; RNAi, RNA interference; ROS, reactive oxygen species; qRT-PCR, quantitative real time PCR; siRNA, short interfering RNA.

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Figures

Figure 1.
Figure 1.
FHIT siRNA significantly reduces expression of FHIT mRNA and protein. HBEC3-TK cells were transfected with control or FHIT-targeting siRNAs for 48 hours. Cells were then exposed to 1% CSE for 4 hours or were left untreated. (A) Decreased FHIT expression was determined by qRT-PCR. Data represent the results of five experiments. (B) Fhit protein abundance was assessed by western blot. Treatment with 2 FHIT-targeting siRNAs significantly reduces Fhit protein levels and treatment with 2 negative control siRNAs does not affect Fhit protein levels. β-actin serves as an internal standard for loading control. Data represent the results of five experiments.
Figure 2.
Figure 2.
Canonical pathways affected by cigarette smoke exposure. Gene expression changes between cells treated with siControl (48 h) plus 1% CSE (4 h) and cells treated with siControl (48 h) alone were determined by microarray. IPA core analysis was used to generate a list of top biological functions affected by CSE exposure. The significant canonical pathways affected by CSE exposure are represented. x-axis bars represent the [–log(p-value)] of the pathway, calculated by Fisher's exact test. The threshold for this analysis was set to P-value≤0.05. x-axis points (orange) represent the ratio calculated by dividing the number of differentially expressed genes that map to a particular pathway by the total number of genes in that pathway.
Figure 3.
Figure 3.
FHIT knockdown enhances expression of oxidative stress response genes after cigarette smoke treatment. HBEC3-TK cells were transfected with control or FHIT-targeting siRNAs for 48 hours. Cells were then exposed to 1% CSE for 4 hours or were left untreated. (A) Gene expression after siRNA treatment was analyzed with microarray. The left panel demonstrates that enhanced expression of a set of oxidative stress response genes was detected in FHIT knockdown cells after exposure to 1% CSE. All of the genes in this set pass FDR correction after ANOVA, P≤ 0.05. The right panel demonstrates enhanced expression of genes in FHIT knockdown cells. In these cases, upregulation passed FDR in FHIT knockdown cells but did not pass this statistical screen in siControl treated cells. (B) qRT-PCR validation of the microarray data. Data represent the results of at least three independent experiments. A 2-tailed paired t-test was used to assess significance of the data; *P ≤ 0.05; **P ≤ 0.01. Error bars represent standard deviations.
Figure 4.
Figure 4.
FHIT knockdown enhances the expression of HMOX1 after cigarette smoke. HBEC3-TK cells were transfected with control or FHIT-targeting siRNAs. After transfection, cells were either treated for 4 hours with 1% CSE or were left untreated. (A) HMOX1 was quantified by qRT-PCR. Data represent results of 6 independent experiments. A 2-tailed paired t-test was used to assess significance; ***P≤ 0.001. (B) Hmox1 protein abundance was assessed by western blot. Treatment with 2 FHIT-targeting siRNAs increases Hmox1 expression relative to cells treated with 2 negative control siRNAs. Western blot data are representative of 4 independent experiments.
Figure 5.
Figure 5.
FHIT loss enhances Hmox1 expression in TERT-HFK and TERT-T-106 cells. (A) Human foreskin keratinocytes or (B) tracheal airway epithelial (T106) cells were transfected with control or FHIT-targeting siRNAs. After knockdown, cells were exposed to 1% CSE for 4 hours. Hmox1 protein abundance was assessed by western blot. The data show that Hmox1 expression is enhanced by FHIT knockdown in these cells, demonstrating that the role of FHIT in modulating Hmox1 expression does not require a specific genetic background.
Figure 6.
Figure 6.
FHIT knockdown induces early and sustained expression of Hmox1. HBEC3-TK cells were transfected with control or FHIT siRNAs and exposed to 1% CSE for increasing time points, up to 4 hours. After 4 hours, cells were recovered in fresh media for increasing amounts of time. (A) Hmox1 protein is detected earlier in FHIT knockdown cells and (B) Hmox1 expression is enhanced at all time points by FHIT loss. Data represent the results of three independent experiments. At each time point, there is approximately 2-fold greater Hmox1 in FHIT knockdown cells.
Figure 7.
Figure 7.
PI3K inhibition does not affect expression of FHIT. HBEC3-TK cells were treated with control or FHIT siRNA for 48 hours. Cells were pretreated with 30 μM LY294002 or DMSO vehicle for 1 hour and then treated with 1% CSE supplemented with 30 μM LY294002 or DMSO for the indicated time intervals. (A) Western blot demonstrates that FHIT knockdown does not affect phosphorylation of AKT. AKT is phosphorylated in response to CSE but is not altered in degree of activation by FHIT knockdown. (B) Densitometry measurements of three independent experiments. p-values were determined with a 2-tailed t-test; * P≤ 0.05. Error bars indicate standard deviations of triplicate samples.
Figure 8.
Figure 8.
PI3K inhibition blunts the induction of Hmox1 but does not effect FHIT mediated repression of Hmox1. Treatment with LY294002 inhibits phosphorylation of AKT and blunts induction of Hmox1 in response to CSE. Hmox1 remains superinduced in FHIT knockdown cells.
Figure 9.
Figure 9.
NFkB inhibition blunts the induction of Hmox1 but does not effect FHIT-mediated repression of Hmox1. SC-514 is a selective inhibitor of IKK2 and blocks NF-kB dependent gene expression. HBEC3-TK cells were treated with control or FHIT siRNA for 48 hours. Cells were pretreated with 15 μM SC-514, 30 μM SC-514, or DMSO vehicle for 2 hours. Cells were then treated with 1% CSE supplemented with DMSO vehicle or indicated concentrations of SC-514. Western blot was used to detect levels of Hmox1 in the samples. While IKK2 inhibition blunts the induction of Hmox1, the protein is still superinduced in FHIT knockdown cells.
Figure 10.
Figure 10.
Calcium chelation does not affect FHIT-mediated repression of HMOX1. HBEC3-TK cells were treated with control or FHIT siRNA for 48 hours. Cells were treated with DMSO vehicle, 10 μM BAPTA-AM, or 5 μM BAPTA-AM for 30 minutes. Cells were then treated with 1% CSE for 4.5 hours. qRT-PCR was used to detect HMOX1 expression in the cells. While BAPTA-AM blunts the induction of HMOX1 in response to CSE, HMOX1 expression is still superinduced by FHIT knockdown.
Figure 11.
Figure 11.
FHIT-mediated repression of HMOX1 does not require Nrf2. HBEC3-TK cells were transfected with control, FHIT, or NFE2L2 siRNAs. After transfection, cells were either treated for 4 hours with 1% CSE or left untreated. (A) NFE2L2 (Nrf2) expression was quantified by qRT-PCR. FHIT knockdown does not effect NFE2L2 mRNA levels before or after CSE treatment. The data represent the results of three independent experiments. (B) Nrf2 protein abundance was assessed by protein gel blot. Nrf2 migrates at the expected molecular weight in both CSE and non-CSE treated samples. A slow migrating species of Nrf2 (∼ 75 kD) appears in CSE treated cells. Treatment with NFE2L2 siRNA confirms both isoforms as Nrf2. FHIT knockdown has no effect on Nrf2 protein abundance. (C) RNA was collected from cells transfected with control, FHIT siRNA, or NFE2L2 siRNA alone, or FHIT and NFE2L2 siRNA together. Knockdown of NFE2L2 blunts the induction of HMOX1 after CSE exposure. Concurrent knockdown of FHIT and NFE2L2 yields a superinduction of HMOX1 to a magnitude similar to that when only FHIT is depleted. Data represent the results of three independent experiments.
Figure 12.
Figure 12.
FHIT loss is associated with reduced Bach1 protein levels. HBEC3-TK cells were transfected with control or FHIT-targeting siRNAs. After transfection, cells were either treated with 1% CSE for 4 hours or left untreated. (A) BACH1 mRNA was quantified with qRT-PCR. FHIT knockdown does not significantly affect BACH1 mRNA levels before or after CSE treatment. Data represent the results of three independent experiments. (B) Bach1 protein abundance was assessed by western blot. Bach1 protein levels are not affected by FHIT knockdown in the absence of CSE. In CSE treated cells, Bach1 migrates as 2 species. A fast migrating species exists at the expected molecular weight (92 kD), and a slow migrating species exists at a higher molecular weight (∼ 150 kD). Total Bach1 protein levels are reduced in FHIT knockdown CSE-treated cells. Western data represent the results of 4 independent experiments (C) BACH1 siRNA treatment confirms both bands to be Bach1. (D) Relative protein quantification. Error bars represent mean +/− SEM of results from three independent experiments; ***P < 0.001.
Figure 13.
Figure 13.
Fhit and Bach1 expression levels are inversely correlated. (A) HBEC3-TK cells were transfected with control or BACH1 siRNA. After transfection, cells were treated with 1% CSE for 4 hours. Protein abundance was measured by western blot. Hmox1 and Fhit protein levels are inversely correlated with Bach1 protein abundance. (B) HBEC3-TK cells were transfected with control, BACH1, or FHIT siRNA. FHIT expression was measured by qRT-PCR. BACH1 knockdown increases FHIT expression approximately 2-fold. Data represent the results of three independent experiments. Error bars represent standard deviations.
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