Introduction
It has been postulated that the functional plasticity of the adult mammary epithelium, which is required for response to hormonal changes during pregnancy and lactation, may make the breast tissue more susceptible to malignancy.Identifying mechanisms of normal mammary epithelial cell (MEC) biology that contribute to tumorigenesis is critically important for furthering our understanding of breast cancer and extending the survival of breast cancer patients. Of particular interest are postpartum breast cancers (PPBCs), defined as breast cancers diagnosed in women within 10 years of most recent childbirth, as these cancers are nearly three times more likely to result in lethal metastasis .
The majority of mammary morphogenesis occurs postnatally beginning at puberty and culminating with lactation following pregnancy. As such, differentiation of the mammary epithelium is not complete until a full pregnancy-lactation cycle. Upon weaning, or after pregnancy in the absence of nursing, the mammary gland regresses to resemble the pre-pregnant architecture through a process termed postpartum involution. In mice, postpartum involution occurs in two primary phases . In the first phase, milk proteins accumulate in the mammary ducts and alveoli resulting in downstream phosphorylation and activation of signal transducer and activator of transcription (pSTAT3), and subsequent lysosomal-mediated cell death . In mice, if pups are returned within 48 h of weaning, this process can be reversed, and lactation can continue. If pups are not returned, however, the irreversible second phase of involution begins. This phase is characterized by degradation of the mammary basement membrane by matrix metalloproteinases and anoikis-mediated cell death in the absence of laminin-mediated activation of integrin alpha-6/beta-1 (α6β1) and downstream pro-survival signaling .
SEMA7A, or CD108w, is a member of the semaphorin protein family, named after the English verb "semaphore", which means to transmit a signal across a long distance. SEMA7A was first identified for its expression on lymphocytes , but has since been shown to exert diverse roles in neuronal development , cell differentiation , ECM deposition , various components of the immune system , and pathologies including fibrosis  and cancer . Though elevated SEMA7A is observed in breast cancer, where we have established a role for it in conferring cell survival, in part, via promotion of anoikis resistance , the role of SEMA7A in normal MECs has not previously been investigated. SEMA7A is known to signal through β1-integrin in immune cells, neurons, and cancer; β1-integrin also mediates MEC survival during development, in part, via phosphorylation and activation of downstream pro-survival signaling mediated by PI3-kinase and protein kinase B, or Akt1 . Though it is known that redundant secretory epithelial cells die during postpartum involution, the factor(s) that allow some cells to survive and regenerate a lactation-competent gland for future pregnancies remain unknown. Herein, we have identified enrichment for SEMA7A expressing α6β1 positive MECs during the matrix remodeling period of postpartum involution and a role for SEMA7A in specifically promoting the survival of α6-integrin + luminal progenitor cells. We provide evidence that exogenous stimulation of immortalized MECs with purified SEMA7A protein promotes anoikis resistance via activation of α6 and β1-integrins and activation of phosphorylated AKT (pAKT). Consistent with a role for SEMA7A in promoting anoikis resistance, SEMA7A knockout mice (SEMA7Atmalk1/J) exhibit phenotypes associated with accelerated involution including early presence of apoptotic cell bodies in mammary lumens, increased pSTAT3, and premature reappearance of adipocytes. α6-integrin also drives maintenance of tissue resident stem cells and therapy resistant cancer stem cells  and our findings reveal that SEMA7A drives stem cell phenotypes and chemoresistance in cultured MECs in a manner that is dependent on α6 and β1. Finally, we provide evidence for SEMA7A in driving transformed phenotypes in normal MECs and reversing transformed phenotypes in genetically identical, ras-transformed, variants of the same cell line. Our results are the first to identify a role for SEMA7A in anoikis resistance and promotion of stem and cancer cell phenotypes via activation of α6 and β1-integrins.
Methods
Cell culture
MCF10A/MCF12A cells were obtained from, and cultured, according to ATCC. MCF10DCIS.com cells were obtained from K. Polyak (University of Harvard, Cambridge, MA). Cells were validated by the DNA sequencing core at the University of Colorado Anschutz Medical Campus and found to be pure populations of their respective cell lines. Cells were regularly tested for mycoplasma throughout the studies. Cells were sub-cultured as previously described (reference), or according to ATCC standards. Exogenous SEMA7A protein utilized in our studies was purified from media of cells stably transfected with an Fc-tagged version of SEMA7A by published methods . Control plasmid (pcDNA3.1) was obtained from H. Ford (University of Colorado Anschutz Medical Campus, Denver, CO).
shRNA knockdown and overexpression
shRNA plasmids targeting Sema7a, and a negative control shRNA (SABiosciences), were amplified in E. coli and plasmid DNA was isolated by Plasmid Maxi-Prep (Qiagen). MCF10DCIS.com-GFP cells were cultured overnight to ~80% confluence. 1 μg/μL of each shRNA was added to Transfectagro Reduced-Serum Medium (Corning) and incubated for 15 min with 4 μL of X-treme Gene HP DNA transfection reagent (Roche). Transfected cells were selected for hygromycin resistance. Stable knockdown was confirmed by qPCR as shown in Black et al . Negative control cells were transfected with a scrambled artificial sequence not matching human, mouse, or rat. Overexpression plasmid (SEMA7A-Fc) was a generous gift from R. Medzhitov (Yale University, New Haven, CT). All other over-expression plasmids (p304-V5-Blasticidin and V5-SEMA7A)were obtained from the Functional Genomics Core at the CUAnschutz Medical Campus and overexpression was confirmed via qPCR and western blot analysis. KD  and overexpression  as previously described.
Animal studies
All animal procedures were approved by the University of Colorado Anschutz Medical Campus Institutional Animal Care and Use Committee. Sema7atm1Alk/J mice (a generous gift from Alex Kolodkin at John's Hopkins University) and C57Bl/6 (Jax) were housed and bred as previously described . Briefly, C57Bl/6 SEMA7Atm1Alk females were bred with WT C57Bl/6 males to induce pregnancy, lactation, and postpartum involution. Pup numbers were normalized to 6–8 per dam after birth to ensure for adequate lactation. Postpartum involution was initiated by forced weaning of pups at the peak of lactation (L10-L12). #4 right and left mammary glands were harvested from age-matched nulliparous animals and postpartum dams at involution days 1,2,4,6,8,10,14, and 28. For flow cytometry, inguinal lymph nodes were removed prior to tissue digestion. #3 mammary glands were also harvested, formalin-fixed, paraffin-embedded, and 5 μm sections generated for histologic/immunohistochemical analaysis. See Supplemental Table 1 for animal study sample sizes.
In vitro survival assays
MCF10A and MCF12A cells were cultured with 200 ng/ml purified SEMA7A or PBS vehicle control in adherent (tissue culture plastic) or forced suspension conditions (ultra-low attachment plates (Corning, Corning, NY, USA: #3473)). Cells were seeded at a density of 1000 cells/well in a 96-well plate plus media only wells as controls. Cell death was measured 24 h post seeding via luminescence using the Caspase Glo assay (Promega, Madison, WI, USA: #G8090). Annexin-V /7AAD staining (Biolegend, San Diego, CA, USA: #640930) and pAKTS473 (ThermoFisher: Waltham, MA, USA, #17-9715-42) were also used to confirm cell viability by flow cytometry. See flow cytometry methods for staining protocol. Function blocking antibodies for b1 (CD29- 9EG7; BD Biosciences: #550531) and a6 integrin (ThermoFisher: #14-0495-82) were used to disrupt integrin signaling in the presence of SEMA7A or as antibody alone controls to determine off-target effects. IgG controls were also used. 9EG7 was used at a concentration of 0.6 μg/ml while GoH3 was used at a concentration of 100 μg/mL for inhibition studies consistent with previous reports . Cells were cultured with anti-integrin inhibitors at time of seeding.
Label retention stem cell assay
MCF10A and MCF12A cells were labeled with CellTrace Violet (ThermoFisher: #C34557) for 20 min at 37 °C according to the manufacturer's instructions. A sample of labeled cells was analyzed by flow cytometry to determine labeling efficiency and fluorescent intensity at time of seeding (Supplemental Fig. 1) Single cells were seeded at a density of 4000 cells/well in ultra-low attachment 24-well plates to induce mammosphere formation. Cells were cultured in normal culture media for 7 days and were subsequently counted, dissociated enzymatically in Accutase, (Stem Cell Technologies, Vancouver, Canada: #07922), and analyzed by flow cytometry. See flow cytometry methods for detailed information.
Paclitaxel resistance studies
MCF10DCIS.com cells were treated with 100 nM paclitaxel (Millipore Sigma, Burling, MA, USA #T7402-5MG) for 24 h prior to analysis by flow cytometry. Dose was determined experimentally based on cell viability and cancer stem cell enrichment. Drug resistance was measured by label-retention and mammosphere formation as described above and %CD44+ (Biolegend: #103018) CD24- (Biolegend: #311104) of singlets was used as a molecular definition of drug-resistant stem cells. See label retention stem cell assay and flow cytometry methods for additional details.
Flow cytometry
Single cell suspensions were generated from cultured cells via enzymatic harvest with Accutase. Mouse mammary tissues were minced with scalpels, digested in Click's media containing 500 units/mL Collagenase II (Worthington, Columbus, OH, USA; LS004174) and Collagenase IV (Worthington, LS004186) and 20 μg/mL DNAse (Worthington, LS002004) for 1 h at 37 °C with occasional trituration and strained through 70um filters to generate single cell suspensions. Single cells were stained at 4 °C for 30 min and washed with PBS + 2%FBS. Cells were filtered through 30 μm filters, diluted to <106 cells/ml, and analyzed with the ZE5-YETI flow cytometer. Fixation/Permeabilization kit (BD Biosciences, Franklin Lakes, NJ, USA: #554715) was used for intracellular antigen staining. Data were analyzed with Kaluza software. Single stain, FMO, and unstained controls were used to set gates. Single cells were lineage depleted using CD45- (BD Biosciences: #564279)/CD31- (BD Biosciences: #745436) gating. Full gating strategy is included as Supplemental Fig. 2.
Immunohistochemistry
Tissues were formalin-fixed and paraffin-embedded as previously described . Hematoxylin and Eosin staining was used to define morphological features. Antigen retrieval was performed using target retrieval solution (Dako, Glostrup, Denmark; #S1699) for CC3(Cell Signaling Technologies, Danvers, MA, USA; #9661) and Perilipin(Cell Signaling: #3470) or (EDTA Dako cat# S2367) for pSTAT3 (Cell Signaling Technologies; #9145). ImmPRESS polymer anti-rabbit IgG secondary reagent (Vector Labs, San Francisco, CA, USA t#MP-7401-15) was used for secondary staining of CC3 and pSTAT3 stained tissues and anti-rabbit secondary (Dako #K4003) for perilipin stains. DAB (Vector Labs; #SK-4105) and counterstaining were performed with hematoxylin (Vector Labs #H-3401). Alveolar area was measured on H&E-stained tissues in ImageJ on 10 alveoli/field; 5 fields/tissue. 10 fields/tissue were analyzed for positive CC3, pSTAT3, and perilipin stained tissues using CellSense Dimension software count and measure feature on regions of interest.
Data mining
Analysis of SEMA7A in breast cancer versus normal was performed on http://ualcan.path.uab.edu/index.html by selecting breast cancer samples from The Cancer Genome Atlas (TCGA) . Five-year relapse-free survival (RFS) curves for Sema7a (Affy ID: 230345_at) and Itga6 (Affy ID: 201656_at) were generated in KM plot using default parameters and no restrictions. Patients split by median survival. For multi-gene analyses, genes were filtered by median expression .
Statistical analysis
Unpaired t-test and one-way ANOVA were performed in GraphPad Prism. Nonparametric equivalent analyses were used for samples with uneven distribution or unequal standard deviations. p values of <0.05 were considered significant. Error bars represent mean ± standard error of the mean. All in vivo studies were replicated in at least three mice and in vitro studies performed in technical and biological triplicate with representative data presented.
Results
Semaphorin-7a promotes anoikis resistance in normal MECs
To identify whether SEMA7A promotes survival of normal MECs we utilized two methods: transfection of FC-tagged SEMA7A into immortalized human (MCF10A) cells (Supplemental Fig. 3) and addition of exogenous SEMA7A, which we purified from FC-tagged SEMA7A expressing cells, to wild-type MCF10A and MCF12A cells—another non-transformed MEC line. We found that genetic SEMA7A overexpression resulted in resistance to cell death, as measured by cleaved caspase 3/7, in a forced suspension assay, but not in attached conditions suggesting that SEMA7A specifically mediates resistance to cell death by anoikis (Fig. 1a). To determine the contribution of endogenous SEMA7A protein to anoikis resistance, we measured SEMA7A + MECs by flow over time in attached and suspended conditions and found that SEMA7A + MECs in attached conditions remain unchanged while SEMA7A + cells become significantly enriched as early as 24 h in detached conditions (Fig. 1b). We also show that stimulation with exogenous purified SEMA7A protein in both MCF10A and MCF12A cells is sufficient to significantly decrease cell death and inhibition of a known SEMA7A receptor, β1-integrin, significantly abrogated SEMA7A-mediated anoikis resistance (Fig. 1c and Supplemental Figs. 4 and 5).