A functional in vivo screen for regulators of tumor progression identifies HOXB2 as a regulator of tumor growth in breast cancer

Abstract

Microarray profiling in breast cancer patients has identified genes correlated with prognosis whose functions are unknown. The purpose of this study was to develop an in vivo assay for functionally screening regulators of tumor progression using a mouse model. Transductant shRNA cell lines were made in the MDA-MB-231 breast cancer line. A pooled population of 25 transductants was injected into the mammary fat pads and tail veins of mice to evaluate tumor growth, and experimental metastasis. The proportions of transductants were evaluated in the tumor and metastases using barcodes specific to each shRNA transductant. We characterized the homeobox 2 transcription factor as a negative regulator, decreasing tumor growth in MDA-MB-231, T47D, and MTLn3 mammary adenocarcinoma cell lines. Homeobox genes have been correlated with cancer patient prognosis and tumorigenesis. Here we use a novel in vivo shRNA screen to identify a new role for a homeobox gene in human mammary adenocarcinoma.

Figure 1. Experimental design of screen

The pool of transductants was injected into the right 4^th mammary fat pad of female SCID mice (Tumor Growth Assay) to screen for in vivo tumor growth regulators and into the tail vein of female SCID mice (Experimental Metastasis Assay) to screen for metastasis regulators. The pool was also grown in vitro in parallel and genomic DNA isolated at the same time point as the in vivo samples. For mammary fat pad injections, after 6 weeks the primary tumor was removed and tumor cells were dissociated and plated in puromycin containing medium to select for tumor cells. For tail vein injections, after 4 weeks the lungs were removed and lung metastases were dissociated and tumor cells plated and plated in puromycin containing medium to select for tumor cells. The parallel in vitro grown cells were also puromycin treated. Genomic DNA was isolated from puromycin resistant cultures, and specific barcode primers were used to evaluate the change in proportion of each of the individual transductants in the samples compared to the proportions in the initial injected pool sample.

Figure 2. In vivo screen for regulators of tumor growth and metastasis

Bars of potential growth/metastasis enhancers are black, potential growth/metastasis suppressors are white, controls (previously published positive enhancers of metastasis) are in a diagonal stripe pattern, and the non-targeting shRNA CT-1 is in a horizontal stripe pattern. (A.) Average fold change of shRNA transductants after growth in the primary tumor. Nine female SCID mice were injected in the mammary fat pad with 1×10⁶ cells of the initial pool. After 6 weeks tumors were removed, dissociated with collagenase, hyaluronidase, and DNase I and plated under puromycin selection. Genomic DNA was isolated from the cells and used to determine the average fold change in proportion to the initial pool using qRT-PCR with specific barcode primer sets. Means and SEM of results from primary tumors from 9 mice are plotted. (B.) Average fold change of shRNA transductants after seeding and growth in lung metastases. 1×10⁶ cells of the initial pool were injected in PBS into the lateral tail vein of female SCID mice. After 4 weeks, lungs were extracted and dissociated with collagenase, hyaluronidase, and DNase I and metastases plated under puromycin selection. Genomic DNA was isolated from the cells and used to determine the average fold change in proportion to the initial pool using qRT-PCR with specific barcode primer sets. Mean and SEM are plotted for results from 3 separate mice. (C.) Average fold change to injected pool proportions after in vitro growth. The initial pool was grown in vitro and split 1:5, 3 times per week over 6 weeks (two separate 6 week in vitro growth measurements were done of the initial pool, over the time period of the two separate in vivo tumor injections). Genomic DNA was isolated at 4 and 6 weeks, and used to determine the average fold change in proportion to the initial pool using qRT-PCR with specific barcode primer sets. The average was taken of the fold change from the 4 week, and two 6 week isolations. Means and SEM of the data are plotted.

Figure 3. Relationships between transductants with significant changes in representation

For genes that were significantly altered in the screen, the effect of knockdown on transductant representation in the pool is indicated by arrows (down indicates reduced representation and up indicates increased representation). The genes are organized according to the selection under which their representation is altered.

Figure 4. The CBFB shRNA cell line is not altered in lung metastasis

The MDA-MB-231 parental cell line, SERPINE1-A, and CBFB-C shRNA cell lines were evaluated for seeding and growth in the lungs after tail vein injection. 2.5×10⁵ cells were injected into the lateral tail vein of female SCID mice for each cell line. After 3 weeks lungs were formalin fixed, sectioned and H&E stained, and the number of lung metastases was counted. Mean and SEM are plotted for measurements from 5 mice for each cell line. There were no significant differences.

Figure 5. HOXB2 negatively regulates tumor growth

(A.) HOXB2-A shRNA cell line tumors grew faster than the control SERPINE1-A pGipz cell line. 1×10⁶ cells were injected into the mammary fat pads of female SCID mice. Two separate injections of 5 and/or 4 mice were performed for each cell line. Tumors were measured and volumes calculated in mm³ every 3 days for 45 days. Data represent the mean and SEM of 10 mice for the SERPINE1-A shRNA transductant line and 9 mice for the HOXB2-A shRNA transductant line. Stars represent p≤ 0.05 at the indicated time points. At 45 days, p≤ 0.000181. (B.) HOXB2 has no effect on in vitro growth over 96 hours. The HOXB2-ORF, JP1520 vector control, HOXB2-A shRNA, and SERPINE1-A cell lines were seeded in 6 well dishes at 50,000 cells per well and cells were counted in triplicate at 0 (first time point), 24, 48, 72, and 96 hours from the first time point. Relative growth was calculated as a ratio of the average cell number at each time point to the initial average cell number at the first time point. In vitro growth assays were done in triplicate and repeated three times. Data are mean and SEM. There were no significant differences. (C.) MDA-MB 231 HOXB2-ORF overexpression cell line tumors grew slower than the JP1520 vector control cell line. 1×10⁶ cells were injected into the mammary fat pads of 10 female SCID mice, (two separate injections of 5 mice each, for each cell line). Tumors were measured and volumes calculated in mm³ every 3 days for 45 days. Data represent the mean and SEM. Stars represent p≤ 0.05. At 45 days, p≤ 0.00884. (D.) HOXB2 overexpression in MTLn3 rat adenocarcinoma cells decreased tumor growth. 5 × 10⁵ cells were injected into the mammary fat pads of 5 female SCID mice for each cell line. Tumors were measured and volume was calculated over 30 days. Data shown are mean and SEM, stars represent p values <.05. At 31 days p value = 0.000328049.

Figure 6. HOXB2 affects the mitotic index of MDA-MB 231 tumors

Mitotic figures were counted in H&E tumor sections using a 40x objective in 10 random fields. N=5 tumors for each cell line. For the shRNA lines: SERPINE1-A control vs HOXB2-A p value = 0.015648318. For the overexpressor lines: JP1520 empty vector control vs. HOXB2 ORF p value = 0.00537099. Scale bar = 25um.