Maria Capra

EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer

Recent experiments have demonstrated that the Polycomb group (PcG) gene EZH2 is highly expressed in metastatic prostate cancer and in lymphomas. EZH2 is a component of the PRC2 histone methyltransferase complex, which also contains EED and SUZ12 and is required for the silencing of HOX gene expression during embryonic development. Here we demonstrate that both EZH2 and EED are essential for the proliferation of both transformed and non‐transformed human cells. In addition, the pRB‐E2F pathway tightly regulates their expression and, consistent with this, we find that EZH2 is highly expressed in a large set of human tumors. These results raise the question whether EZH2 is a marker of proliferation or if it is actually contributing to tumor formation. Significantly, we propose that EZH2 is a bona fide oncogene, since we find that ectopic expression of EZH2 is capable of providing a proliferative advantage to primary cells and, in addition, its gene locus is specifically amplified in several primary tumors.

Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis

The biological antagonism between Notch and Numb controls the proliferative/differentiative balance in development and homeostasis. Although altered Notch signaling has been linked to human diseases, including cancer, evidence for a substantial involvement of Notch in human tumors has remained elusive. Here, we show that Numb-mediated control on Notch signaling is lost in ∼50% of human mammary carcinomas, due to specific Numb ubiquitination and proteasomal degradation. Mechanistically, Numb operates as an oncosuppressor, as its ectopic expression in Numb-negative, but not in Numb-positive, tumor cells inhibits proliferation. Increased Notch signaling is observed in Numb-negative tumors, but reverts to basal levels after enforced expression of Numb. Conversely, Numb silencing increases Notch signaling in normal breast cells and in Numb-positive breast tumors. Finally, growth suppression of Numb-negative, but not Numb-positive, breast tumors can be achieved by pharmacological inhibition of Notch. Thus, the Numb/Notch biological antagonism is relevant to the homeostasis of the normal mammary parenchyma and its subversion contributes to human mammary carcinogenesis.

The ubiquitin ligase HectH9 regulates transcriptional activation by Myc and is essential for tumor cell proliferation.

The Myc oncoprotein forms a binary activating complex with its partner protein, Max, and a ternary repressive complex that, in addition to Max, contains the zinc finger protein Miz1. Here we show that the E3 ubiquitin ligase HectH9 ubiquitinates Myc in vivo and in vitro, forming a lysine 63-linked polyubiquitin chain. Miz1 inhibits this ubiquitination. HectH9-mediated ubiquitination of Myc is required for transactivation of multiple target genes, recruitment of the coactivator p300, and induction of cell proliferation by Myc. HectH9 is overexpressed in multiple human tumors and is essential for proliferation of a subset of tumor cells. Our results suggest that site-specific ubiquitination regulates the switch between an activating and a repressive state of the Myc protein, and they suggest a strategy to interfere with Myc function in vivo.

Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity

ABSTRACT Human tumors are believed to harbor a disabled p53 tumor suppressor pathway, either through direct mutation of the p53 gene or through aberrant expression of proteins acting in the p53 pathway, such as p14ARF or Mdm2. A role for Mdmx (or Mdm4) as a key negative regulator of p53 function in vivo has been established. However, a direct contribution of Mdmx to tumor formation remains to be demonstrated. Here we show that retrovirus-mediated Mdmx overexpression allows primary mouse embryonic fibroblast immortalization and leads to neoplastic transformation in combination with HRasV12. Furthermore, the human Mdmx ortholog, Hdmx, was found to be overexpressed in a significant percentage of various human tumors and amplified in 5% of primary breast tumors, all of which retained wild-type p53. Hdmx was also amplified and highly expressed in MCF-7, a breast cancer cell line harboring wild-type p53, and interfering RNA-mediated reduction of Hdmx markedly inhibited the growth potential of these cells in a p53-dependent manner. Together, these results make Hdmx a new putative drug target for cancer therapy.

Tip60 is a haplo-insufficient tumour suppressor required for an oncogene-induced DNA damage response

The acetyl-transferase Tip60 might influence tumorigenesis in multiple ways. First, Tip60 is a co-regulator of transcription factors that either promote or suppress tumorigenesis, such as Myc and p53. Second, Tip60 modulates DNA-damage response (DDR) signalling, and a DDR triggered by oncogenes can counteract tumour progression. Using Eμ–myc transgenic mice that are heterozygous for a Tip60 gene (Htatip) knockout allele (hereafter denoted as Tip60+/– mice), we show that Tip60 counteracts Myc-induced lymphomagenesis in a haplo-insufficient manner and in a time window that is restricted to a pre- or early-tumoral stage. Tip60 heterozygosity severely impaired the Myc-induced DDR but caused no general DDR defect in B cells. Myc- and p53-dependent transcription were not affected, and neither were Myc-induced proliferation, activation of the ARF–p53 tumour suppressor pathway or the resulting apoptotic response. We found that the human TIP60 gene (HTATIP) is a frequent target for mono-allelic loss in human lymphomas and head-and-neck and mammary carcinomas, with concomitant reduction in mRNA levels. Immunohistochemical analysis also demonstrated loss of nuclear TIP60 staining in mammary carcinomas. These events correlated with disease grade and frequently concurred with mutation of p53. Thus, in both mouse and human, Tip60 has a haplo-insufficient tumour suppressor activity that is independent from—but not contradictory with—its role within the ARF–p53 pathway. We suggest that this is because critical levels of Tip60 are required for mounting an oncogene-induced DDR in incipient tumour cells, the failure of which might synergize with p53 mutation towards tumour progression.

ATAD2 Is a Novel Cofactor for MYC, Overexpressed and Amplified in Aggressive Tumors

The E2F and MYC transcription factors are critical regulators of cell proliferation and contribute to the development of human cancers. Here, we report on the identification of a novel E2F target gene, ATAD2, the predicted protein product of which contains both a bromodomain and an ATPase domain. The pRB-E2F pathway regulates ATAD2 expression, which is limiting for the entry into the S phase of the cell cycle. We show that ATAD2 binds the MYC oncogene and stimulates its transcriptional activity. ATAD2 maps to chromosome 8q24, 4.3 Mb distal to MYC, in a region that is frequently found amplified in cancer. Consistent with this, we show that ATAD2 expression is high in several human tumors and that the expression levels correlate with clinical outcome of breast cancer patients. We suggest that ATAD2 links the E2F and MYC pathways and contributes to the development of aggressive cancer through the enhancement of MYC-dependent transcription.

Frequent Alterations in the Expression of Serine/Threonine Kinases in Human Cancers

Protein kinases constitute a large family of regulatory enzymes involved in the homeostasis of virtually every cellular process. Subversion of protein kinases has been frequently implicated in malignant transformation. Within the family, serine/threonine kinases (STK) have received comparatively lesser attention, vis-a-vis tyrosine kinases, in terms of their involvement in human cancers. Here, we report a large-scale screening of 125 STK, selected to represent all major subgroups within the subfamily, on nine different types of tumors ( approximately 200 patients), by using in situ hybridization on tissue microarrays. Twenty-one STK displayed altered levels of transcripts in tumors, frequently with a clear tumor type-specific dimension. We identified three patterns of alterations in tumors: (a) overexpression in the absence of expression in the normal tissues (10 kinases), (b) overexpression in the presence of expression by normal tissues (8 kinases), and (c) underexpression (3 kinases). Selected members of the three classes were subjected to in-depth analysis on larger case collections and showed significant correlations between their altered expression and biological and/or clinical variables. Our findings suggest that alteration in the expression of STK is a relatively frequent occurrence in human tumors. Among the overexpressed kinases, 10 were undetectable in normal controls and are therefore ideal candidates for further validation as potential targets of molecular cancer therapy.

Breast cancer metastases are molecularly distinct from their primary tumors

Metastases have been widely thought to arise from rare, selected, mutation-bearing cells in the primary tumor. Recently, however, it has been proposed that breast tumors are imprinted ab initio with metastatic ability. Thus, there is a debate over whether ‘phenotypic’ disease progression is really associated with ‘molecular’ progression. We profiled 26 matched primary breast tumors and lymph node metastases and identified 270 probesets that could discriminate between the two categories. We then used an independent cohort of breast tumors (81 samples) and unmatched distant metastases (32 samples) to validate and refine this list down to a 126-probeset list. A representative subset of these genes was subjected to analysis by in situ hybridization, on a third independent cohort (57 primary breast tumors and matched lymph node metastases). This not only confirmed the expression profile data, but also allowed us to establish the cellular origin of the signals. One-third of the analysed representative genes (4 of 11) were expressed by the epithelial component. The four epithelial genes alone were able to discriminate primary breast tumors from their metastases. Finally, engineered alterations in the expression of two of the epithelial genes (SERPINB5 and LTF) modified cell motility in vitro, in accordance with a possible causal role in metastasis. Our results show that breast cancer metastases are molecularly distinct from their primary tumors.

An Atlas of Altered Expression of Deubiquitinating Enzymes in Human Cancer

Background Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin (Ub) or ubiquitin-like gene products, remodel polyubiquitin(-like) chains on target proteins, and counteract protein ubiquitination exerted by E3 ubiquitin-ligases. A wealth of studies has established the relevance of DUBs to the control of physiological processes whose subversion is known to cause cellular transformation, including cell cycle progression, DNA repair, endocytosis and signal transduction. Altered expression of DUBs might, therefore, subvert both the proteolytic and signaling functions of the Ub system. Methodology/Principal Findings In this study, we report the first comprehensive screening of DUB dysregulation in human cancers by in situ hybridization on tissue microarrays (ISH-TMA). ISH-TMA has proven to be a reliable methodology to conduct this kind of study, particularly because it allows the precise identification of the cellular origin of the signals. Thus, signals associated with the tumor component can be distinguished from those associated with the tumor microenvironment. Specimens derived from various normal and malignant tumor tissues were analyzed, and the “normal” samples were derived, whenever possible, from the same patients from whom tumors were obtained. Of the ∼90 DUBs encoded by the human genome, 33 were found to be expressed in at least one of the analyzed tissues, of which 22 were altered in cancers. Selected DUBs were subjected to further validation, by analyzing their expression in large cohorts of tumor samples. This analysis unveiled significant correlations between DUB expression and relevant clinical and pathological parameters, which were in some cases indicative of aggressive disease. Conclusions/Significance The results presented here demonstrate that DUB dysregulation is a frequent event in cancer, and have implications for therapeutic approaches based on DUB inhibition.

RaLP, a New Member of the Src Homology and Collagen Family, Regulates Cell Migration and Tumor Growth of Metastatic Melanomas

The Src homology and collagen (Src) family of adaptor proteins comprises six Shc-like proteins encoded by three loci in mammals (Shc, Rai, and Sli). Shc-like proteins are tyrosine kinase substrates, which regulate diverse signaling pathways and cellular functions, including Ras and proliferation (p52/p46Shc), phosphatidylinositol 3-kinase and survival (p54Rai), and mitochondrial permeability transition and apoptosis (p66Shc). Here, we report the identification, cloning, and sequence characterization of a new member of the Shc family that we termed RaLP. RaLP encodes a 69-kDa protein characterized by the CH2-PTB-CH1-SH2 modularity, typical of the Shc protein family, and expressed, among adult tissues, only in melanomas. Analysis of RaLP expression during the melanoma progression revealed low expression in normal melanocytes and benign nevi, whereas high levels of RaLP protein were found at the transition from radial growth phase to vertical growth phase and metastatic melanomas, when tumor cells acquire migratory competence and invasive potential. Notably, silencing of RaLP expression in metastatic melanomas by RNA interference reduced tumorigenesis in vivo. Analysis of RaLP in melanoma signal transduction pathways revealed that (a) when ectopically expressed in RaLP-negative melanocytes and nonmetastatic melanoma cells, it functions as a substrate of activated insulin-like growth factor-1 and epidermal growth factor receptors and increases Ras/mitogen-activated protein kinase (MAPK) signaling and cell migration, whereas (b) its silencing in RaLP-positive melanoma cells abrogates cell migration in vitro, without affecting MAPK signaling, suggesting that RaLP activates both Ras-dependent and Ras-independent migratory pathways in melanomas. These findings indicate that RaLP is a specific marker of metastatic melanomas, a critical determinant in the acquisition of the migratory phenotype by melanoma cells, and a potential target for novel anti-melanoma therapeutic strategies.