Patient-derived xenografts of triple-negative breast cancer reproduce molecular features of patient tumors and respond to mTOR inhibition

Abstract

Introduction: Triple-negative breast cancer (TNBC) is aggressive and lacks targeted therapies. Phosphatidylinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways are frequently activated in TNBC patient tumors at the genome, gene expression and protein levels, and mTOR inhibitors have been shown to inhibit growth in TNBC cell lines. We describe a panel of patient-derived xenografts representing multiple TNBC subtypes and use them to test preclinical drug efficacy of two mTOR inhibitors, sirolimus (rapamycin) and temsirolimus (CCI-779).

Methods: We generated a panel of seven patient-derived orthotopic xenografts from six primary TNBC tumors and one metastasis. Patient tumors and corresponding xenografts were compared by histology, immunohistochemistry, array comparative genomic hybridization (aCGH) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) sequencing; TNBC subtypes were determined. Using a previously published logistic regression approach, we generated a rapamycin response signature from Connectivity Map gene expression data and used it to predict rapamycin sensitivity in 1,401 human breast cancers of different intrinsic subtypes, prompting in vivo testing of mTOR inhibitors and doxorubicin in our TNBC xenografts.

Results: Patient-derived xenografts recapitulated histology, biomarker expression and global genomic features of patient tumors. Two primary tumors had PIK3CA coding mutations, and five of six primary tumors showed flanking intron single nucleotide polymorphisms (SNPs) with conservation of sequence variations between primary tumors and xenografts, even on subsequent xenograft passages. Gene expression profiling showed that our models represent at least four of six TNBC subtypes. The rapamycin response signature predicted sensitivity for 94% of basal-like breast cancers in a large dataset. Drug testing of mTOR inhibitors in our xenografts showed 77 to 99% growth inhibition, significantly more than doxorubicin; protein phosphorylation studies indicated constitutive activation of the mTOR pathway that decreased with treatment. However, no tumor was completely eradicated.

Conclusions: A panel of patient-derived xenograft models covering a spectrum of TNBC subtypes was generated that histologically and genomically matched original patient tumors. Consistent with in silico predictions, mTOR inhibitor testing in our TNBC xenografts showed significant tumor growth inhibition in all, suggesting that mTOR inhibitors can be effective in TNBC, but will require use with additional therapies, warranting investigation of optimal drug combinations.

Figure 1

Histology of patient TNBC samples and corresponding patient-derived orthotopic xenografts. A. H&E staining of patient tumors; B. H&E staining of the corresponding xenograft tumors; C. ER staining of xenograft tumors; D. PR staining of xenograft tumors; and E. HER2 staining of xenograft tumors. Pictures were taken with 200× magnification. The scale bar is 100 μm in length. ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; PR, progesterone receptor.

Figure 2

Array CGH profiling. Data for SUTI110 and SUTI151 show matching variations on chromosomes 5 and 14 for patient tumors and corresponding xenografts. Green represents loss and red represents gain for each probe aligned along the chromosome. Other chromosome profiles are provided in Figure S1 in Additional file 4. CGH, comparative genomic hybridization.

Figure 3

PIK3CA sequence variations. A. This is a table of PIK3CA exon mutations in patient and corresponding xenograft samples. The exon number, mRNA position and allele change, and protein position and residue change for each mutation are indicated. B. A sequencing image of patient and corresponding xenograft tumors of the SUTI097 and SUTI110 at the mutated sites. MDA-MB-231 cell line is shown as a normal control. C. Table of PIK3CA intron SNPs that flank sequenced exons in patient and corresponding xenograft samples. Note the conservation of sequence variations between primary tumors and their xenografts, and also between different xenograft passages, before and after rapamycin treatment. SNPs, single nucleotide polymorphisms.

Figure 4

Rapamycin response signature. A. Heatmap of rapamycin response signature gene expression of training set samples with 18 control samples on the left and 5 rapamycin treated samples on the right. Each row is a probe set in the signature. Red indicates up-regulation and blue indicates down-regulation of the gene. B. LOOCV from the Connectivity Map training set samples. On the y-axis, 0 = predicted as untreated; 1 = predicted as treated. Control samples are in blue, and rapamycin treated samples in red. Note that only one control sample was misclassified. C. Heatmap of predicted rapamycin response of 1,401 human breast tumors with a color scaled from red to blue indicating a high to low predicted sensitivity. Each column represents an individual tumor sample, grouped by intrinsic subtypes. D. The percent of samples with predicted rapamycin sensitivity of >0.5 for each intrinsic subtype. The background color represents the overall sensitivity of each subtype at the same scale used in 1C. LOOCV, leave-one-out cross-validation.

Figure 5

In vivo growth curves of seven patient-derived orthotopic xenografts of TNBC. Treatment with vehicle control in blue; doxorubicin in purple; rapamycin in red; CCI-779 in green. Tumor volumes in mm³. Each data point represents the mean tumor volume of each treatment group. Error bars represent standard error of the mean. CCI-779, temsirolimus; TNBC, triple-negative breast cancer.

Figure 6

Protein expression and phosphorylation of PTEN, mTOR, S6K1, 4EBP1, and eIF4E in xenografts. Western blot images were cropped at the molecular weight of each of the target proteins. Pretreatment samples were collected prior to initiation of treatment; control samples (vehicle control), rapamycin-treated and CCI-779-treated samples were collected at the end of the treatment period. Tubulin was used as a loading control. See also Table S5 in Additional file 7. CCI-779, temsirolimus.