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PTEN Loss in E-Cadherin-Deficient Mouse Mammary Epithelial Cells Rescues Apoptosis and Results in Development of Classical Invasive Lobular Carcinoma

Summary Invasive lobular carcinoma (ILC) is an aggressive breast cancer subtype with poor response to chemotherapy. Besides loss of E-cadherin, a hallmark of ILC, genetic inactivation of PTEN is frequently observed in patients. Through concomitant Cre-mediated inactivation of E-cadherin and PTEN in mammary epithelium, we generated a mouse model of classical ILC (CLC), the main histological ILC subtype. While loss of E-cadherin induced cell dissemination and apoptosis, additional PTEN inactivation promoted cell survival and rapid formation of invasive mammary tumors that recapitulate the histological and molecular features, estrogen receptor (ER) status, growth kinetics, metastatic behavior, and tumor microenvironment of human CLC. Combined inactivation of E-cadherin and PTEN is sufficient to cause CLC development. These CLCs showed significant tumor regression upon BEZ235-mediated inhibition of PI3K signaling. In summary, this mouse model provides important insights into CLC development and suggests inhibition of phosphatidylinositol 3-kinase (PI3K) signaling as a potential therapeutic strategy for targeting CLC.


Adenoviral delivery of Cre recombinase
Before embedding in Matrigel, Adcre was added to isolated mammary organoids to drive Cre-recominase activity during subsequent organoid development in matrigel (1×10 8 Transducing Units (TU); Gene Transfer Vector Core, University of Iowa). Seven days later, DNA was isolated using direct PCR lysis buffer (301-C; Viagen) to analyze Cre-driven recombination efficiency, and protein was isolated using NP40 lysis buffer (20 mM Tris pH 7.4, 100 mM NaCl, 1% NP40, 10% glycerol, 10 mM EDTA complemented with protease inhibitors (Roche) to analyze inactivation of E-cadherin and/or PTEN.

Histopathology
Mouse tissues were formalin-fixed in 10% neutral buffered formalin for 48 hours, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) or Masson's trichrome. IHC was performed as previously described (Henneman et al., 2015). Primary antibody details and antigen retrieval methods are described in Supplemental information Table S1. Secondary antibodies that were used are HRP anti-rabbit Envision (DAKO K4011), Biotin anti-rabbit (DAKO E0432), and Biotin anti-rat (Santa Cruz sc-2041). All slides were digitally processed using the Aperio ScanScope (Aperio, Vista, CA, USA) and captured using ImageScope software version 12.0.0 (Aperio).

RNA sequencing
Total tumor tissues were homogenized in TRIzol reagent (15596-018, Ambion life technologies) using a polytron (DI 18 Disperser, IKA) according to the manufactures protocol. Briefly, 0.2x volumes of chloroform (Chloroform stab./Amylene, Biosolve) was added to the homogenate and the tube(s) were shaken vigorously. The tube(s) were incubated for 2-3 minutes at room temperature and centrifuged (Hettich, rotanta 46 RS) for 1 hour (4120 RCF, 4 °C) . Approximately 70% of the upper aqueous phase was transferred to a clean 15 mL tube and 0.5x volume of isopropanol (33539, Sigma-Aldrich, ) was added. The tube(s) were incubated overnight at -20 °C and centrifuged for 30 minutes (4120 RCF, 4 °C). The supernatant was removed and the pellet was washed twice with 80% ethanol (32221-2.5L, Sigma-Aldrich). The total RNA pellet was air-dried and dissolved in an appropriate volume of nuclease free water (AM9937, Ambion life technologies) and quantified using Nanodrop UV-VIS Spectrophotometer (Thermo Fisher Scientific Inc.). Total RNA was purified using the MinElute Cleanup Kit (74204, Qiagen) according to the manufactures instructions. Quality and quantity of the total RNA was assessed by the 2100 Bioanalyzer using a Nano chip (Agilent, Santa Clara, CA). Total RNA samples having RIN>8 were subjected to library generation.
Illumina TruSeq mRNA libraries were generated using the TruSeq RNA Library Preparation Kit v2 sample preparation kit (Illumina Inc., San Diego, Cat.No RS-122-2001/2) according to the manufacturer's instruction (Part # 15026495 Rev. B) Briefly, polyadenylated RNA from intact total RNA was purified using oligo-dT beads. Following purification the RNA was fragmented, random primed and reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen, part # 18064-014). Second strand synthesis was accomplished by using Polymerase I and RNaseH. The generated cDNA fragments were 3' end adenylated and ligated to Illumina Pairedend sequencing adapters and subsequently amplified by 15 cycles of PCR. The libraries were analyzed on a 2100 Bioanalyzer using a 7500 chip (Agilent, Santa Clara, CA), diluted and pooled equimolar into a 10nM sequencing pool containing 8-10 samples each. The libraries were sequenced with 50 base single reads on a HiSeq2000 using V3 chemistry (Illumina Inc., San Diego). The obtained data will available for future analysis at www.xxx.org.

Generating PAM50 signature of mCLC
In order to investigate molecular similarities between mCLC from Wcre;Cdh1 F/F ;Pten F/F mice and human breast cancer samples, the TCGA BRCA dataset was integrated with RNAseq data of mouse mammary tumor samples from Wcre;Cdh1 F/F ;Pten F/F and K14cre;Brca1 F/F ;Trp53 F/F mice. Gene expression profiles for each sample were collected from RNAseq data normalized by TMM (Robinson and Oshlack, 2010). Batch effects were corrected using ComBat (Johnson et al., 2007). Unsupervised hierarchical clustering was performed using genes from the PAM50 signature (Parker et al., 2009).

Whole-exome sequencing
Due to high stromal contamination in mCLC, the tumor cell population was enriched through FACS-sorting based on expression of epithelial marker EpCAM. Tumor tissues of Wcre;Cdh1 F/F ;Pten F/F mice were processed as described above. Single cells were stained with directly conjugated EpCAM-FITC (eBioscience 11-5791) for 30 min at 4 °C in the dark in PBS/1% BSA. DAPI (1:20) was added to exclude dead cells. EpCAM-positive cells were collected using a BD FACSAria IIu flow cytometer. Genomic DNA was isolated from this cell population using QIAamp DNA micro kit (Qiagen) according to manufacturer's instructions. For exponentially growing non-CLC tumor tissue, genomic DNA was isolated using proteinase K lysis and phenol-chloroform organic extraction. For whole-exome sequencing, up to 2 ug of genomic DNA samples was sheared to a length between the 200-300 bp using the covaris S2 system. The sheared DNA samples were cleaned using 1.8x of the Agencourt Ampure XP beads (part.no A63881). The DNA library preparation was performed with the Kapa HTP DNA library preparation kit (KK8234), according to protocol. Input library prep sample of FACS-sorted CLC tumor samples was 5-150 ng dsDNA; Input library prep sample of non-CLCs was 1000 ng dsDNA. The enrichment assay was performed using the SureSelect XT2 Mouse All Exon kit (part no. 5190-4681). One adjustment to the hybridization protocol was made, we used a 1:1 diluted Mouse All Exon capture library. The obtained data will available for future analysis at www.xxx.org

Extraction of mutations from whole-exome sequencing data
Sequence reads from the exome capture were mapped to the mouse reference genome (mm10), using BWA (Li and Durbin, 2009). The mapped sequence reads were further processed according to GATK best practices using Picard (version 1.128) and GATK (version 3.4) (Van der Auwera et al., 2013) to perform duplicate marking, indel realignment and base quality score recalibration. SNPs and indels were called from these processed alignments using HaplotypeCaller (version 3.4.0). Variants were filtered to remove calls from sequencing artefacts using GATKs recommended practice for hard-filtering of variant calls. To remove variants that are part of the mouse background, we filtered variants that were known FVB variants (Wong et al., 2012) and variants that occurred with a variant allele frequency greater than 0.1 in at least one sample in a panel of four normal (spleen) samples. The remaining variants were then annotated using VEP and filtered to select only deleterious variants (missense variants, frame shift variants, stop gained/lost variants and UTR variants). To prioritize cancer-related variants, these variants were annotated for their presence in the Cancer Gene Census (Futreal et al., 2004).

CNV analysis
We used the CopywriteR tool (Kuilman et al., 2015) to obtain copy number profiles from the exome alignments, which effectively extracts off-target reads from the exome capture and uses these to estimate copy number profiles. The log2 copy number estimates from CopywriteR were smoothed and segmented using the circular binary segmentation (CBS) algorithm as implemented in the DNACopy R package (Venkatraman and Olshen, 2007). In this segmentation, segments that were smaller than 5 probes or that deviated less than 3 standard deviations were pruned to reduce the number of spurious segments. For each sample, we then centered the (log2) copy number estimates of these segments by subtracting the segment copy number value in each sample. Using these centered values, we then determined the number of chromosomal aberrations in each sample as the number of segments with copy number values above 0.1 or below -0.1. To remove systematic artefacts from the copy number calling and/or segmentation, we did not count any segments that were present in more than 15 samples. Similarly, to further reduce the noise in the counts, we removed segments that spanned less than 10 probes, as these were considered more likely to represent noise than actual focal amplifications/deletions. Arrows highlight E-cadherin or PTEN proficient cells. Bar=25µm. (E) Representative IHC staining for CK8, Ecadherin and PTEN in mammary tumors from 12-week-old Wcre;Cdh1 F/F ;Pten F/+ mice. The arrow highlights CK8marked luminal cells expressing E-cadherin and PTEN in the mammary duct, in contrast to CK8-marked tumor cells in the adjacent lesion, which lost E-cadherin and PTEN protein expression. Bar=100µm. (F) Esr1 mRNA expression levels extracted from RNA sequencing data of mammary tumors from K14cre;Brca1 F/F ;Trp53 F/F (n=22) and Wcre;Cdh1 F/F ;Pten F/F (n=20) mice and compared to FACS-sorted Epcam-positive tumor cells from Wcre;Cdh1 F/F ;Pten F/F mammary tumors (n=18). Figure S4. Characterization of mammary tumors from mice transplanted with precancerous Wcre;Cdh1 F/F ;Pten F/F mammary gland fragments (A) Representative images of IHC staining of CK8, E-cadherin and PTEN in tumors from mice transplanted with precancerous Wcre;Cdh1 F/F ;Pten F/F mammary gland fragments. Bar=100µm. (B) DNA copy-number profiles of linear-growing mCLCs (n=14) and exponential-growing solid ILCs and EMT tumors (n=7) from mice transplanted with precancerous Wcre;Cdh1 F/F ;Pten F/F mammary gland fragments.