Abstract
CDK4/6 inhibitors are used in the treatment of advanced estrogen receptor (ER)(+) breast cancer. Their efficacy in ER(−) and early-stage breast cancer is currently under investigation. Here, we show that palbociclib, a CDK4/6 inhibitor, can inhibit both progression of ductal carcinoma in situ (DCIS) and growth of invasive disease in both an ER(−) basal breast cancer model (MCFDCIS) and an ER(+) luminal model (MCF7 intraductal injection). In MCFDCIS cells, palbociclib repressed cell-cycle gene expression, inhibited proliferation, induced senescence, and normalized tumorspheres formed in Matrigel while the formation of acini by normal mammary epithelial cells (MCF10A) was not affected. Palbociclib treatment of mice with MCFDCIS tumors inhibited their malignant progression and reduced proliferation of invasive lesions. Transcriptomic analysis of the tumor and stromal cell compartments showed that cell cycle and senescence genes, and MUC16, an ovarian cancer biomarker gene, were repressed during treatment. Knockdown of MUC16 in MCFDCIS cells inhibited proliferation of invasive lesions but not progression of DCIS. After cessation of palbociclib treatment genes associated with differentiation, for example, P63, inflammation, IFNγ response, and antigen processing and presentation remained suppressed in the tumor and surrounding stroma. We conclude that palbociclib can prevent progression of DCIS and is antiproliferative in ER(−) invasive disease mediated in part via MUC16. Lasting effects of CDK4/6 inhibition after drug withdrawal on differentiation and the immune response could impact the approach to treatment of early-stage ER(−) breast cancer.
Introduction
Entry into the cell cycle is mediated by a restriction point controlled by the CDK4/6-RB axis, which protects against aberrant proliferation (1–3). Cancer cells frequently subvert this restriction point using a variety of signaling cascades that converge on enhanced CDK4/6 activity and inhibition of RB to facilitate cell-cycle entry (4–6). Consequently, the ability to take therapeutic control of the restriction point to block proliferation has been long sought after.
Palbociclib is a second-generation CDK inhibitor, and unlike previous inhibitors in this class, palbociclib demonstrates high affinity to CDK4/6 with little influence on other CDK family members and low toxicity (7). CDK4/6 inhibitors, when used in conjunction with standard-of-care hormone therapy, provide substantial improvements in progression-free survival compared with hormone therapy alone in cases of advanced ER(+)/HER2(−) breast cancer (8–10). Although approved use is currently limited to advanced ER(+) breast cancer, these drugs have the potential for widespread application due to the central nature of cell-cycle escape in carcinogenesis (11). Clinical trials in a variety of other cancer types are underway such as squamous cell lung cancer (NCT02785939), pancreatic neuroendocrine tumors (NCT02806648), oligodendroglioma and oligoastrocytoma (NCT02530320), along with others.
Though efficacious in ER(+) breast cancer, the use of palbociclib in earlier stages of breast cancer and in ER(−) disease is currently under investigation. One lesion type that is frequently diagnosed alongside invasive disease is ductal carcinoma in situ (DCIS). DCIS is considered an in-obligate precursor to invasive ductal carcinoma (IDC) in that it has the potential to become aggressive and potentially life-threatening but will not progress to IDC with 100% penetrance (12–16). Here we examine the effects of inhibition of CDK4/6 on DCIS and early-stage IDC. A challenge in these type of studies is that there are few models of early-stage breast cancer progression (17, 18). The best model of DCIS progression was derived from the MCF10A series of basal-like triple negative [ER(−), PR(−), HER2 non-amplified] cell lines known as MCFDCIS (19, 20). These cells exhibit bi-polar progenitor properties and are able to give rise to luminal and myoepithelial cell populations in xenografts, forming mammary acinar structures that will progress through DCIS to IDC with a predictable time course (21, 22).
Here we demonstrate the palbociclib is able to inhibit proliferation and induce senescence in both normal MCF10A and MCFDCIS cells mediated through the downregulation of cell-cycle driving genes and senescence genes. In 3D culture systems, palbociclib was able to normalize the architecture of MCFDCIS spheres but interestingly has no effect on normal MCF10A mammary acini. In vivo treatment of MCFDCIS tumors inhibited DCIS progression and reduced proliferation of invasive lesions. Palbociclib was also able to delay the invasive transition of a luminal model of DCIS, the mouse mammary intraductal (MIND) model, in which MCF7 cells are injected intraductally. The inhibition of proliferation of invasive lesions was in part mediated by MUC16, a protein that also serves as an ovarian cancer biomarker. Drug withdrawal experiments demonstrate that palbociclib is predominantly cytostatic in MCFDCIS, although lasting effects of CDK4/6 inhibition on differentiation and the immune response after drug withdrawal is of potential interest for rational design of therapeutic strategies targeting early-stage ER(−) breast cancer.
Materials and Methods
Cell culture
MCF10A and MCFDCIS cell lines were obtained from Dr. Susette Mueller (Georgetown University) and were cultured as described previously (22, 23). HEK293 cells were obtained from Dr. Rabindra Roy (Georgetown University) and were cultured in DMEM (GIBCO/Invitrogen) with 10% FBS. All cell lines were fingerprinted to confirm identity and mycoplasma tested regularly (most recent test 6/2018). Cells were used between passages 4 and 15. The FOXM1-12D phospho-mimic plasmid was obtained from Dr. Peter Sicinski (Dana Farber; ref. 24) and was used to transfect HEK293 cells alongside an empty-vector control using the Fugene 6 reagent as recommended by the manufacturer (Promega). Forty-eight hours after transfection, G418 selection agent was added to cells at 800 μg/mL and maintained for 14 days generate stable HEK-12D and HEK-EV cell lines. shMUC16 MCFDCIS lines were established using unique shRNA and nonsilencing control sequences inserted into the pGIPz (Dharmacon) as described previously (25).
In vitro drug sensitivity assays
Effects on cell phenotype were assessed after cells were treated for 72 hours with various concentrations of palbociclib (PALBOCICLIB-0332991; Taizhou Crene Biotechnology—NMR was performed by Dr. Milton Brown at Georgetown University to confirm compound structure prior to use). Treatment was initiated 24 hours after cells were seeded. Changes in cell morphology were captured with an Olympus IX-71 inverted epifluorescence microscope. Cell proliferation was assessed via crystal violet as described previously (25). Total protein and RNA were extracted from cells for analysis via Western blot analysis and qRT-PCR. Cell-cycle analysis was done on one million cells per condition using the Vindelov method (26). Apoptosis was assessed in an aliquot of the cell-cycle cell sample suspension via an Annexin V staining and flow cytometry.
Western blot analysis
Cells were cold lysed in an NP40-based lysis buffer whereas frozen tumor samples were homogenized in lysis buffer using the Roche MagNA Lyser instrument (Roche). Following protein extraction, cellular and tumor lysates were prepared and separated on an SDS-PAGE gel prior transfer and immunoblotting as described previously (25). Information on primary and secondary antibodies used can be found in Supplementary Table S1.
qRT-PCR
Total RNA was extracted from cells using the RNeasy Kit (Qiagen). RNA was extracted from tissues using the RNeasy Kit as well but following a homogenization step using the MagNA Lyser. qRT-PCR was performed on extracted RNA as described previously (27). Primers were purchased from Integrated DNA technologies, sequences can be found in Supplementary Table S2.
Senescence assays
To assess the senescence-related phenotype of increased cell size, cells were treated for 72 hours with different concentrations of palbociclib prior to collection and measurement of cell diameter utilizing a Multisizer3 Coulter Counter. After 72 hours of treatment with either vehicle or a dose of palbociclib, cells were stained for the senescence marker β-galactosidase (Cell Signaling Technologies: 9860) following manufacturer's instructions. Stain was allowed to develop for 48 hours. Images were acquired using the Olympus IX-71 microscope as described above, and the number of positive cells per field was quantified using the cell counting feature in ImageJ.
3D culture
Matrigel sphere formation assay was conducted as described previously (23, 28) with a modification to the protocol to add 100 nmol/L palbociclib or sterile water control to the assay media in which the tumor spheres were seeded. Spheres were photographed at days 7 and 10 for MCF10A and MCFDCIS respectively, using an Olympus IX-71 Inverted epifluorescence microscope. Sphere sizes were quantified using Fiji software. Sphere isolation and immunofluorescent staining were also performed as described previously (23) using Laminin 5 (details in Supplementary File S1; Table S1) and mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Images were acquired using the Leica SP8 Confocal microscope and fluorescent intensity was quantified using Fiji software. To isolate RNA from spheres, Matrigel was dissolved by adding dispase 5 U/mL (Stem Cell Technologies) to chamber slides and incubating at 37°C for 45 minutes. Solubilized tumorspheres were collected, spun, and washed with sterile PBS prior to RNA extraction using the Qiagen RNeasy Kit (Qiagen).
Animal models
Subcutaneous models: Six-week-old female athymic nude mice (average weight, 24 g) were purchased from Envigo. MIND Model: Six-week-old female NOD-SCID mice (average weight, 25 g) were purchased from Jackson Labs. Animals were housed in a pathogen-free environment with controlled temperature and humidity. All animal experiments were conducted in accordance with Protocol Number = 2016−1138.
Xenograft experiments
For subcutaneous xenograft experiments, MCFDCIS cells were injected as described previously (22). Animals received sterile water or 50 mg/kg palbociclib diluted in a water-based vehicle containing 0.5% methylcellulose and 0.2% Tween 80 for 1 or 2 weeks total depending on cohort, as outlined in Figs. 3A and 5A. Upon tumor collection, samples were divided for hematoxylin and eosin (H&E) and IHC, qRT-PCR (see Materials and Methods) and Western blot analysis (see Materials and Methods). Blood was collected to assess the impact of treatment on white blood cell counts using a Giemsa Stain Solution (Sigma-Aldrich) according to manufacturer's instructions. shMUC16 MCFDCIS cells were injected as previously described and tracked as outlined in Supplementary Fig. S5A. Serum was taken from animals for circulating MUC16 expression using an ELISA for detection of the human protein following manufacturer's instructions (R&D Systems). For the mammary intraductal method (MIND model), MCF7-luc2-DSred cells (kindly donated by Dr. Cathryn Briskin—Swiss Federal Institute of Technology in Lausanne) were prepared and injected into 6-week-old female NOD-SCID mice purchased from Jackson Labs as described previously (29), outlined in Supplementary Fig. S4A. Mammary glands were collected and H&E stained.
Histologic analysis
H&E and IHC analyses were performed on paraffin-embedded 5-μm sections using standard protocols described elsewhere (30). Information on primary antibodies (Phospho-RB, RB, KI67, P63, MUC16 and cleaved-CASPASE-3) for IHC can be found in Supplementary Table S1. Images were captured using an Olympus BX40 microscope and quantification of histologic areas and staining positivity were performed using ImageJ.
cDNA array and RNA-seq analysis
Total tumor RNA was extracted in triplicate, as described above. RNA with an integrity number greater than 8.7 were sent to the UCLA Neuroscience Genomics Core (UNGC). Microarray analysis which was carried out by the UNGC using Illumina HumanHT-12 v4 Expression BeadChip representing 47,000 well-annotated genes according to the manufacturer's instructions on RNA isolated from the xenograft described in Fig. 3A (Illumina, Inc.). Data were analyzed in R using the Bioconductor software. Greater than 2-fold difference in expression and a P-value <0.05 were set as the threshold for significance. Differential gene expression lists were generated by comparing palbociclib-treated tumor expression to vehicle-treated tumor expression and ranking genes by log2-fold change. RNA from the xenograft described in Fig. 5A was used for RNA library construction and sequencing (RNA-seq). Mouse genome GRCm38.p6 and human genome GRCh38.p10 were concatenated into a meta-genome. A STAR index was generated using GTF annotation from GENCODE for mouse and human genome features (version v18 and v27, respectively). Samples were aligned using single sample two-pass alignment, limiting multimapping reads to not more than one unique location. Unique alignment rates were 80% to 85%, with 10% to 15% reads mapping to more than once location. Only unique alignments were used to quantify expression at mouse and human genome features. Feature counts were used in downstream comparisons. Data available on GEO: GSE130903.
Statistical analysis
RStudio and Prism 7 (Graphpad Inc.) were used for statistical analysis and graphing. ANOVA was used for multiple comparisons and t tests or chi-square tests were used for paired comparisons, with P < 0.05 as the threshold for statistical significance in all tests.
Results
Effects of palbociclib on normal breast and DCIS cells in vitro
We compared the effects of palbociclib on MCF10A, an immortalized normal breast epithelial cell line, and MCFDCIS cells, a DCIS model cell line (20). Palbociclib, and a related CDK4/6 inhibitor abemaciclib, inhibited cellular proliferation in a concentration-dependent manner with similar IC50 in MCF10A and MCFDCIS (Supplementary Fig. S1A and S1B), accompanied by diminished phosphorylation of the canonical CDK4/6 target RB (Supplementary Fig. S1C) and reduced expression of known cell-cycle target genes (Fig. 1A; refs. 20, 31, 32). Palbociclib-treated cells accumulated in G1 with a reduction in both G2- and S-phase (Supplementary Fig. S1D).
Palbociclib (PD) treatment in 2D. PD elicits canonical antiproliferation and senescence responses in both MCF10A and MCFDCIS that are reversible upon discontinuation of treatment. A, qPCR analysis of cell-cycle–regulated mRNA targets in MCF10A (left) and MCFDCIS (right) cells treated with PD as indicated. Mean fold change ± SEM (one-way ANOVA followed by Dunnett multiple comparisons test, relative to vehicle). B, Quantification of β-galactosidase–stained MCF10A (left) and MCFDCIS (right) cells. Mean ± SEM (one-way ANOVA followed by Dunnett multiple comparisons test, relative to vehicle, 72-hour treatment). C, qPCR analysis of Lamin B1 (LMNB1) expression in MCF10A (left) and MCFDCIS (right) treated as indicated. Mean fold change ± SEM (one-way ANOVA followed by Dunnett multiple comparisons test, relative to vehicle, 72-hour treatment). D, qPCR analysis of FOXM1 expression in MCF10A (left) and MCFDCIS (right) treated as indicated. Mean fold change ± SEM (one-way ANOVA followed by Dunnett multiple comparisons test, relative to vehicle, 72-hour treatment). E, Crystal violet proliferation curves for MCFDCIS cells in vitro treated for 6 days with vehicle (constant vehicle), 6 days with 0.1 μmol/L palbociclib (constant palbociclib), or 3 days with 0.1 μmol/L palbociclib followed by 3 days with vehicle (reversal). F, qPCR analysis of cell-cycle–regulated mRNA targets in MCFDCIS cells treated with PD as indicated. Mean fold change ± SEM (Student T test, relative to vehicle). G, Cell-cycle analysis of MCFDCIS cells treated with PD as indicated. H, ANNEXIN-5 apoptosis assay on MCFDCIS cells treated as indicated; all apoptotic death is represented as a fold change from the normalized vehicle-treated baseline. I, Quantification of β-galactosidase–stained MCFDCIS cells in vitro treated with PD as indicated. Mean ± SEM (Student T test, relative to vehicle). J, qPCR analysis of LMNB1 (left) and FOXM1 (right) expression in MCFDCIS treated as indicated. Mean fold change ± SEM (Student T test, relative to vehicle). K, Quantification of PD senescence challenge in HEK293 cells transfected with constitutively active FOXM1-12D or empty vector. HEK293-EV and HEK293-12D were treated with indicated concentrations of PD for 72 hours prior to staining for β-galactosidase activity. Mean ± SEM (Student t test, relative to vehicle). For all: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. n.s., not significant.
Palbociclib (PD) treatment in 2D. PD elicits canonical antiproliferation and senescence responses in both MCF10A and MCFDCIS that are reversible upon discontinuation of treatment. A, qPCR analysis of cell-cycle–regulated mRNA targets in MCF10A (left) and MCFDCIS (right) cells treated with PD as indicated. Mean fold change ± SEM (one-way ANOVA followed by Dunnett multiple comparisons test, relative to vehicle). B, Quantification of β-galactosidase–stained MCF10A (left) and MCFDCIS (right) cells. Mean ± SEM (one-way ANOVA followed by Dunnett multiple comparisons test, relative to vehicle, 72-hour treatment). C, qPCR analysis of Lamin B1 (LMNB1) expression in MCF10A (left) and MCFDCIS (right) treated as indicated. Mean fold change ± SEM (one-way ANOVA followed by Dunnett multiple comparisons test, relative to vehicle, 72-hour treatment). D, qPCR analysis of FOXM1 expression in MCF10A (left) and MCFDCIS (right) treated as indicated. Mean fold change ± SEM (one-way ANOVA followed by Dunnett multiple comparisons test, relative to vehicle, 72-hour treatment). E, Crystal violet proliferation curves for MCFDCIS cells in vitro treated for 6 days with vehicle (constant vehicle), 6 days with 0.1 μmol/L palbociclib (constant palbociclib), or 3 days with 0.1 μmol/L palbociclib followed by 3 days with vehicle (reversal). F, qPCR analysis of cell-cycle–regulated mRNA targets in MCFDCIS cells treated with PD as indicated. Mean fold change ± SEM (Student T test, relative to vehicle). G, Cell-cycle analysis of MCFDCIS cells treated with PD as indicated. H, ANNEXIN-5 apoptosis assay on MCFDCIS cells treated as indicated; all apoptotic death is represented as a fold change from the normalized vehicle-treated baseline. I, Quantification of β-galactosidase–stained MCFDCIS cells in vitro treated with PD as indicated. Mean ± SEM (Student T test, relative to vehicle). J, qPCR analysis of LMNB1 (left) and FOXM1 (right) expression in MCFDCIS treated as indicated. Mean fold change ± SEM (Student T test, relative to vehicle). K, Quantification of PD senescence challenge in HEK293 cells transfected with constitutively active FOXM1-12D or empty vector. HEK293-EV and HEK293-12D were treated with indicated concentrations of PD for 72 hours prior to staining for β-galactosidase activity. Mean ± SEM (Student t test, relative to vehicle). For all: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. n.s., not significant.
CDK4/6 inhibition can also induce cellular senescence (Supplementary Fig. S1E), which likely contributes to the observed increased cell size in dose–response assays and the measured concentration-dependent increases in median cell diameter (Supplementary Fig. S1F). We also observed a dose-dependent accumulation of β-galactosidase positivity in both MCF10A and MCFDCIS cells with >20% stained for the senescence marker at IC50 concentrations of palbociclib (Fig. 1B; Supplementary Fig. S1G) and significantly reduced expression of an early senescence response gene, nuclear envelope factor Lamin B1 (LMNB1; ref. 33) in MCFDCIS cells (Fig. 1C). Notably, palbociclib was able to significantly reduce phosphorylation and expression of FOXM1, a CDK4/6 target involved in the senescence response (Supplementary Fig. S1H; Fig. 1D; ref. 24). These data indicate that palbociclib inhibits proliferation by regulating cell-cycle machinery and also induces senescence in both normal mammary cells and early-stage breast cancer.
To determine the durability of palbociclib response, we designed reversal experiments in which MCFDCIS cells were treated with palbociclib for 72 hours followed by a recovery period of an additional 72 hours. In reversal experiments, cell proliferation rate did not recover to vehicle baseline following discontinuation of treatment despite the recovery of cell-cycle genes to pretreatment expression levels, with the exception of MCM2 which remained significantly downregulated (Fig. 1E and F). Consistent with this, we found that G1 arrest is transient and dependent on the continued presence of drug (Fig. 1G). However, recovered cells experienced 2.5-fold higher levels of apoptosis than control cells, which could explain the lag in proliferative rate recovery (Fig. 1E and H). In terms of senescence during recovery, β-galactosidase staining returned to baseline, as did expression of LMNB1, although there was a significant induction of FOXM1 following recovery (Fig. 1I and J). These suggested that palbociclib might influence both the proliferative and senescent responses through effects on FOXM1. To investigate this, we overexpressed, in HEK293 cells, a constitutively active FOXM1 “phospho-mimic” in which 12 CDK4/6 consensus sites have been altered to aspartic acid residues (Supplementary Fig. S1I; ref. 24). Expression of the FOXM1-12D did not alter the palbociclib IC50 for proliferation (Supplementary Fig. S1J) but did significantly inhibit palbociclib-induced senescence as measured by β-galactosidase staining (Fig. 1K). FOXM1-12D also attenuated the repression of several cell-cycle genes including CCNB1 and KIF20A (Supplementary Fig. S1K). These suggest that FOXM1 may play an important role in the senescence response to CDK4/6 inhibition in the context of early breast cancer.
Effects of palbociclib on MCFDCIS cells in 3D culture
The effects of palbociclib on normal mammary cells and early breast cancer could be modified by their stromal environment. To investigate this, we examined palbociclib effects on both MCF10A and MCFDCIS cells grown on top of Matrigel and found that MCF10A cells form normal mammospheres whereas MCFDCIS cells form abnormal tumorspheres (22, 23, 28). The MCF10A mammosphere phenotype is largely unchanged over 7 to 10 days of treatment in the presence of palbociclib, whereas MCFDCIS tumorspheres are significantly smaller with a more normalized phenotype when compared with vehicle controls (Supplementary Fig. S2; Fig. 2A and B). MCFDCIS but not MCF10A spheres showed significant reductions in the cell-cycle gene genes and the senescence marker, LMNB1 (Fig. 2C). LAMININ 5, a basement membrane marker (23), is deposited at the periphery of MCF10A mammospheres and remains unaltered after palbociclib treatment (Fig. 2D and E). In contrast, LAMININ 5 shows a disorganized expression across MCFDCIS tumorspheres, but reverted to a normal mammosphere pattern after palbociclib treatment (Fig. 2D and E). Together these results indicate potential selectivity for highly proliferative transformed cells in the 3D context as differentiated MCF10A mammospheres were unaffected by palbociclib.
Palbociclib (PD) treatment in 3D. PD selectively influences MCFDCIS spheres in 3D culture, normalizing their architecture. A, MCF10A (top) and MCFDCIS (bottom) spheres grown in 3D in the presence of vehicle or 0.1 μmol/L PD. ×20 magnification, scale, 200 μm. B, Quantification of A; sphere areas calculated using Fiji software. Mean ± SEM (Student t test, ****P < 0.0001 relative to vehicle). C, qPCR analysis of cell-cycle/senescence-regulated mRNA targets in MCF10A (top) and MCFDCIS (bottom) spheres treated with PD as indicated. Mean fold change ± SEM (Student t test, **P < 0.01, ***P < 0.001, ****P < 0.0001 relative to vehicle). D, Vehicle-treated MCF10A (top) and MCFDCIS (bottom) spheres stained for LAMININ 5 (red) and DAPI (blue) along with quantification of the distribution of LAMININ 5 signal intensity measured along the yellow line indicated in the photos. ×63 magnification, scale, 25 μm. E, PD-treated MCF10A (top) and MCFDCIS (bottom) spheres stained for LAMININ 5 (red) and DAPI (blue) along with quantification of the distribution of LAMININ 5 signal intensity measured along the yellow line indicated. ×63 magnification, scale, 25 μm.
Palbociclib (PD) treatment in 3D. PD selectively influences MCFDCIS spheres in 3D culture, normalizing their architecture. A, MCF10A (top) and MCFDCIS (bottom) spheres grown in 3D in the presence of vehicle or 0.1 μmol/L PD. ×20 magnification, scale, 200 μm. B, Quantification of A; sphere areas calculated using Fiji software. Mean ± SEM (Student t test, ****P < 0.0001 relative to vehicle). C, qPCR analysis of cell-cycle/senescence-regulated mRNA targets in MCF10A (top) and MCFDCIS (bottom) spheres treated with PD as indicated. Mean fold change ± SEM (Student t test, **P < 0.01, ***P < 0.001, ****P < 0.0001 relative to vehicle). D, Vehicle-treated MCF10A (top) and MCFDCIS (bottom) spheres stained for LAMININ 5 (red) and DAPI (blue) along with quantification of the distribution of LAMININ 5 signal intensity measured along the yellow line indicated in the photos. ×63 magnification, scale, 25 μm. E, PD-treated MCF10A (top) and MCFDCIS (bottom) spheres stained for LAMININ 5 (red) and DAPI (blue) along with quantification of the distribution of LAMININ 5 signal intensity measured along the yellow line indicated. ×63 magnification, scale, 25 μm.
Palbociclib treatment reduces MCFDCIS xenograft growth and malignant progression
The selective “normalization” effects of palbociclib on MCFDCIS cells in Matrigel suggested that this CDK4/6 inhibitor could have beneficial effects on DCIS lesions in vivo. Therefore, we tested palbociclib efficacy over 2 weeks in vivo on xenograft tumors in athymic nude mice (Fig. 3A). We used a low dose of 50 mg/kg relative to previous preclinical studies (34–39) to minimize potential toxicity such as neutropenia, which is a common adverse event associated with palbociclib (8). We saw a small, nonsignificant reduction in white blood cell counts following 2 weeks of treatment relative to control (Supplementary Fig. S3A) and observed no difference in animal weight between groups over the course of our study indicating that this dose of palbociclib was well tolerated (Supplementary Fig. S3B). Tumors showed a significantly reduced growth rate after palbociclib leading to stasis in some cases but not to regression (Fig. 3B).
Palbociclib (PD) treatment in vivo. PD delays growth and progression of MCFDCIS xenograft tumors in nude mice. A, Schematic of experimental design and time points. B, Tumor growth curves for vehicle- or PD-treated MCFDCIS tumors following subcutaneous injection of cells, represented as the change (delta) in tumor size normalized to initial tumor size (2-way ANOVA and Bonferroni posttests, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 relative to vehicle). C, Cohort 2 tumor sections stained with hematoxylin and eosin as well as IHC for phosphorylated RB (S807/811), total RB, KI67, and P63, treated with PD as indicated and associated quantifications (chi-square tests, ****P < 0.0001, ×20 magnication, scale, 100 μm). D, qPCR analysis of cell-cycle regulated mRNA targets in MCFDCIS tumors treated with PD as indicated. Mean fold change ± SEM (Student t test, **P < 0.01, ***P < 0.001, ****P < 0.0001 relative to vehicle). E, Western blot analysis for expression of FOXM1 and actin in tumor lysates at both 1 and 2 weeks of treatment with PD as indicated. F, qPCR analysis of senescence-regulated mRNA targets in MCFDCIS tumors treated with PD as indicated. Mean fold change ± SEM (Student t test, ****P < 0.0001 relative to vehicle). n.s., not significant.
Palbociclib (PD) treatment in vivo. PD delays growth and progression of MCFDCIS xenograft tumors in nude mice. A, Schematic of experimental design and time points. B, Tumor growth curves for vehicle- or PD-treated MCFDCIS tumors following subcutaneous injection of cells, represented as the change (delta) in tumor size normalized to initial tumor size (2-way ANOVA and Bonferroni posttests, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 relative to vehicle). C, Cohort 2 tumor sections stained with hematoxylin and eosin as well as IHC for phosphorylated RB (S807/811), total RB, KI67, and P63, treated with PD as indicated and associated quantifications (chi-square tests, ****P < 0.0001, ×20 magnication, scale, 100 μm). D, qPCR analysis of cell-cycle regulated mRNA targets in MCFDCIS tumors treated with PD as indicated. Mean fold change ± SEM (Student t test, **P < 0.01, ***P < 0.001, ****P < 0.0001 relative to vehicle). E, Western blot analysis for expression of FOXM1 and actin in tumor lysates at both 1 and 2 weeks of treatment with PD as indicated. F, qPCR analysis of senescence-regulated mRNA targets in MCFDCIS tumors treated with PD as indicated. Mean fold change ± SEM (Student t test, ****P < 0.0001 relative to vehicle). n.s., not significant.
The MCFDCIS model is unique in that xenograft tumors transition from in situ carcinoma to invasive cancer between 4 and 6 weeks after injection (21). Thus, we were able to monitor the impact of palbociclib on malignant progression of MCFDCIS lesions and found a significant delay of progression in palbociclib-treated tumors. At 4 weeks, the tumor area of about half of the control group showed invasive lesions, whereas about 90% in the palbociclib-treated group remained noninvasive DCIS (Fig. 3C, first panel). Palbociclib treatment resulted in significantly diminished expression of phosphorylated RB without significantly impacting total RB staining by IHC, although Western blot analysis demonstrates a reduction in treated tumor lysates (Fig. 3C; Supplementary Fig. S3C). Palbociclib treatment also significantly reduced KI67 proliferative index from >25% of nuclei stained to <10% within the tumor (Fig. 3C). P63 expression, which marks the invasive transition in the MCFDCIS model (21), was also significantly reduced (Fig. 3C), corroborating the above conclusion from the surgical pathology analysis. Despite the smaller size of tumors and reduced proliferation, CASPASE-3 cleavage was unaltered within the tumors, which suggests that palbociclib does not induce apoptosis in this context (Supplementary Fig. S3D). As expected, palbociclib-responsive cell-cycle genes also showed significant mRNA downregulation in tumors (Fig. 3D) and FOXM1 expression was reduced at both the protein and mRNA levels, mirroring in vitro assays (Fig. 3E and F). These observation, along with significant loss of LMNB1, suggest that the DCIS tumors are undergoing both proliferative arrest and senescence induction following 2 weeks of palbociclib treatment (Fig. 3E and F).
Effect of palbociclib in the MIND model of DCIS
To determine if palbociclib effects on DCIS tumors were dependent on the tumor microenvironment we utilized the MIND model, in which tumor cells are directly injected into the mammary duct. In this model, early-stage DCIS-like breast cancer lesions develop after injection of MCF7 luminal ER(+) breast cancer cells (Supplementary Fig. S4A; ref. 29). Following injection of MCF7-luc2-DSred cells (kindly donated by Dr Cathryn Briskin—Swiss Federal Institute of Technology in Lausanne) into mammary ducts, successful injection was verified by IVIS imaging (Supplementary Fig. S4B). DCIS lesions were established over 2 months prior to treatment with palbociclib for 28 days by oral gavage. IVIS imaging and histology of the mammary duct lesions revealed that palbociclib-treated mice had reduced tumor burden and bore fewer invasive lesions than their vehicle-treated counterparts (Supplementary Fig. S4C–S4E). Palbociclib is therefore effective in postponing the invasive transition not only in basal-like DCIS but also in hormone-dependent luminal DCIS.
Role of MUC16 in the palbociclib response
To assess global gene expression changes in DCIS following 2 weeks of palbociclib treatment, mRNA isolated from MCFDCIS tumors was analyzed using Illumina bead chip array. Overall, 1,515 genes were found to be significantly (P < 0.05) up- or downregulated by at least 1.5-fold at 2 weeks on treatment with the majority of highly regulated genes playing a role in the cell cycle (Fig. 4A and B). MUC16 (CA-125), a cancer antigen that has been used as a biomarker and therapy response marker in ovarian cancer (40–43), stood out in this analysis as it is not a cell-cycle regulatory gene. We verified by PCR that significant reductions in MUC16 mRNA occurred after 1 and 2 weeks of treatment and this was validated at the protein level by IHC (Fig. 4C and D). MUC16 expression appears to increase with MCFDCIS tumor progression as 2-week vehicle-treated tumor sections stained more intensely than 1-week treated (Fig. 4D). To evaluate a functional role of MUC16 in MCFDCIS tumor growth and progression, we generated 3 independent shMUC16 MCFDCIS cell lines along with a nonsilencing control. Subcutaneous tumor growth of the shMUC16 cell lines (Supplementary Fig. S5A) was reduced relative to nonsilenced control tumors with both shMUC16_2 and shMUC16_3 tumors demonstrating significantly reduced growth (Fig. 4E). Despite some differences in growth rate, all shMUC16 MCFDCIS tumors had significantly less MUC16 expression than nonsilenced controls (Fig. 4F and G). Tumor-derived circulating MUC16 in serum was easily detectable in the controls but below detection threshold in the majority of shMUC16 tumors, with blood-borne expression correlating tightly with tumor growth rate across independent shMUC16 lines (Fig. 4H). Although shMUC16 tumors grew more slowly than their control counterparts, loss of MUC16 did not delay the transition from DCIS to invasive lesions and tumor histology was indistinguishable between the groups (Fig. 4I). These suggest that MUC16 does not drive MCFDCIS to invasive transition but does contribute to overall tumor growth. Of note is that shMUC16 tumors demonstrate more widespread staining for cleaved CASPASE-3, suggesting that the reduction in tumor size relative to control tumors is due to an increased rate of cell death (Supplementary Fig. S5B).
MUC16′s role in palbociclib (PD) response. MUC16 can serve as a biomarker of palbociclib treatment in MCFDCIS tumors and may play a functional role in tumor growth. A, Cigar plot of Log2 expression of cDNA array data generated from RNA isolated from vehicle- and palbociclib-treated tumors of cohort 2. Points highlighted in red are upregulated by palbociclib, whereas those in blue are downregulated by drug. B, Top 15 most PD-regulated genes from the cDNA array at 2 weeks of treatment, ranked by Log2-fold change and significance of P < 0.05. Upregulated genes are depicted in red, and downregulated genes in blue. C, qPCR analysis of MUC16 expression in MCFDCIS tumors treated as indicated. Mean fold change ± SEM (Student t test, ***P < 0.001, ****P < 0.0001 relative to vehicle). D, Representative cohorts 1 and 2 tumor sections stained by IHC for MUC16, treated with PD as indicated. ×10 magnification, scale, 200 μm. E, Tumor growth curves for nonsilenced (NS) or shMUC16 MCFDCIS tumors following subcutaneous injection of cells, represented as the delta in tumor size normalized to initial tumor size (two-way ANOVA and Bonferroni posttests, *P < 0.05, **P < 0.01 relative to vehicle). F, Representative NS and shMUC16 MCFDCIS tumor sections stained by IHC for MUC16. ×10 magnification, scale, 200 μm. G, Quantification of MUC16 IHC staining as a percent of tumor area. Mean ± SEM (1-way ANOVA followed by Dunnett multiple comparisons test, ***P < 0.001, relative to vehicle). H, ELISA for circulating MCFDCIS tumor-derived MUC16 in serum taken at day 28 from mice bearing NS or shMUC16 MCFDCIS tumors. I, Representative NS and shMUC16 MCFDCIS tumor sections stained using the PAS method. ×10 magnification, scale, 200 μm. n.s., no significant.
MUC16′s role in palbociclib (PD) response. MUC16 can serve as a biomarker of palbociclib treatment in MCFDCIS tumors and may play a functional role in tumor growth. A, Cigar plot of Log2 expression of cDNA array data generated from RNA isolated from vehicle- and palbociclib-treated tumors of cohort 2. Points highlighted in red are upregulated by palbociclib, whereas those in blue are downregulated by drug. B, Top 15 most PD-regulated genes from the cDNA array at 2 weeks of treatment, ranked by Log2-fold change and significance of P < 0.05. Upregulated genes are depicted in red, and downregulated genes in blue. C, qPCR analysis of MUC16 expression in MCFDCIS tumors treated as indicated. Mean fold change ± SEM (Student t test, ***P < 0.001, ****P < 0.0001 relative to vehicle). D, Representative cohorts 1 and 2 tumor sections stained by IHC for MUC16, treated with PD as indicated. ×10 magnification, scale, 200 μm. E, Tumor growth curves for nonsilenced (NS) or shMUC16 MCFDCIS tumors following subcutaneous injection of cells, represented as the delta in tumor size normalized to initial tumor size (two-way ANOVA and Bonferroni posttests, *P < 0.05, **P < 0.01 relative to vehicle). F, Representative NS and shMUC16 MCFDCIS tumor sections stained by IHC for MUC16. ×10 magnification, scale, 200 μm. G, Quantification of MUC16 IHC staining as a percent of tumor area. Mean ± SEM (1-way ANOVA followed by Dunnett multiple comparisons test, ***P < 0.001, relative to vehicle). H, ELISA for circulating MCFDCIS tumor-derived MUC16 in serum taken at day 28 from mice bearing NS or shMUC16 MCFDCIS tumors. I, Representative NS and shMUC16 MCFDCIS tumor sections stained using the PAS method. ×10 magnification, scale, 200 μm. n.s., no significant.
Residual effects after cessation of treatment with palbociclib
Palbociclib's effects in vitro are largely but not entirely reversible as shown above (see Fig. 1E and F). Thus, we sought to determine if similar residual effects of CDK4/6 inhibition could be detected in vivo in either the tumor cells or stroma after discontinuation of therapy. To study this, we allowed mice an 11-day drug-recovery period following 2 weeks of palbociclib treatment (Fig. 5A). Upon treatment termination, palbociclib-treated tumors resumed growth but final tumor sizes in the palbociclib recovery group remained significantly smaller than vehicle-treated tumors (Fig. 5B). Growth rates of tumors during the 11 days of recovery paralleled those of the vehicle-treated tumors (Fig. 5B); notably, in tumors recovering from palbociclib treatment, almost 30% of tumor area remained DCIS whereas vehicle-treated tumors progressed to invasive disease with only 8.5% of tumor area remaining DCIS (Fig. 5C). We observed partial re-expression of phosphorylated RB, KI67, and P63 upon palbociclib withdrawal (Fig. 5C). P63 expression remained lower in palbociclib-treated and recovered tumors than in vehicle-treated tumors (Figs. 5C and 3C), suggesting that palbociclib has some lasting effects on tumor differentiation status and progression to invasive lesions. Interestingly, the panel of cell-cycle genes completely returned to baseline as did the senescence markers LMNB1 and FOXM1 (Fig. 5D).
Palbociclib (PD) treatment in vivo is reversible. Most, but not all of, PD's effects on tumor growth and progression are reversible upon discontinuation of drug treatment. A, Schematic of experimental design, time points, and comparisons made using RNA isolated from tumors. B, Tumor growth curves for vehicle and PD-treated MCFDCIS tumors, represented as the change (delta) in tumor size normalized to initial day of treatment (two-way ANOVA and Bonferroni posttests, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 relative to vehicle). C, Cohort 4 tumor sections stained with hematoxylin and eosin as well as IHC for phosphorylated RB (S807/811), total RB, KI67, and P63, treated with PD as indicated and associated quantifications (chi-squared tests, *P < 0.05, ***P < 0.001, ****P < 0.0001, ×20 magnification, scale, 100 μm). D, Fold change values of the cell-cycle gene panel in palbociclib-treated (cohort 3) and -recovered (cohort 4) tumors relative to a vehicle baseline, represented by the dotted line. E, Fold change values of genes significantly upregulated during treatment (palbo) and their expression following recovery (palbo-recovered) relative to a vehicle baseline indicated by the dotted line. F, Fold change values of genes significantly downregulated during treatment (palbo) and their expression following recovery (palbo-recovered) relative to a vehicle baseline indicated by the dotted line. D–F are based on values derived from RNA-seq. All values were filtered for minimum expression (CPM > 2) and significance (P < 0.05). G, ELISA for circulating MCFDCIS tumor-derived MUC16 in serum taken from cohort 4 mice. n.s., not significant.
Palbociclib (PD) treatment in vivo is reversible. Most, but not all of, PD's effects on tumor growth and progression are reversible upon discontinuation of drug treatment. A, Schematic of experimental design, time points, and comparisons made using RNA isolated from tumors. B, Tumor growth curves for vehicle and PD-treated MCFDCIS tumors, represented as the change (delta) in tumor size normalized to initial day of treatment (two-way ANOVA and Bonferroni posttests, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 relative to vehicle). C, Cohort 4 tumor sections stained with hematoxylin and eosin as well as IHC for phosphorylated RB (S807/811), total RB, KI67, and P63, treated with PD as indicated and associated quantifications (chi-squared tests, *P < 0.05, ***P < 0.001, ****P < 0.0001, ×20 magnification, scale, 100 μm). D, Fold change values of the cell-cycle gene panel in palbociclib-treated (cohort 3) and -recovered (cohort 4) tumors relative to a vehicle baseline, represented by the dotted line. E, Fold change values of genes significantly upregulated during treatment (palbo) and their expression following recovery (palbo-recovered) relative to a vehicle baseline indicated by the dotted line. F, Fold change values of genes significantly downregulated during treatment (palbo) and their expression following recovery (palbo-recovered) relative to a vehicle baseline indicated by the dotted line. D–F are based on values derived from RNA-seq. All values were filtered for minimum expression (CPM > 2) and significance (P < 0.05). G, ELISA for circulating MCFDCIS tumor-derived MUC16 in serum taken from cohort 4 mice. n.s., not significant.
To better understand lasting versus reversible effects of CDK4/6 inhibition, we performed RNA-seq on RNA isolated from treated and recovered MCFDCIS tumors. We confirmed that the majority of regulated genes are involved in cell-cycle regulation and that nearly all of them recover to a baseline level of expression following treatment cessation (Supplementary Fig. S6A). Interestingly, several genes uninvolved with the cell cycle broke this pattern. Some genes were upregulated >1.5-fold by palbociclib and remained upregulated following drug withdrawal such as the differentiation factor NELL2, which experienced even further upregulation in recovery (Fig. 5E). High expression of NELL2 is associated with improved recurrence-free survival in basal breast cancers (Supplementary Fig. S6B). Other factors initially upregulated by palbociclib were significantly downregulated following palbociclib withdrawal (Fig. 5E) and some genes that experienced significant downregulation during palbociclib treatment did not recover to baseline following discontinuation of treatment, including several RNA processing factors, matrix metalloproteinases, and inflammatory factors (Fig. 5F). In the RNA-seq analysis, we also saw that MUC16 was significantly downregulated by treatment; its mRNA levels increased somewhat following drug withdrawal but did not return to baseline. Consistent with this, MUC16 protein in the circulation remained significantly reduced even after treatment cessation (Fig. 5G).
Palbociclib has residual effects on stromal immune and IFN-related genes
To gain insight into the effects of palbociclib on the tumor host, we simultaneously aligned the RNA-seq reads from tumors to both the human and mouse genomes (see Materials and Methods). Mouse stromal cells contributed 15% to 25% of the mRNA isolated from tumor samples that were harvested at different points during treatment (Fig. 6A and B). The cell cycle and senescence gene panel regulated in tumor cells was also impacted in the mouse stroma, although the downregulation of these genes was smaller than seen in the tumor cell compartment. Following recovery from treatment, several genes exceeded the expression observed at baseline (Fig. 6C). Using the same method described above to identify lasting influence of human tumor genes, we identified mouse stromal genes that experienced a lasting impact of palbociclib (Fig. 6D and E). Interestingly, the genes that showed significant downregulation during palbociclib treatment that persists after treatment cessation are almost entirely immune-related, specifically those related to interferon signaling with roles in transcriptional regulation and antigen processing and presentation (Fig. 6E). Xenografts of human cells do impact stromal gene expression and signaling in the host, but the repression of these immunity-related genes was only noted in palbociclib-treated animals indicating it is a drug-specific effect. Quite strikingly, reduced expression of these genes is associated with a highly significant reduction in recurrence-free survival of patients with basal breast cancers (Supplementary Fig. S7A).
Palbociclib (PD)'s influence on the tumor microenvironment. PD affects gene expression in mouse stromal cells with a residual effect after treatment cessation on immune-related signaling pathways. A, Percent of RNA-seq reads for each tumor that aligned to the murine or human genome. Plotted as mean ± SEM with individual tumors plotted as separate symbols. B, Schematic of experimental design, time points, and comparisons made using mouse RNA isolated from tumors. C, Fold change values of the cell-cycle gene panel in palbociclib-treated (cohort 3) and palbociclib recovered-treated (cohort 4) tumor stroma relative to a vehicle baseline, represented by the dotted line. D, Fold change values of genes significantly upregulated during treatment (palbo) and their expression following recovery (palbo-recovered) relative to a vehicle baseline indicated by the dotted line. E, Fold change values of genes significantly downregulated during treatment (palbo) and their expression following recovery (palbo-recovered) relative to a vehicle baseline indicated by the dotted line. C–E are based on values derived from RNA-seq. All values were filtered for minimum expression (CPM > 2) and significance (P < 0.05). F, Normalized enrichment scores for hallmark pathways identified in both the human tumor and mouse stromal RNA, and the dotted line indicates a significance cutoff based on P values < 0.05 and FDR Q values < 0.25.
Palbociclib (PD)'s influence on the tumor microenvironment. PD affects gene expression in mouse stromal cells with a residual effect after treatment cessation on immune-related signaling pathways. A, Percent of RNA-seq reads for each tumor that aligned to the murine or human genome. Plotted as mean ± SEM with individual tumors plotted as separate symbols. B, Schematic of experimental design, time points, and comparisons made using mouse RNA isolated from tumors. C, Fold change values of the cell-cycle gene panel in palbociclib-treated (cohort 3) and palbociclib recovered-treated (cohort 4) tumor stroma relative to a vehicle baseline, represented by the dotted line. D, Fold change values of genes significantly upregulated during treatment (palbo) and their expression following recovery (palbo-recovered) relative to a vehicle baseline indicated by the dotted line. E, Fold change values of genes significantly downregulated during treatment (palbo) and their expression following recovery (palbo-recovered) relative to a vehicle baseline indicated by the dotted line. C–E are based on values derived from RNA-seq. All values were filtered for minimum expression (CPM > 2) and significance (P < 0.05). F, Normalized enrichment scores for hallmark pathways identified in both the human tumor and mouse stromal RNA, and the dotted line indicates a significance cutoff based on P values < 0.05 and FDR Q values < 0.25.
Gene set enrichment analysis (GSEA) revealed that similar hallmark pathways were significantly regulated by palbociclib both in the human tumor and the surrounding stroma (Fig. 6F). Although both the tumor and stroma experience a downregulation of cell-cycle driver pathways including E2F Targets, G2M Checkpoint, and the Mitotic Spindle, the stroma is less impacted and better able to recover following treatment cessation, as indicated by upregulation of these pathways (Fig. 6F). However, unlike the cell-cycle pathways, a more dramatic regulation of hallmark inflammatory and IFNγ signaling pathways is seen in the stroma compared with the tumor cell compartment. These pathways remain significantly downregulated in the tumor microenvironment even after cessation of palbociclib treatment, reflecting a lasting gene signature in the mouse stroma (Fig. 6E and F).
Discussion
In this study we show that CDK4/6 inhibition delays malignant progression to invasive disease in both basal MCFDCIS subcutaneous xenograft and luminal MCF7 intraductal injection models of DCIS. Even after cessation of palbociclib treatment, the residual effects of the drug result in less aggressive tumors and reduced overall recurrence. In vitro, palbociclib caused canonical cell-cycle arrest in normal and transformed breast cells and in parallel also displayed classic features of senescence with no discernible increase in apoptosis. In 3D assays, which are more faithful predictors of cell behavior and drug responsiveness in vivo (44), there was differential sensitivity of MCF10A and MCFDCIS spheres to palbociclib. This is likely explained by the fact that MCF10A cells grown as spheres undergo a well-described process of differentiation, quickly enter a maintenance phase marked by long-term quiescence (23), and are relatively insensitive to the effects of CDK4/6 inhibition. Conversely, MCFDCIS spheres do not experience differentiation or quiescence as they grow in 3D; palbociclib is able to significantly reduce expression of E2F target genes while simultaneously inducing senescence as evidenced by loss of LMNB1. Consistent with these observations, palbociclib treatment had no discernable effects on normal, differentiated mammary gland architecture over the course of therapy in both subcutaneous xenografts and in MIND model experiments. Cell-cycle inhibition and G1 arrest following palbociclib is mainly cytostatic as the majority of MCFDCIS tumor cells were able to re-express cell-cycle driving genes and reinitiate growth following treatment cessation, similar to other studies (34, 37, 45).
To our knowledge, this is the first interrogation of early-stage breast cancer cells in parallel with the immediate mouse stromal cell transcriptomes during palbociclib treatment and after recovery from treatment. The majority of the 97 genes that recover expression after cessation of palbociclib treatment contribute to hallmark pathways related to the cell cycle. The tight regulation of cell-cycle genes on and after treatment highlights the specificity of CDK4/6 inhibition in targeting proliferation. Many genes identified in our signature overlap with those previously identified as responding to CDK4/6 inhibition (35). This work suggests that there is a core set of targets that are influenced by palbociclib treatment across different contexts that may be useful indicators when assessing drug responsiveness in patients. The mouse stroma also experienced similar changes in canonical cell-cycle driver genes but to a lesser magnitude, accompanied by a more complete expression recovery following treatment cessation. Stromal tissue is not as proliferative as the tumor, which likely provides some resistance to palbociclib's primary effects.
The discovery that a non–cell-cycle regulatory gene, the cancer-specific antigen MUC16 (also known as CA-125), is one of the most significantly downregulated genes by palbociclib is surprising. MUC16 is well-known in the ovarian cancer field where it has been used clinically as a biomarker of disease in tracking the efficacy of therapy (40–42). Interestingly, it has been reported that ovarian cancer and basal breast cancer have significant genomic similarities, and MUC16 is frequently mutated in breast cancer cases (46). In our model, MUC16 is not only a biomarker of palbociclib responsiveness but also contributes to the growth of invasive lesions. MUC16 has been linked to cancer progression and aggressive behaviors in both ovarian and breast cancers (43), although the mechanism MUC16 utilizes to increase proliferation of cancer cells is not clear. Nevertheless, it appears that CDK4/6-mediated repression of MUC16 is partially responsible for the efficacy of palbociclib in our MCFDCIS model. MUC16 has been previously implicated in triple-negative breast cancer as a potential driver of progression and may play a key role in early disease (47). It will be interesting to determine if, in a subset of human basal breast cancers, MUC16 expression could indicate patient response to palbociclib therapy. It is of note that establishment of predictive biomarkers of response to CDK4/6 inhibitors in advanced stages of disease has been challenging with many parameters related to cell cycle such as Cyclin D1 or p16INK4A expression levels demonstrating no correlation with response (48).
Other genes of interest revealed in the transcriptomic analyses of palbociclib-treated and recovered MCFDCIS tumors were those that did not return to baseline following cessation of treatment. It is notable that in both the human tumor cells and the surrounding mouse stroma the majority of these genes have no known role in cell-cycle regulation. In the tumor cells, both NELL2 and KLK11 were upregulated by palbociclib that was further increased following termination of drug treatment, which may suggest that treatment selects for populations of tumor cells expressing high levels of these genes. KLK11 is a serine protease and NELL2 is a glycoprotein with several epidermal growth factor-like domains. Although the former has no known role basal breast cancer survival, high expression of NELL2 is associated with significantly prolonged recurrence-free survival in patients with basal breast cancers (Supplementary Fig. S6B). Therefore, treatment with palbociclib could generate lasting benefits. Many genes that were downregulated within the tumor cells by palbociclib did not return to baseline levels after cessation of treatment are involved in processes such as extracellular reorganization, RNA processing, and inflammatory signaling. The contributions of these genes to long-term CDK4/6 inhibitor response is worthy of further study.
Genes with lasting downregulation in the mouse stroma were more homogeneous and comprised almost entirely of factors involved in IFN-mediated immune signaling. CDK4/6 inhibition and its relationship to immune regulation has been a topic of both interest and controversy, as groups have reported both pro- and anti-immune responses which may impact the overall outcome of treatment with this class of drugs (49–52). One proposed mechanism of antitumor immune regulation driven by CDK4/6 inhibition is the induction of an IFN-driven viral mimicry response initiated by cell-intrinsic factors, mediated through repression of the E2F target gene DMNT1 (50). However, in our study we did not observe significant DMNT1 repression in either the human tumor or the mouse stroma in response to palbociclib.
Furthermore, several of the genes involved in antigen processing and presentation such as Tap1, Ifit2, Oas2, Oasl2, and Stat1 that were previously reported to be upregulated in tumor cells by CDK4/6 inhibition (50) actually showed lasting downregulation in the stromal analysis in our mouse model (Fig. 6E). These differences could be due to the cell types examined, treatment duration or the breast cancer models used. Our xenograft experiments were performed in athymic nude mice that lack functional T-cell populations and it is difficult to assess the full impact of palbociclib on antitumor immunity in this model. The suppression of antigen-processing genes is associated with a significant reduction in recurrence-free survival of patients with basal breast cancer and indicates that palbociclib in some contexts may have long-term and potentially detrimental immunosuppressive effects (Supplementary Fig. S7). Future studies could address this question in early stage breast cancer models from syngeneic immune-competent animals.
Three CDK4/6 inhibitors are now FDA approved for the treatment of advanced stage breast cancer either alone or in combination with aromatase inhibitor treatment (8–10). Our study demonstrates that short-term treatment with palbociclib is able to slow the growth and progression of both basal DCIS and early invasive tumor models, generating tumor stasis without significant side effects, and that the persistent influence of the drug even after treatment cessation may result in less aggressive tumors and reduced overall recurrence. Based on these findings, palbociclib might be best applied as a neoadjuvant therapy following diagnosis with DCIS and prior to surgical removal of lesions as a means of preventing further disease progression and potential dissemination. An ongoing phase II clinical trial at Georgetown University titled “Preoperative Palbociclib in Patients with DCIS of the Breast that are Candidates for Surgery (WI223281)” (NCT03535506) will investigate this potential. The results of this study and other future studies should address whether different doses, increased duration of treatment, or combination therapies with palbociclib could contribute to a more lasting impact of CDK4/6 in the treatment of earlier stage breast cancer.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: W.B. Kietzman, V. Ory, G. Sharif, A. Wellstein, A.T. Riegel
Development of methodology: W.B. Kietzman, V. Ory, G. Sharif, A. Wellstein, A.T. Riegel
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): W.B. Kietzman, B. Kallakury, A.T. Riegel
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): W.B. Kietzman, G.T. Graham, M.H. Kushner, B. Kallakury, A. Wellstein, A.T. Riegel
Writing, review, and/or revision of the manuscript: W.B. Kietzman, G.T. Graham, V. Ory, M.H. Kushner, G.T. Gallanis, B. Kallakury, A. Wellstein, A.T. Riegel
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): W.B. Kietzman, G.T. Graham, G.T. Gallanis
Study supervision: A.T. Riegel
Acknowledgments
We thank Drs. Deb Berry, Elena Tassi, and Marcel Schmidt for their valuable discussions. We thank Maria Idalia Cruz for her technical assistance with in vivo experiments. We thank Dr. Paula Pohlmann, lead investigator of NCT03535506, for her collaboration and insight on this project. T32 Training Grant in Tumor Biology CA009686, F31 CA232664 (to M. Kushner), R01 CA 231291 (to A. Wellstein), R01 CA 205632 (to A. Riegel), and P30CA051008 (PI: Weiner): usage of the following shared resources: microscopy and imaging, tissue culture, flow cytometry, histopathology, and animal models.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.