Chromatin insulation orchestrates matrix metalloproteinase gene cluster expression reprogramming in aggressive breast cancer tumors.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive subtype that exhibits a high incidence of distant metastases and lacks targeted therapeutic options. Here we explored how the epigenome contributes to matrix metalloprotease (MMP) dysregulation impacting tumor invasion, which is the first step of the metastatic process. METHODS: We combined RNA expression and chromatin interaction data to identify insulator elements potentially associated with MMP gene expression and invasion. We employed CRISPR/Cas9 to disrupt the CCCTC-Binding Factor (CTCF) binding site on an insulator element downstream of the MMP8 gene (IE8) in two TNBC cellular models. We characterized these models by combining Hi-C, ATAC-seq, and RNA-seq with functional experiments to determine invasive ability. The potential of our findings to predict the progression of ductal carcinoma in situ (DCIS), was tested in data from clinical specimens. RESULTS: We explored the clinical relevance of an insulator element located within the Chr11q22.2 locus, downstream of the MMP8 gene (IE8). This regulatory element resulted in a topologically associating domain (TAD) boundary that isolated nine MMP genes into two anti-correlated expression clusters. This expression pattern was associated with worse relapse-free (HR = 1.57 [1.06 - 2.33]; p = 0.023) and overall (HR = 2.65 [1.31 - 5.37], p = 0.005) survival of TNBC patients. After CRISPR/Cas9-mediated disruption of IE8, cancer cells showed a switch in the MMP expression signature, specifically downregulating the pro-invasive MMP1 gene and upregulating the antitumorigenic MMP8 gene, resulting in reduced invasive ability and collagen degradation. We observed that the MMP expression pattern predicts DCIS that eventually progresses into invasive ductal carcinomas (AUC = 0.77, p < 0.01). CONCLUSION: Our study demonstrates how the activation of an IE near the MMP8 gene determines the regional transcriptional regulation of MMP genes with opposing functional activity, ultimately influencing the invasive properties of aggressive forms of breast cancer.

Calreticulin is a Critical Cell Survival Factor in Malignant Neoplasms.

Calreticulin (CRT) is a high-capacity Ca2+ protein whose expression is up-regulated during cellular transformation and is associated with disease progression in multiple types of malignancies. At the same time, CRT has been characterized as an important stress-response protein capable of inducing immunogenic cell death (ICD) when translocated to the cell surface. It remains unclear why CRT expression is preserved by malignant cells during the course of transformation despite its immunogenic properties. In this study, we identify a novel, critical function of CRT as a cell survival factor in multiple types of human solid-tissue malignancies. CRT knockdown activates p53, which mediates cell-death response independent of executioner caspase activity and accompanied full-length poly ADP ribose polymerase (PARP) cleavage. Mechanistically, we show that down-regulation of CRT results in mitochondrial Ca2+ overload and induction of mitochondria permeability transition pore (mPTP)-dependent cell death, which can be significantly rescued by the mPTP inhibitor, Cyclosporin A (CsA). The clinical importance of CRT expression was revealed in the analysis of the large cohort of cancer patients (N = 2,058) to demonstrate that high levels of CRT inversely correlates with patient survival. Our study identifies intracellular CRT as an important therapeutic target for tumors whose survival relies on its expression.

CD271 is a molecular switch with divergent roles in melanoma and melanocyte development.

Dysregulation of signaling networks controlling self-renewal and migration of developmental cell lineages is closely linked to the proliferative and invasive properties of tumors. Identification of such signaling pathways and their critical regulators is vital for successful design of effective targeted therapies against neoplastic tissue growth. The neurotrophin receptor (CD271/NGFR/p75NTR) is a key regulator of the melanocytic cell lineage through its ability to mediate cell growth, survival, and differentiation. Using clinical melanoma samples, normal melanocytes and global gene expression profiling we have investigated the role of CD271 in rewiring signal transduction networks of melanoma cells during neoplastic transformation. Our analysis demonstrates that depending on the cell fate of tumor initiation vs normal development, elevated levels of CD271 can serve as a switch between proliferation/survival and differentiation/cell death. Two divergent arms of neurotrophin signaling hold the balance between positive regulators of tumor growth controlled by E2F, MYC, SREBP1 and AKT3 pathways on the one hand, and differentiation, senescence, and apoptosis controlled by TRAF6/IRAK-dependent activation of AP1 and TP53 mediated processes on the other hand. A molecular network map revealed in this study uncovers CD271 as a context-specific molecular switch between normal development and malignant transformation.

Down-regulation of FZD3 receptor suppresses growth and metastasis of human melanoma independently of canonical WNT signaling.

Frizzled 3 receptor (FZD3) plays an important role in the homeostasis of the neural crest and its derivatives, which give rise to pigment-synthesizing cells, melanocytes. While the role for FZD3 in specification of the melanocytic lineage from neural crest is well established, its significance in the formation of melanoma, its associated malignancy, is less understood. In this study we identified FZD3 as a critical regulator of human melanoma tumorigenesis. Down-regulation of FZD3 abrogated growth, colony-forming potential, and invasive capacity of patient-derived melanoma cells. Xenotransplantation of tumor cells with down-regulated FZD3 levels originating from melanomas carrying the BRAF(V600) mutation uniformly suppressed their capacity for tumor and metastasis formation. FZD3 knockdown leads to the down-regulation of the core cell cycle protein components (cyclins D1, E2, B1, and CDKs 1, 2, and 4) in melanomas with a hyperactive BRAF oncogene, indicating a dominant role of this receptor during melanoma pathogenesis. Enriched pathway analysis revealed that FZD3 inhibits transcriptional networks controlled by CREB5, FOXD1, and ATF3, which suppress the activity of MAPK-mediated signaling. Thus, FZD3 establishes a positive-feedback mechanism that activates MAPK signal transduction network, critical to melanoma carcinogenesis. Importantly, high levels of FZD3 mRNA were found to be correlated with melanoma advancement to metastatic stages and limited patient survival. Changes in gene-expression patterns mediated by FZD3 activity occur in the absence of nuclear ß-catenin function, thus representing an important therapeutic target for the melanoma patients whose disease progresses independent of canonical WNT signaling.

Antibody Therapy Targeting CD47 and CD271 Effectively Suppresses Melanoma Metastasis in Patient-Derived Xenografts.

The high rate of metastasis and recurrence among melanoma patients indicates the existence of cells within melanoma that have the ability to both initiate metastatic programs and bypass immune recognition. Here, we identify CD47 as a regulator of melanoma tumor metastasis and immune evasion. Protein and gene expression analysis of clinical melanoma samples reveals that CD47, an anti-phagocytic signal, correlates with melanoma metastasis. Antibody-mediated blockade of CD47 coupled with targeting of CD271(+) melanoma cells strongly inhibits tumor metastasis in patient-derived xenografts. This therapeutic effect is mediated by drastic changes in the tumor and metastatic site immune microenvironments, both of whichwhich exhibit greatly increased density of differentiated macrophages and significantly fewer inflammatory monocytes, pro-metastatic macrophages (CCR2(+)/VEGFR1(+)), and neutrophils, all of which are associated with disease progression. Thus, antibody therapy that activates the innate immune response in combination with selective targeting of CD271(+) melanoma cells represents a powerful therapeutic approach against metastatic melanoma.

Therapeutic implications of melanoma heterogeneity.

Over the last decade, the treatment of metastatic melanoma has been revolutionized by the translation of molecular insights into therapeutic benefit for patients. These include advances in immunotherapeutic and small-molecule approaches aimed at destroying cells with immunogenic antigens or gene mutations. Despite these advances, the limited durability of clinical response and eventual disease progression underscores a need for better understanding of mechanisms underlying tumor development. Current targeted therapies are developed partly based on the rationale that tumors are primarily clonal with respect to mutant oncogene or cell surface antigen target. However, with the advancement of cell isolation and transplantation approaches coupled with deep sequencing and mutation detection techniques, it has become increasingly clear that tumors are polyclonal. As a result, sensitive malignant cells are eradicated by treatment while the remaining tumor cell populations are conferred varying degrees of resistance and survival advantages by harbouring or acquiring certain epigenetic and genetic abnormalities. Tumor heterogeneity thus represents a major obstacle to the successful application of current therapies. Gaining insights into the cellular and molecular aspects of tumor diversity will not only facilitate the development and selection of therapeutic targets but also promote the evolution of precision medicine. In this viewpoint, we will discuss the implications of tumor heterogeneity for the treatment of metastatic melanoma and propose approaches to accelerate the translation of scientific discovery into improved clinical outcomes.

Profiling Melanoma Heterogeneity Using Microwell RNA Cytometry.

Unraveling heterogeneity of melanoma to discover new subpopulations of cells within the tumor has been fundamental to many advances in cancer biology, including identification of tumor initiating subsets and cells resisting immune-therapeutic approaches (Boiko et al., Nature 466:133-137, 2010; Civenni et al., Cancer Res 71:3098-3109, 2011; Schatton et al., Nature 451:345-349, 2008; Landsberg et al., Nature 490:412-416, 2012; Fang et al., Cancer Res 65:9328-9337, 2005). Traditionally, these discoveries were made possible due to the existence of well-characterized antibodies that enabled identification of cells homogeneous for the expression of specific cell surface antigen. However, further unwinding of heterogeneous cell populations into homogenous subsets in order to more precisely define their functional profile is limited by the availability of highly specific antibodies. Here we describe a technique capable of identifying homogeneous cell populations in heterogeneous sample based on the transcriptome profile. This approach enables semiquantitative measurement of gene expressions in hundreds to thousands of single cells in one step, paving the way to identify homogenous subpopulations of melanoma cells based on gene transcripts, independent of the availability of antibodies.

Isolation of melanoma tumor-initiating cells from surgical tissues.

A new model of cancer progression has been put forward that predicts existence of tumor stem cells (TSCs) in the heterogeneous bulk tumor mass that self-renew, are resistant to chemo- and radiotherapies, and sustain tumor growth during the course of its progression or relapse (Ailles and Weissman, Curr Opin Biotechnol 18:460-466, 2007; Chan et al., Proc Natl Acad Sci U S A 106:14016-14021, 2009; D'Angelo and Wicha, Prog Mol Biol Transl Sci 95:113-158, 2010; O'Brien, Semin Radiat Oncol 19:71-77, 2009; Park et al., Mol Ther 17:219-230, 2009). Using most advanced methods of cell purification and transplantation, our laboratory and another independent study identified melanoma stem cells as CD271(NFGR/p75)+ cells from surgical human specimens (Boiko et al., Nature 466:133-137, 2010; Civenni et al., Cancer Res 71:3098-3109, 2011). Here we describe in great detail an approach for isolating tumor-initiating cells from freshly resected melanomas (Boiko et al., Nature 466:133-137, 2010).

VHL loss in renal cell carcinoma leads to up-regulation of CUB domain-containing protein 1 to stimulate PKC{delta}-driven migration.

A common genetic mutation found in clear cell renal cell carcinoma (CC-RCC) is the loss of the von Hippel-Lindau (VHL) gene, which results in stabilization of hypoxia-inducible factors (HIFs), and contributes to cancer progression and metastasis. CUB-domain-containing protein 1 (CDCP1) was shown to promote metastasis in scirrhous and lung adenocarcinomas as well as in prostate cancer. In this study, we established a molecular mechanism linking VHL loss to induction of the CDCP1 gene through the HIF-1/2 pathway in renal cancer. Also, we report that Fyn, which forms a complex with CDCP1 and mediates its signaling to PKCδ, is a HIF-1 target gene. Mechanistically, we found that CDCP1 specifically regulates phosphorylation of PKCδ, but not of focal adhesion kinase or Crk-associated substrate. Signal transduction from CDCP1 to PKCδ leads to its activation, increasing migration of CC-RCC. Furthermore, patient survival can be stratified by CDCP1 expression at the cell surface of the tumor. Taken together, our data indicates that CDCP1 protein might serve as a therapeutic target for CC-RCC.

Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271.

The question of whether tumorigenic cancer stem cells exist in human melanomas has arisen in the last few years. Here we show that in melanomas, tumour stem cells (MTSCs, for melanoma tumour stem cells) can be isolated prospectively as a highly enriched CD271(+) MTSC population using a process that maximizes viable cell transplantation. The tumours sampled in this study were taken from a broad spectrum of sites and stages. High-viability cells isolated by fluorescence-activated cell sorting and re-suspended in a matrigel vehicle were implanted into T-, B- and natural-killer-deficient Rag2(-/-)gammac(-/-) mice. The CD271(+) subset of cells was the tumour-initiating population in 90% (nine out of ten) of melanomas tested. Transplantation of isolated CD271(+) melanoma cells into engrafted human skin or bone in Rag2(-/-)gammac(-/-) mice resulted in melanoma; however, melanoma did not develop after transplantation of isolated CD271(-) cells. We also show that in mice, tumours derived from transplanted human CD271(+) melanoma cells were capable of metastatsis in vivo. CD271(+) melanoma cells lacked expression of TYR, MART1 and MAGE in 86%, 69% and 68% of melanoma patients, respectively, which helps to explain why T-cell therapies directed at these antigens usually result in only temporary tumour shrinkage.

A systematic search for downstream mediators of tumor suppressor function of p53 reveals a major role of BTG2 in suppression of Ras-induced transformation.

Factors that mediate p53 tumor suppressor activity remain largely unknown. In this study we describe a systematic approach to identify downstream mediators of tumor suppressor function of p53, consisting of global gene expression profiling, focused short hairpin RNA (shRNA) library creation, and functional selection of genetic elements cooperating with oncogenic Ras in cell transformation. This approach is based on our finding that repression of gene expression is a major event, occurring in response to p53 inactivation during transformation and immortalization of primary cells. Functional analysis of the subset of genes universally down-regulated in the cells that lacked functional p53 revealed BTG2 as a major downstream effector of p53-dependent proliferation arrest of mouse and human fibroblasts transduced with oncogenic Ras. shRNA-mediated knockdown of BTG2 cooperates with oncogenic Ras to transform primary mouse fibroblasts containing wild-type transcriptionally active p53. Repression of BTG2 results in up-regulation of cyclins D1 and E1 and phosphorylation of Rb and, in cooperation with other oncogenic elements, induces neoplastic transformation of primary human fibroblasts. BTG2 expression was found to be significantly reduced in a large proportion of human kidney and breast carcinomas, suggesting that BTG2 is a tumor suppressor that links p53 and Rb pathways in human tumorigenesis.

Secreted transforming growth factor beta2 activates NF-kappaB, blocks apoptosis, and is essential for the survival of some tumor cells.

The basis of constitutive activation of NF-kappaB, essential for survival and resistance to apoptosis in many tumors, is not well understood. We find that transforming growth factor beta2 (TGFbeta2), predominantly in its latent form, is secreted by several different types of tumor cell lines that exhibit constitutively active NF-kappaB and that TGFbeta2 potently stimulates the activation of NF-kappaB in reporter cells. Suppression of TGFbeta2 expression by small interfering RNA kills prostate cancer PC3 cells, indicating that the TGFbeta2-NF-kappaB pathway is important for their viability. These findings identify TGFbeta2 as a potential target for therapeutic strategies to inhibit the growth of tumor cells that depend on constitutively active NF-kappaB, or to sensitize them to treatment with cytotoxic drugs.

Cdk4 disruption renders primary mouse cells resistant to oncogenic transformation, leading to Arf/p53-independent senescence.

A large number of human cancers display alterations in the Ink4a/cyclin D/Cdk4 genetic pathway, suggesting that activation of Cdk4 plays an important role in oncogenesis. Here we report that Cdk4-null mouse embryonic fibroblasts are resistant to transformation in response to Ras activation with dominant-negative (DN) p53 expression or in the Ink4a/Arf-null background, judged by foci formation, anchorage-independent growth, and tumorigenesis in athymic mice. Cdk4-null fibroblasts proliferate at normal rates during early passages. Whereas Cdk4(+/+)Ink4a/Arf(-/-) cells are immortal in culture, Cdk4(-/-)Ink4a/Arf(-/-) cells undergo senescence during continuous culture, as do wild-type cells. Activated Ras also induces premature senescence in Cdk4(-/-)Ink4a/Arf(-/-) cells and Cdk4(-/-) cells with DNp53 expression. Thus, Cdk4 deficiency causes senescence in a unique Arf/p53-independent manner, which accounts for the loss of transformation potential. Cdk4-null cells express high levels of p21(Cip1/Waf1) with increased protein stability. Suppression of p21(Cip1/Waf1) by small interfering RNA (siRNA), as well as expression of HPV-E7 oncoprotein, restores immortalization and Ras-mediated transformation in Cdk4(-/-)Ink4a/Arf(-/-) cells and Cdk4(-/-) cells with DNp53 expression. Therefore, Cdk4 is essential for immortalization, and suppression of Cdk4 could be a prospective strategy to recruit cells with inactive Arf/p53 pathway to senescence.