October Core Spotlight: Rodent Histopathology

October, 2017

In this DF/HCC News Spotlight Edition, we present the DF/HCC Rodent Histopathology Core, which provides DF/HCC members with high quality professional, technical, and educational pathology services, supporting investigator research that leads to the identification of pathologic processes in mice and other animal models that can be directly translatable to human disease.

Key Services

Services provided by the Core include whole mouse necropsies, tissue processing, sectioning frozen and paraffin embedded tissues, and conventional and special histological staining.

Project Highlights

Sox2 Suppresses gastric tumorigenesis in mice

PIs: K. HochedlingerMGH (Cancer Cell Biology and Cancer Genetics Programs); R. MostoslavskyMGH (Cancer Genetics Program); and A. BassDFCI (Cancer Genetics and Gastrointestinal Malignancies Programs)

Sox2 expression marks gastric stem and progenitor cells, raising important questions regarding the genes regulated by Sox2 and the role of Sox2 itself during stomach homeostasis and disease. By using ChIP-seq analysis, we have found that the majority of Sox2 targets in gastric epithelial cells are tissue specific and related to functions such as endoderm development, Wnt signaling, and gastric cancer. Unexpectedly, we found that Sox2 itself is dispensable for gastric stem cell and epithelial self-renewal, yet Sox2(+) cells are highly susceptible to tumorigenesis in an Apc/Wnt-driven mouse model. Moreover, Sox2 loss enhances, rather than impairs, tumor formation in Apc-deficient gastric cells in vivo and in vitro by inducing Tcf/Lef-dependent transcription and upregulating intestinal metaplasia-associated genes, providing a mechanistic basis for the observed phenotype. Together, these data identify Sox2 as a context-dependent tumor suppressor protein that is dispensable for normal tissue regeneration but restrains stomach adenoma formation through modulation of Wnt-responsive and intestinal genes.

Efficient Tumor Formation following Wnt-Driven Transformation of Adult Sox2+ Gastric Cells

(A)   Breeding strategy to determine tumor-initiating potential of Sox2+ cells.

(B) IHC for β-catenin in Apc KO antrum 3 days, 1 month, and 7 months after tamoxifen induction. Arrows point to cells or tumors with nuclear accumulation of β-catenin. Blue dotted line highlights microadenoma.

(C) Stomachs from Apc WT and Apc KO mice 1 month and 1 year after tamoxifen induction. Arrow indicates enlarged antrum in Apc KO stomach. Blue dotted line highlights large tumors in antrum of Apc KO stomach.

(D and E) IHC for YFP (D) and Sox2 (E) on adenoma from Apc KO antrum 7 months after tamoxifen induction.

(F) Breeding strategy to compare tumor-initiating potential of Sox2+ and Lgr5+ cells.

(G) Quantification of adenomas developing in Lgr5CreER and Sox2CreER mice 1 week (p = 0.0006; t test) and 1 month (p < 0.0001; t test) after tamoxifen induction.

(H) IHC for β-catenin in Lgr5CreER and Sox2CreER antrum 1 week after induction. Abbreviations: FS, forestomach; C, corpus; A, antrum; I, intestine.

Contribution of the Core: Tissue and slide preparation and H&E staining was performed by Core technologists. Histopathological interpretation was performed by R. BronsonHMS. 

Publication: Sarkar A, Huebner AJ, Sulahian R, Anselmo A, Xu X, Flattery K, Desai N, Sebastian C, Yram MA, Arnold K, Rivera M, Mostoslavsky R, Bronson R, Bass AJ, Sadreyev R, Shivdasani RA, Hochedlinger K. Sox2 Suppresses Gastric Tumorigenesis in Mice. Cell Rep. 16:1829-1921, 2016.

Deletion of interstitial genes between TMPRSS2 and ERG promotes prostate cancer progression

PIs: Z. LiBWH (Breast Cancer and Prostate Cancer Programs) and L. MucciHSPH (Cancer Epidemiology, and Prostate Cancer Programs) 

TMPRSS2-ERG gene fusions that occur frequently in human prostate cancers can be generated either through insertional chromosomal rearrangement or by intrachromosomal deletion. Genetically, a key difference between these two mechanisms is that the latter results in deletion of a ∼3-Mb interstitial region containing genes with unexplored roles in prostate cancer. In this study, we characterized two mouse models recapitulating TMPRSS2-ERG insertion or deletion events in the background of prostate-specific PTEN deficiency. We found that only the mice that lacked the interstitial region developed prostate adenocarcinomas marked by poor differentiation and epithelial-to-mesenchymal transition. Mechanistic investigations identified several interstitial genes, including Ets2 and Bace2, whose reduced expression correlated in the gene homologs in human prostate cancer with biochemical relapse and lethal disease. Accordingly, PTEN-deficient mice with prostate-specific knockout of Ets2 exhibited marked progression of prostate adenocarcinomas that was partly attributed to activation of MAPK signaling. 

Collectively, our findings established that Ets2 is a tumor suppressor gene in prostate cancer, and its loss along with other genes within the TMPRSS2-ERG interstitial region contributes to disease progression. 

Reduced dosage of Ets2 contributes to PCa progression under the Pten-null background

(A) Prostate regeneration assay using PtenL/L or Ets2L/+;PtenL/L prostate cells infected with Ad-CMV-Cre adenovirus prior to implantation. Red arrow denotes a lesion resembling HG-PIN. Scale bars are 50μm.

(B) H&E staining showing enhanced HG-PIN phenotype in a 6-month old Pb-Cre;Ets2L/+;PtenL/L male compared to its age-matched Pb-Cre;PtenL/L control male in the dorsolateral and ventral lobes. Scale bars are 100μm.

(C) H&E staining showing invasive PCa and poorly differentiated prostate adenocarcinoma phenotype in a 9-month old Pb-Cre;Ets2L/L;PtenL/L male compared to its age-matched Pb-Cre;PtenL/L control male in the dorsolateral and ventral lobes. Scale bars are 100μm.

Contribution of the Core: Tissue and slide preparation and H&E staining was performed by Core technologists. Histopathological interpretation was performed by R. BronsonHMS.

Publication: Linn DE, Penney KL, Bronson RT, Mucci LA, Li Z. Deletion of interstitial genes between TMPRSS2 and ERG promotes prostate cancer progression. Cancer Res. 76:1869-1881, 2016. 574.

The APC/C E3 Ligase Complex Activator FZR1 Restricts BRAF Oncogenic Function  

PI: P.P. PandolfiBIDMC (Breast Cancer and Prostate Cancer Program)

BRAF drives tumorigenesis by coordinating the activation of the RAS/RAF/MEK/ERK oncogenic signaling cascade. However, upstream pathways governing BRAF kinase activity and protein stability remain undefined. Here, we report that in primary cells with active APCFZR1, APCFZR1 earmarks BRAF for ubiquitination-mediated proteolysis, whereas in cancer cells with APC-free FZR1, FZR1 suppresses BRAF through disrupting BRAF dimerization. Moreover, we identified FZR1 as a direct target of ERK and CYCLIN D1/CDK4 kinases. Phosphorylation of FZR1 inhibits APCFZR1, leading to elevation of a cohort of oncogenic APCFZR1 substrates to facilitate melanomagenesis. Importantly, CDK4 and/or BRAF/MEK inhibitors restore APCFZR1 E3 ligase activity, which might be critical for their clinical effects. Furthermore, FZR1 depletion cooperates with AKT hyperactivation to transform primary melanocytes, whereas genetic ablation of Fzr1 synergizes with Pten loss, leading to aberrant coactivation of BRAF/ERK and AKT signaling in mice. Our findings therefore reveal a reciprocal suppression mechanism between FZR1 and BRAF in controlling tumorigenesis.Significance: FZR1 inhibits BRAF oncogenic functions via both APC-dependent proteolysis and APC-independent disruption of BRAF dimers, whereas hyperactivated ERK and CDK4 reciprocally suppress APCFZR1 E3 ligase activity. Aberrancies in this newly defined signaling network might account for BRAF hyperactivation in human cancers, suggesting that targeting CYCLIN D1/CDK4, alone or in combination with BRAF/MEK inhibition, can be an effective anti-melanoma therapy. 

Contribution of the Core: Tissue and slide preparation and H&E staining was performed by Core technologists. Histopathological interpretation was performed by R. BronsonHMS.

Publication: Wan L, Chen M, Cao J, Dai X, Yin Q, Zhang J, Song SJ, Lu Y, Liu J, Inuzuka H, Katon JM, Berry K, Fung J, Ng C, Liu P, Song MS, Xue L, Bronson R, Kirschner MW, Cui R, Pandolfi PP, Wei W. The APC/C E3 Ligase Complex Activator FZR1 Restricts BRAF Oncogenic Function. Cancer Discov 7:424-441. 2017. 

G1 cyclins link proliferation, pluripotency and differentiation of embryonic stem cells

PIs: P. SicinskiDFCI (Breast Cancer and Leukemia Programs); K. LigonDFCI (Neuro-Oncology Program)

Progression of mammalian cells through the G1 and S phases of the cell cycle is driven by the D-type and E-type cyclins. According to the current models, at least one of these cyclin families must be present to allow cell proliferation. Here, we show that several cell types can proliferate in the absence of all G1 cyclins. However, following ablation of G1 cyclins, embryonic stem (ES) cells attenuated their pluripotent characteristics, with the majority of cells acquiring the trophectodermal cell fate. We established that G1 cyclins, together with their associated cyclin-dependent kinases (CDKs), phosphorylate and stabilize the core pluripotency factors Nanog, Sox2 and Oct4. Treatment of murine ES cells, patient-derived glioblastoma tumour-initiating cells, or triple-negative breast cancer cells with a CDK inhibitor strongly decreased Sox2 and Oct4 levels. Our findings suggest that CDK inhibition might represent an attractive therapeutic strategy by targeting glioblastoma tumour-initiating cells, which depend on Sox2 to maintain their tumorigenic potential.

Contribution of the Core: Tissue and slide preparation and H&E staining was performed by Core technologists. Histopathological interpretation was performed by R. BronsonHMS.

Publication: Liu L, Michowski W, Inuzuka H, Shimizu K, Nihira NT, Chick JM, Li N, Geng Y, Meng AY, Ordureau A, Kołodziejczyk A, Ligon KL, Bronson RT, Polyak K, Harper JW, Gygi SP, Wei W, Sicinski P. G1 cyclins link proliferation, pluripotency and differentiation of embryonic stem cells. Nat Cell Biol. 19:177-188, 2017. 

More Information

For more information or to contact the Rodent Histopathology, visit the core website here.