Limiting Self-Renewal of the Basal Compartment by PKA Activation Induces Differentiation and Alters the Evolution of Mammary Tumors

Abstract

Differentiation therapy utilizes our understanding of the hierarchy of cellular systems to pharmacologically induce a shift toward terminal commitment. While this approach has been a paradigm in treating certain hematological malignancies, efforts to translate this success to solid tumors have met with limited success. Mammary-specific activation of PKA in mouse models leads to aberrant differentiation and diminished self-renewing potential of the basal compartment, which harbors mammary repopulating cells. PKA activation results in tumors that are more benign, exhibiting reduced metastatic propensity, loss of tumor-initiating potential, and increased sensitivity to chemotherapy. Analysis of tumor histopathology revealed features of overt differentiation with papillary characteristics. Longitudinal single-cell profiling at the hyperplasia and tumor stages uncovered an altered path of tumor evolution whereby PKA curtails the emergence of aggressive subpopulations. Acting through the repression of SOX4, PKA activation promotes tumor differentiation and represents a possible adjuvant to chemotherapy for certain breast cancers.

Keywords: epithelial-mesenchymal transition; mammary development; tumor differentiation; tumor evolution; tumor metastasis.

Figure 1:. Activation of PKA impairs mammary development and repopulating ability.

(A) PKA-CαR mice were crossed to MMTV-Cre mice to activate the CαR constitutively active allele. (B) FACS plots showing proportions of EpCAM^hiCD49f^med luminal and EpCAM^medCD49f^hi basal compartments upon constitutive activation of PKA and a summary of relative proportions of these subpopulations across multiple mice (C). Carmine alum stained whole mounts of mammary glands from and Prkaca^CαR/fl control mice displaying ductal outgrowth (D). Bar, 3mm. (E) Fluorescence imaging of Krt8 (luminal) and Krt14 (basal) layers in mammary glands from Prkaca^CαR/fl and Prkaca^fl/fl mice. Bar, 150μm. (F) Hematoxylin-eosin staining of FFPE sections of mammary glands depicting terminal ductal lobular units from Prkaca^CαR/fl and Prkaca^fl/fl mice. Bar, 150μm. Organoid assays were carried out to assay for differences in self-renewal potential upon activation of PKA (G, H). Data are shown as mean ± SD (n =10 mice). p value was determined by Student’s two-tailed t test (unpaired). *p < 0.01. Limiting dilution transplantation into cleared fat pads estimated the frequency of mammary repopulating units in Prkaca^CαR/fl and Prkaca^fl/fl controls (I). Schematic representation of breeding strategy to generate mammary-specific Gα_s active mutants (J). FACS was carried out to capture differences in representation of luminal and basal subpopulations (K). Organoid assays carried out to test for differences in self-renewal potential (L) (n = 5 mice). Statistical significance was calculated by a Student t-test (two-tailed) to compare two groups (P < 0.05 was considered significant).

Figure 2:. Specific activation of PKA in basal, but not luminal cells, impairs ductal outgrowth.

Schematic of breeding strategy to generate Krt5-Prkaca^CαR/fl mice that specifically express activated PKA in basal cells (A). FACS plots (B) and fluorescence imaging of ducts (C) show the extent of GFP-expressing cells within the basal compartment and ducts that have undergone Cre recombination in Krt5-Prkaca^CαR/fl mice and Krt5-Prkaca^fl/fl controls at 8 weeks. Bar, 500μm (D) Fluorescence imaging of a cross-section of a mammary duct of Krt5-mTmG-Prkaca^CαR/fl mice to assess histology and extent of tdTomato vs GFP recombination. Bar, 150μm. Schematic of breeding strategy to generate Krt8-Prkaca^CαR/fl mice that specifically express activated PKA in luminal cells (E). FACS plots (F) and fluorescence imaging of ducts (G) show the extent of GFP-expressing that have undergone Cre recombination in Krt8-PrkacaCαR/fl mice and Krt8-Prkaca^fl/fl controls at 8 weeks. Bar, 500μm. (H) Fluorescence imaging of a cross-section of mammary ducts of Krt8-mTmG-Prkaca^CαR/fl mice to assess histology and extent of tdTomato vs GFP recombination. Bar, 150μm.

Figure 3:. Recurrent genomic amplification of the PKA locus in human breast cancer.

Analysis of 1085 breast tumors from the TCGA dataset revealing amplifications in the regulatory (PRKAR) and catalytic (PRKAC) genes that encode PKA subunits (A, B). (C) Box plots show the mRNA expression levels of genes encoding each PKA subunit and their relative expression levels across human breast cancer subtypes. Summary of PRKAR1A and PRKACA amplifications in the TCGA and METABRIC datasets in basal and luminal B subtypes (D). mRNA expression of PRKAR1A, the most amplified PRKA gene, across tumors with and without amplifications in the PRKAR1A locus (E). Kaplan-Meier curves outline the ability of high or low levels of PRKAR1A (F) or PRKACA (G) mRNA levels to stratify prognosis of patients over ten years. PRKACA activation was measured by immunohistochemistry (IHC) using a p-PKA substrate antibody and quantifying the percentage of cells with nuclear signal in primary breast cancer patient samples that harbor amplifications in PRKAR1A compared to controls (H). Statistical significance was calculated by a Student t-test (two-tailed) to compare two groups (P < 0.05 was considered significant) (n=35 patient tumors). IHC using p-PKA substrate antibody showing samples that express different levels of PRKACA activation (I). Bar, 25μm.

Figure 4:. Induction of tumor differentiation upon activation of PKA signaling.

(A) Schematic of breeding strategy to generate MMTV-PyMT mice with active PKA. Differences in survival (B; n = 24 mice) and primary tumor volumes (C; n = 11 mice) of Prkaca^CαR/fl PyMT mice compared to Prkaca^fl/fl PyMT controls. Differences in lung metastatic burden was captured from H&E stained FFPE lung sections (D, E), red arrows indicate macrometastases. Bar, 2mm (n = 13 mice). Treatment with Adriamycin showing differences in response to chemotherapy (F, G) between Prkaca^CαR/fl PyMT mice and Prkaca^fl/fl PyMT controls (n = 20 tumors). Tumor initiating potential of Prkaca^CαR/fl PyMT mice and Prkaca^fl/fl PyMT controls was assessed by limiting dilution transplantation analyses (H). Fluorescence imaging of tumor sections stained with antibodies against E-cadherin, Vimentin, Krt8 and Krt14 revealing epithelial-mesenchymal (I,J) and luminal-basal heterogeneity (K,L). Yellow arrows highlight Vimentin (I) - and Krt14 (K)-expressing tumor cells. Bar, 10μm and 4μm. Error bars represent ± standard deviations of the mean. Statistical significance was calculated by a Student t-test (two-tailed) to compare two groups (P < 0.05 was considered significant) except for survival analyses where significance was determined using a Log-rank (Mantel-Cox) test (P < 0.05 was considered significant) and chemotherapy treatment where a Wald Z-test was used to compute the p-value for the difference of slopes in two treatment groups.

Figure 5:. Differentiated histology induced by PKA across tumor models.

(A) Schematic of mouse crossing strategy to express the doxycycline-inducible Gα_s^R201C/fl allele in the mammary glands of MMTV-PyMT mice following which tumors were harvested and FFPE sections were stained with hematoxylin and eosin (B) to study their histopathology and differentiation status. (C) Schematic of mouse crossing strategy to express the PKA-CαR allele in the mammary glands of C3(1)-Tag mice following which tumors were harvested and FFPE sections stained with hematoxylin and eosin to study their histopathology (D). Fluorescence imaging of Prkaca^CαR/fl C3(1)-Tag and control Prkaca^fl/fl C3(1)-Tag tumors using Vimentin and E-cadherin to assess EMT status (E). Kaplan-Meier curve comparing the survival of Prkaca^CαR/fl C3(1)-Tag and control Prkaca^fl/fl C3(1)-Tag mice (F). Bar values as shown. Statistical significance was determined using a Log-rank (Mantel-Cox) test (P < 0.05 was considered significant; n = 22 mice).

Figure 6:. Altered evolution of tumor cell subpopulations upon activation of PKA.

(A) Schematic of experimental set up to carry out sequential scRNA-Seq of hyperplastic and overt tumor samples from matched mice. tSNE plots show similar cellular constituents in hyperplastic glands of Prkaca^CαR/fl PyMT mice and Prkaca^fl/fl PyMT controls (B, D) but divergent evolution of subpopulations during the transition from hyperplasia to tumor stages of development (C, E). (F) tSNE plots highlighting the expression levels of the Lalba (left) and Sox4 (right) genes in tumors from Prkaca^fl/fl PyMT (top) and Prkaca^CαR/fl PyMT tumors (bottom) The lumino-basal subpopulation is highlighted by the red ellipse. Pseudotime analyses enabled the capture of the directionality of evolution from hyperplasia to tumor in the Prkaca^fl/fl PyMT tumors (G) that is altered upon PKA activation in Prkaca^CαR/fl PyMT tumors (H).

Figure 7:. Suppression of Sox4 activity by PKA leads to tumor differentiation.

CRISPR-Cas9 mediated knockout of Sox4 in PB3 cells to assess differences in cell morphology as captured by bright-field images (A) and the expression of epithelial-mesenchymal markers by immunofluorescence (B). Bar, 5, 10μm. H&E stained FFPE sections of transplanted PB3 tumors were carried to estimate changes in histopathology which would reflect altered differentiation state (C). Bar, 50μm (D) Brightfield images of immunohistochemical staining of Sox4 in FFPE sections from Prkaca^fl/fl PyMT (top) and Prkaca^CαR/fl PyMT (bottom) tumors. Bar, 25μm. PKA phosphorylation sites on Sox4 identified by in-vitro kinase assays (E) including sites in the HMG-box containing DNA binding domain that are conserved across evolution (F). Electrophoretic mobility shift assays carried out in the presence of purified PKA-Cα and Sox4 (right) or PKA-Cα, Sox4 and ATP (left) to test for retardation of mobility upon phosphorylation of Sox4 by PKA (G).