Genetic ablation of Bcl-x attenuates invasiveness without affecting apoptosis or tumor growth in a mouse model of pancreatic neuroendocrine cancer

Abstract

Tumor cell death is modulated by an intrinsic cell death pathway controlled by the pro- and anti-apoptotic members of the Bcl-2 family. Up-regulation of anti-apoptotic Bcl-2 family members has been shown to suppress cell death in pre-clinical models of human cancer and is implicated in human tumor progression. Previous gain-of-function studies in the RIP1-Tag2 model of pancreatic islet carcinogenesis, involving uniform or focal/temporal over-expression of Bcl-x(L), demonstrated accelerated tumor formation and growth. To specifically assess the role of endogenous Bcl-x in regulating apoptosis and tumor progression in this model, we engineered a pancreatic beta-cell-specific knockout of both alleles of Bcl-x using the Cre-LoxP system of homologous recombination. Surprisingly, there was no appreciable effect on tumor cell apoptosis rates or on tumor growth in the Bcl-x knockout mice. Other anti-apoptotic Bcl-2 family members were expressed but not substantively altered at the mRNA level in the Bcl-x-null tumors, suggestive of redundancy without compensatory transcriptional up-regulation. Interestingly, the incidence of invasive carcinomas was reduced, and tumor cells lacking Bcl-x were impaired in invasion in a two-chamber trans-well assay under conditions mimicking hypoxia. Thus, while the function of Bcl-x in suppressing apoptosis and thereby promoting tumor growth is evidently redundant, genetic ablation implicates Bcl-x in selectively facilitating invasion, consistent with a recent report documenting a pro-invasive capability of Bcl-x(L) upon exogenous over-expression.

Figure 1. Bcl-x expression during RIP1-Tag2 tumor progression.

Protein extracts from pools of normal, non-transgenic islets (N), hyperplastic islets (H), angiogenic islets (A), and tumors (T) from RIP1-Tag2 mice were immunoblotted with antibodies to Bcl-x and β-actin as loading control. The 27 kDa band represents Bcl-x_L; the faster migrating band present in angiogenic islet and tumor extracts may represent the alternative splice variant, Bcl-x_β.

Figure 2. RIP-Cre Bclx^fl/fl mice exhibit normal islet morphology and efficient Bcl-x recombination.

(A) H&E stained pancreatic sections from 12 week old mice of the following genotypes: Bcl-x^+/+; RIP-Cre;Bcl-x^+/+; and RIP-Cre;Bcl-x^fl/fll. Multiple sections of each of the animals depicted in photomicrographs were examined. (B) Genomic DNA from pools of pancreatic islets was isolated from mice of distinct genotypes. PCR was carried out using a primer that binds upstream of the 5′ loxP site and another primer that binds 3′ to the downstream loxP site. The PCR fragment of the non-recombined allele and the recombined allele are 2.9 kb and 150 bp, respectively. Pancreatic islets were isolated (Materials and Methods) from 4–6 animals of each genotype and pooled.

Figure 3. RIP1-Tag2 tumorigenesis is not dependent on Bcl-x function.

(A and B) Comparison of tumor volume and number. Macroscopically visible tumors from 13 week old animals were excised, attached exocrine tissue removed and measured with ruler. Tumor volume was calculated using the formula V = (x * y²)×0.52. Data represent Mean±SEM; +/+ and FL/+: n = 7; FL/FL: n = 24; RIP-Cre;Fl/Fl: n = 22. Statistical significance was determined by non-parametric T-test (Mann-Whitney). P = 0.98 (D) Apoptotic rate. Apoptotic cells were quantified via TUNEL labeling of pancreatic sections from 13 week old animals. Data represent Mean±SEM (n = 5 mice/group). The percent TUNEL⁺ cells was calculated from sections of 16–17 individual tumors/group. Statistical significance determined by non-parametric T-test (Mann-Whitney). P = 0.98.

Figure 4. Bcl-x expression is ablated in the majority of RIP1-Tag2; RIP-Cre; Bcl-x^fl/fl tumors.

(A) Efficient recombination of Bcl-x in RIP1-Tag2; RIP-Cre; Bcl-x^fl/fl tumors. Genomic DNA was isolated from individual tumors from RIP1-Tag2; RIP-Cre; Bcl-x^fl/fl and RIP1-Tag2; Bcl-x^{fl/f l} mice. PCR was carried out using a primer that binds upstream of the 5′ loxP site and another primer that binds 3′ to the downstream loxP site. The PCR fragment of the non-recombined allele and the recombined allele are 2.9 kb and 150 bp, respectively. (B) Bcl-x expression is significantly reduced in Bclx-KO tumors. Quantitative RT-PCR (Taqman) was carried out on 1^st strand cDNA synthesized from RNA isolated from individual tumors from RIP1-Tag2; RIP-Cre; Bcl-x^fl/fl and RIP1-Tag2; Bcl-x^fl/fl. A spanning probe that detected all major anti-apoptotic splice variants was used. Gene expression was normalized to and plotted as a function of GAPDH expression. (D) The RIP-Cre transgene is not expressed in RIP1-Tag2; RIP-Cre; Bcl-x^fl/fl tumors that retain Bcl-x expression. Cre mRNA expression was monitored by RT-PCR on 1^st strand cDNA synthesized from RNA isolated from individual tumors from RIP1-Tag2; RIP-Cre; Bcl-x^fl/fl and RIP1-Tag2; Bcl-x^fl/fl mice. Following 27 cycles of PCR, the cDNA was fractionated on agarose gels and visualized by ethidium bromide staining. The PCR product is 520 bp. *non-recombined, Cre-negative tumor. (D) Bcl-x protein levels are significantly reduced in Bcl-x-KO tumors, as revealed by Western blotting as in Figure 1. β-actin is used as a loading control.

Figure 5. Expression of anti-apoptotic Bcl-2 family members in the stages of islet tumor development.

The mRNA levels of the pro-survival Bcl-2 family members Bcl-x, Bcl-2, Bcl-w, A1, Mcl-1 and Boo were assessed using quantitative RT-PCR (Taqman) on first strand cDNA synthesized from total RNA pools (4–6 animals per pool) of normal, non-transgenic islets, and of hyperplastic islets, angiogenic islets, and tumors from RIP1-Tag2 mice. Gene expression was normalized to and plotted as a percentage of GAPDH expression.

Figure 6. Loss of Bcl-x does not elicit transcriptional up-regulation of other anti-apoptotic Bcl-2 family members.

The mRNA levels of Bcl-x, Bcl-2, Bcl-w, A1, Mcl-1 and Boo were assessed using quantitative RT-PCR (Taqman) on first strand cDNA synthesized from total RNA isolated from individual tumors from RIP1-Tag2; RIP-Cre; Bcl-x^fl/fl (n = 9 tumors from 5 mice; 1 tumor found to not express Cre and exhibit wild-type levels of Bcl-x excluded from these data) and RIP1-Tag2; Bcl-x^fl/fl mice (n = 10 tumors from 5 individual mice). Relative gene expression was normalized to and presented as a percentage of GAPDH expression. Data represents mean±SEM. Statistical significance determined by non-parametric T-test (Mann-Whitney). P<0.05 considered significant.

Figure 7. Loss of Bcl-x expression results in an altered proportion of adenomas and carcinomas.

Tumors in H&E-stained sections from 13 wk RIP1-Tag2; Bcl-x^fl/fl (n = 46 tumors from 5 mice) and RIP1-Tag2; RIP-Cre; Bcl-x^fl/fl mice (n = 35 tumors from 5 mice) were scored as either non-invasive islet tumors/adenomas (IT) or invasive carcinomas (either IC1 or IC2) and the proportion of tumors in each class was calculated. Distribution of tumors types was compared between the two groups using Fisher's exact test. p = 0.017.

Figure 8. Effect of Bcl-x gene knockout on in vitro migration and invasion of tumor cells.

βTC-Bcl-x^fl/fl tumor cells were infected with adenovirus expressing Cre or control GFP and plated in triplicate in the presence or absence of cobalt chloride to assess the effect of reduced Bcl-x expression on cell migration and invasion. (A) Western blot of Bcl-x and actin protein levels in βTC-Bcl-x ^fl/fl cells treated +/−Cre at time of migration/invasion assays. (B) Cell growth. 2×10⁵ βTC-Bcl-x^fl/fl cells +/−cre were plated in the presence or absence of CoCl₂ and cells were counted after 48 hours. (C and D) Cell migration and invasion assays. Cells were plated in control trans-well inserts (C) or Matrigel-coated inserts (D). For the migration assay, results are plotted as the mean number of migrating cells/10× field (8 fields/membrane counted), while for the invasion assays the total number of invading cells/membrane are plotted. CoCl₂ treatment resulted in a significant decrease in invasion of βTC-Bcl-x-KO cells compared to wild-type cells (P = 0.005). Results shown represent the mean±SEM of results from three independent experiments performed in triplicate and are corrected for changes in total cell number (from B) compared to control cells (-Cre, -CoCl₂).