Gelman Lab and Members

1) Masashi Muramatsu, Ph.D. (Postdoc): The role of SSeCKS/AKAP12 in metastasis suppression.

Data from our lab indicate that the SSeCKS/AKAP12 gene product functions as a metastasis suppressor, likely by inhibiting tumor-induced expression of pro-angiogenic factors such as VEGF and HIF-1a (work by a previous postdoc, Bing Su, Ph.D.).  We and other have amassed data showing that SSeCKS expression is typically lost in metastatic prostate cancers, in about a third of the cases due to gene deletion, and in two-thirds, due to epigenetic silencing (associated with hypermethylation of promoter CpG islands).  In human prostate cancer cases with metastasis, we published that SSeCKS loss correlates with earlier onset of metastatic disease and poorer long-term survival.  We also showed that SSeCKS re-expression in prostate cancer cells suppresses neovascularization at metastatic sites although it has little effect on growth of tumors at the primary site.  The avascular micrometastases induced by SSeCKS-re-expressing tumor cells exhibit downregulated VEGF expression, and indeed, the forced re-expression of VEGF in these cells could rescue full macrometastasis-forming activity. 

We also noted that higher grade human prostate tumors (Gleason sum >7) exhibited not only downregulated SSeCKS expression in prostate epithelial cells but also in adjoining stroma defining the so-called tumor microenvironment (TME).  To address whether SSeCKS controls metastatic progression through a role in TME cells, we produced AKAP12-null mice and then tested these for increased metastatic potential when challenged with tumor xenografts or carcinogens.  Our data indicate that carcinogens such as DMBA and TPA not only induce much greater skin cancer (squamous cell carcinomas) in AKAP12-null compared to WT hosts, but they also induce increased progression to spontaneous metastasis formation, namely spindle cell carcinomas.  AKAP12-null hosts exhibit increased levels of spontaneous peritoneal and lung metastases arising from orthotopic, subcutaneous B16F10 melanomas (work by a previous postdoc, Shin Akakura, Ph.D.) or from i.v. tumor injections. 

2) Henry Withers (PhD Grad. Student): Identification and characterization of novel metastasis suppressors in breast cancer. 

We are using a so-called 3rd generation genomic shRNA library (cloned into high-tier lentivirus vectors) to identify genes that suppress oncogenic invasiveness in poorly metastatic breast (T47D) or prostate cancer (LNCaP) cells.  Following infection of target cells, Henry isolated clones with increased invasiveness through Matrigel and increased metastasis from orthotopic sites (mammary glands or prostates) in nude mice.  He is characterizing the genes from these screens and elucidating how they suppress metastasis in breast cancer cell lines.  He will then determine whether human breast cancer cases associated with metastasis show decreased levels of these gene products compared to non-metastatic cancer cases. 

3) Karina Miller (PhD Grad. Student)Role of SSeCKS/AKAP12 in prostate cancer progression and metastasis. 

Previous data from our lab indicate that AKAP12-null mouse embryo fibroblasts (MEF) exhibit Rb-dependent premature cell senescence marked by polyploidy and multinucleation.  Moreover, AKAP12-null prostates are hyperplastic and they express markers of increased senescence such as senescence-associated β-galactosidase, p16ink4a or γ-H2AX.  To investigate whether the combined loss of AKAP12 and Rb is sufficient to induce prostate cancer progression, a previous graduate student, Hyun-Kyung Ko, Ph.D. (with the help of Jennifer Peresie and a previous postdoc, Shin Akakura, Ph.D.) crossed our AKAP12-null mice with Pb-Cre;Rbfl/fl mice (or to Pb-Cre; p53fl/fl mice, as a control), which produce prostate-specific (PE) Rb deletions.  The Akap12-/-;RbPE-/- mice displayed high grade prostatic intraepithelial neoplasias (HG-PIN) by 7 months of age, but there was little progression to adenocarcinoma.  In contrast, all the mice showed evidence of lymph node metastasis, consisting of well-differentiated lesions expressing markers of a transitional basal/luminal prostate cell phenotype (cytokeratin 14, 8, AR and p63).  This suggests that rare cells in the HG-PIN lesions are marked by the combined loss of Rb and SSeCKS/Akap12 for metastatic progression.  Currently, we are crossing these mice with lineage-tracing transgenic stocks so that we can determine if the metastatic cells arise from either basal (CK14+) or luminal (CK8+) cells in the prostate, and then characterize the epigenetic signature of early metastatic dissemination.  We are also crossing this model into a prostate-specific conditional loss of the Smad4 suppressor gene, based on evidence that human prostate cancer metastases with decreased SSeCKS and Rb also suffer from Smad4 loss. 

4) Julie McGrath (Masters student)Identification of genes enabling breast cancer dormancy in endosteal bone.

Breast cancers often metastasize to the bone, and after long periods of dormancy, can develop into osteolytic lesions.  Using 3D growths of stromal and bone cells that recapitulate the endosteal niche, Julie showed that a usually aggressive breast cancer cell line, MDA-MB-231, fails to proliferate.  Using MDA-MB-231 cells infected with a genomics shRNA lentivirus library (DECIPHER), Julie is screening by next-gen sequencing protocols for gene knockdowns that facilitate the proliferation of the breast cancer cells in the 3D endosteal niche.  She will then characterize whether the overexpression of such genes is associated with loss of dormancy in the bone environment, leading to aggressive metastatic disease.  The long-term goal is to identify “reactivation” proteins that can therapeutically targeted to treat or prevent breast cancer metastasis in the bone. 

Other ongoing projects:

Genomic Progression Signatures Regulating Castration-Recurrent Prostate Cancer (CR-CaP) or Barrett’s Esophagus.

There is growing evidence that CR-CaP is driven by the activation of the androgen receptor (AR) in the absence of serum androgen levels. Our group and others have shown that AR can be activated by direct phosphorylation by Src family or Ack1 tyrosine kinases.  The current project is to use androgen-dependent (AD)/CR-CaP cell line and tumor pairs to analyze the AR cistrome by AR-ChIP-seq and then to identify differentially-expressed AR-engaged genes by transcriptome-seq.  Bioinformatic analyses of these data will allow us to identify a CR-CaP progression signature that might explain the resistance of CR-CaP to conventional treatments, and that might identify novel targetable pathways.  In parallel, Indranil and collaborators (Kandel, Campbell, Liu, Morrison, Buck) are performing transcriptome-seq, miRNA-seq, microbiome-seq and other next-gen analyses to identify potential genomic signatures that explain the progression of Barrett’s esophagus metaplasias to dysplasias and eventually, esophageal adenocarcinomas. 

The role of FAK/Pyk2 activated of NFκB signaling in tumor-associated macrophages in metastatic progression of breast cancer.

We have studied the role of FAK/Src signaling in the control of cytoskeletal architecture, adhesion and oncogenic motility.  To this end, Sheila has sought to identify novel FAK substrates and to elucidate the FAK phosphorylation motif.  After produced baculovirus FAK, she performed in situ-FAK kinase assays using protein microarrays containing thousands of GST-protein fusions or tyrosine-containing peptides, and has identified many potentially new FAK-specific substrates.  In parallel, she has analyzed how the Src/FAK-induced tyrosine phosphorylation of PRAK/MAPKAP-5 affects adhesion of cancer cells.

The role of FOXO4 in suppressing prostate cancer metastasis

We previously identified with Ryan’s help that the shRNA-mediated knockdown of FOXO4 in LNCaP prostate cancer cells resulted in increased Matrigel invasiveness and increased lymph node metastasis following orthotopic (prostate) injection (work by a previous postdoc, Bing Su, Ph.D.).  In addition to identifying new metastasis suppressor genes in LNCaP cells using the 3rd generation genomic shRNA library, Ryan is investigating which FOXO4-regulated genes are likely to suppress invasiveness and metastatic potential. 

Other projects:

We are collaborating with labs outside RPCI to investigate the role of SSeCKS/AKAP12 in i) suppression of aortic restenosis by retinoids, ii) exercise-induced cardiac failure, iii) learning and memory, iv) B-cell development, v) blood brain barrier formation, vi) brain injury healing, viii) chronic obstructive pulmonary disease, viii) aging, and ix) glomerulonephritis.  We are also collaborating to identify genomic signatures of melanoma metastasis.