PALB2-mutated human mammary cells display a broad spectrum of morphological and functional abnormalities induced by increased TGFβ signaling

Heterozygous mutations in any of three major genes, BRCA1, BRCA2 and PALB2, are associated with high-risk hereditary breast cancer susceptibility frequently seen as familial disease clustering. PALB2 is a key interaction partner and regulator of several vital cellular activities of BRCA1 and BRCA2, and is thus required for DNA damage repair and alleviation of replicative and oxidative stress. Little is however known about how PALB2-deficiency affects cell function beyond that, especially in the three-dimensional setting, and also about its role during early steps of malignancy development. To answer these questions, we have generated biologically relevant MCF10A mammary epithelial cell lines with mutations that are comparable to certain clinically important PALB2 defects. We show in a non-cancerous background how both mono- and biallelically PALB2-mutated cells exhibit gross spontaneous DNA damage and mitotic aberrations. Furthermore, PALB2-deficiency disturbs three-dimensional spheroid morphology, increases the migrational capacity and invasiveness of the cells, and broadly alters their transcriptome profiles. TGFβ signaling and KRT14 expression are enhanced in PALB2-mutated cells and their inhibition and knock down, respectively, lead to partial restoration of cell functions. KRT14-positive cells are also more abundant with DNA damage than KRT14-negative cells. The obtained results indicate comprehensive cellular changes upon PALB2 mutations, even in the presence of half dosage of wild type PALB2 and demonstrate how PALB2 mutations may predispose their carriers to malignancy. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05183-6.

the used guide RNAs were also sequenced using primers listed below and no such off-target indels were detected.To identify PALB2 transcripts, complementary DNA (cDNA) from RNA (extracted with RNeasy Mini kit, Qiagen #74104) was generated using the iScript TM cDNA synthesis kit (Bio-Rad #1708891), and cDNA was PCR-amplified followed by partial purification and sequencing of the amplicons.The used PALB2 primer pairs reached from the first to seventh exon and are listed below.

Quantification of senescence cells
Proportion of senescent cells was quantified using Senescence β-Galactosidase Activity Assay Kit (Cell Signaling Technology #35302) using low-passage parental MCF10A cells as a negative/low-senescence control.Senescent cells were sorted in BD FACSAriaIIIu according to size, granularity, and β-galactosidase activity of the cells (ie., FSC high /SSC high /C12FDG high cells) as described in [9].Results were analysed with FlowJo program package (BD Biosciences, RRID:SCR:008520), and C12FDG-treated cells were imaged using Zoe fluorescent cell imaginer (BioRad).

qRT-PCR
To confirm transcriptome sequencing results and to compare gene expression in all PALB2-compromised cell lines, independent spheroids were grown, and RNA was extracted and quality-checked as for transcriptome sequencing.RNA integrity numbers of the samples ranged from 9.3 to 9.8.Transcripts of 21 selected genes were quantified with qRT-PCR using customized RT 2 Profiler PCR arrays (Qiagen #330171).The selected genes consisted of up-and downregulated genes, expression of which was altered both in #BiAll-93 and #MonoAll-2.13 cells or only in #BiAll-93 cells according to the transcriptome data.These genes also included scarcely and abundantly expressed genes and DEGs with moderate to very high expression difference between the PALB2-compromised and their control cell lines.To compare KRT14 expression between DMSO-and LY2109761-treated, esiRNA-eGFP-and esiRNA-KRT14-treated, as well as 2D-and 3D-grown cells, RNA was extracted in the same manner as for customized arrays, and RT 2 qPCR Primer Assay for human KRT14 (Qiagen #330001: NM_000526) was used for qRT-PCR.To measure the efficiency of PALB2 knock down RNA was isolated from the cells grown as a monolayer and RT 2 qPCR Primer Assay for human PALB2 (Qiagen #330001: NM_024675) was used.
For RT 2 Profiler PCR arrays, 0.5 -0.8 µg RNA was converted to cDNA using RT 2 First Strand kit (Qiagen #330404) and diluted following the manufacturer's RT 2 Profiler instructions.For KRT14 expression analysis, 0.5 µg RNA was used for cDNA synthesis using the same kit, and the synthesis reaction was diluted to 1:2.5.The RT The proteins were then transferred to PVDF membrane with the Trans-Blot Turbo transfer system (Bio-Rad #1704156).
The membranes were blocked with Blotto B (1% BSA and 1% non-fat milk) and incubated with a primary antibody over night at +4 °C.The membranes were washed with TBS buffer, pH 7.5, containing 0.05% Tween® 20 (Sigma-Aldrich #93773) followed by secondary antibody incubation for 1 h at room temperature.The signal was detected using SuperSignal™ West Pico or Femto chemiluminescent substrate (Thermo Scientific™ #34580 and #34095) and imaged with the Fujifilm LAS-3000 or Azure 600 (Azure Biosystems) gel documentation imaging systems.Band intensities were quantified using Azure Spot (Microsoft, RRID:SCR_011880) software.Amount of protein in each band was quantified against total protein on the lane.Sharpness, brightness, and contrast of the images may have been modified up to ± 10% to improve visibility, but it has not affected quantification of samples.The used antibodies and stains are listed below.

Immunocytochemistry
Monolayer cells were grown overnight on poly-L-lysine-coated Nunc TM Lab-Tek™ 8-well chamber slides (Thermo Scientific™ #154534) and were then treated with 2 µM etoposide (Pfizer #391870) for 5 or 10 h when applicable, fixed with 4% PFA, permeabilized with 0.5% Triton X-100 and blocked with a buffer containing 1% BSA, 0.2% Tween® 20 and 0.3 M glycine in PBS.The samples were first incubated with primary antibodies overnight at +4 °C and then with fluorophore-conjugated secondary antibodies for 1 h at room temperature.Nuclei were stained with mounting medium containing DAPI.
Spheroids grown on top of GFR-BME (Corning ® Matrigel ® #354230) -coated, Nunc TM Lab-Tek™ 16-well chamber slides (Thermo Scientific™ #178599) for 8 days were fixed with 2% PFA.For immunostaining of nucleus-and cytosollocated proteins, the spheroids were permeabilized with 0.5% Triton X-100.The spheroids were blocked with primary blocking buffer containing 0.1 M glycine, 0.1% BSA and 10% goat serum and secondary blocking buffer also containing 10 µg/ml goat anti-mouse F(ab')2 fragment [10].The spheroids were then incubated with primary antibodies and with F-actin stain Alexa Fluor 488 Phalloidin at room temperature, followed by PBS-0.1% BSA washes and incubation with fluorophore-conjugated secondary antibodies at room temperature.Finally, the spheroids were further washed and nuclei in non-permeabilized and permeabilized cells were stained with Hoechst® 33342 and mounting medium containing DAPI, respectively.Each staining was repeated at least three times and at least six images of each sample were randomly captured with the same settings for analyses.The used antibodies and stains are listed below.
Antibodies and stains used in 2D and 3D immunocytochemistry and Western blotting.**NS, not significant -p-and q-value is >0.05 for the fold change in RT-qPCR and RNAseq, respectively.***Numerical values for fold changes are not directly comparable between the methods since their sensitivity differs.

Primary antibodies
off-target areas (*) for the guide RNAs used and primers used to PCR-//crispr.mit.edu/guides/ and https://www.deskgen.comN/A; potential off-target sequences with less than 4 mismatches and 10 b-long intact 3' area, were not found Primers used to PCR-amplify and sequence
2Profiler PCR arrays and RT2Primer Assays were analyzed using RT² SYBR® Green qPCR Mastermix (Qiagen #330504) in CFX96 instrument (Bio-Rad, RRID:SCR_018064).The fold changes based on arithmetic mean and p-values based on a Student's t-test of the replicate 2 ^(-Delta CT) values for each gene were calculated with the Qiagen online RT 2 profiler PCR Data Analysis tool (https://dataanalysis.qiagen.com/pcr/arrayanalysis.php) using GAPDH and B2M as reference genes.qPCRcut off value for customized arrays was set to either 37 or 35, depending on using 0.5 µg or 0.8 µg of RNA.Cut off 35 was used for KRT14 Primer Assays.Cells were lysed into NETN-300 buffer (20 mM Tris pH 7.5, 300 mM NaCl, 1 mM EDTA, and 0.5% NP-40) containing cOmplete™ Mini EDTA-free protease inhibitor tablets (Roche Diagnostics #11836170001) and Phosphatase Inhibitor Cocktail Set V (Merck #524629), and protein concentrations were then measured with the Pierce BCA Protein Assay Kit (Thermo Scientific™ #23227).The cell lysates were denatured in Laemmli buffer containing DTT and equal amounts of protein were separated by SDS-PAGE using 4-15% Mini-PROTEAN® TGX™ gels (Bio-Rad #4561083).