A. Emre Sayan

ZEB proteins link cell motility with cell cycle control and cell survival in cancer

Epithelial mesenchymal transitions (EMT), the generation of motile mesenchymal cells from epithelial sheets, are differentiation programs which take place at several critical steps of embryonic development and in metastatic cancer. Recent data have shown that the transcription factors which are master regulators of EMT also regulate cell cycle progression, apoptosis and senescence. In light of these new observations, the role of these factors in human cancer may be broader than previously anticipated. Here we review recent literature on non-EMT functions of EMT-controlling transcription factors. We will mainly focus on transcription factors belonging to the ZEB family, but some important results obtained by investigators studying other key EMT regulators, Snail and Twist are also discussed.

p53 Is Cleaved by Caspases Generating Fragments Localizing to Mitochondria

The p53 tumor suppressor protein exerts most of its anti-tumorigenic activity by transcriptionally activating several pro-apoptotic genes. Accumulating evidence also suggests a transcription-independent function of p53 during apoptosis. It has recently been shown that, when activated, a fraction of p53 translocates to mitochondria, causing cytochrome c release. We now demonstrate a caspase-dependent cleavage of p53 resulting in the generation of four fragments, two of which lack a nuclear localization signal and consequently localize to cytosol. Moreover, these two fragments translocate to mitochondria and induce mitochondrial membrane depolarization in the absence of transcriptional activity. This novel feature of p53 supports the model whereby cytosolic p53 exerts major functions in apoptosis and also suggests the presence of a positive feedback loop in which activated caspases cleave p53 to augment mitochondrial membrane depolarization.

MicroRNA control of invasion and metastasis pathways

Despite recent advances, cancer remains a leading cause of death worldwide. In developed countries, the incidence of colorectal and breast cancer has been stable, but no improvement in prognosis has been observed if the patient presents with metastases at diagnosis. This fact highlights the importance of therapeutic approaches targeting cellular invasion and metastasis programs as the next step in cancer treatment. During carcinoma progression a process called epithelial–mesenchymal transition (EMT) results in enhanced invasion and motility which is directly linked with loss of epithelial polarity and epithelial junctions, migration permissive cytoskeleton alterations, and the acquisition of mesenchymal properties. The recent discovery of microRNAs (miRNAs) controlling key cellular pathways has opened a new era in understanding how EMT pathways are modulated. In this review, we classify EMT regulating proteins according to their cellular localization (membrane, cytoplasmic, and nuclear), and summarize the current knowledge on how they are controlled by miRNAs and propose potential miRNAs for the transcripts that may control their expression.

Mechanism of Induction of Apoptosis by p73 and Its Relevance to Neuroblastoma Biology

Abstract: TP73, as a TP53 homologue, drew the attention of tumor biologists because it is rarely mutated in human cancers and can induce cell cycle arrest and apoptosis by activating genes also regulated by p53. However, TP73 harbors an additional promoter that produces a dominant negative p73 protein (ΔNp73) having the opposite effect of the TAp73 protein. Thus, the regulation of p53 responsive genes in the absence of p53 relies on a critical balance between different p73 gene‐derived proteins. Recent reports have described additional complexity in the mechanism of action of transcriptionally active p73 (TAp73) in the induction of cell death. The molecular mechanism through which p73 induces apoptosis involves (i) expression and changes in subcellular localization of scotin, producing an endoplasmic reticulum (ER) stress; and (ii) transactivation of PUMA and Bax, thus determining cell fate. On the contrary, ΔNp73 inhibits apoptosis, thus contributing to the oncogenic potential of neuroblastoma cells.

Novel monoclonal antibodies detect Smad-interacting protein 1 (SIP1) in the cytoplasm of human cells from multiple tumor tissue arrays.

Smad-interacting protein 1 (SIP1, also known as ZEB2) represses the transcription of E-cadherin and mediates epithelial-mesenchymal transition in development and tumor metastasis. Due to the lack of human SIP1-specific antibodies, its expression in human tumor tissues has not been studied in detail by immunohistochemistry. Hence, we generated two anti-SIP1 monoclonal antibodies, clones 1C6 and 6E5, with IgG1 and IgG2a isotypes, respectively. The specificity of these antibodies was shown by Western blotting studies using siRNA mediated downregulation of SIP1 and ZEB1 in a human osteosarcoma cell line. In the same context, we also compared them with 5 commercially available SIP1 antibodies. Antibody specificity was further verified in an inducible cell line system by immunofluorescence. By using both antibodies, we evaluated the tissue expression of SIP1 in paraffin-embedded tissue microarrays consisting of 22 normal and 101 tumoral tissues of kidney, colon, stomach, lung, esophagus, uterus, rectum, breast and liver. Interestingly, SIP1 predominantly displayed a cytoplasmic expression, while the nuclear localization of SIP1 was observed in only 6 cases. Strong expression of SIP1 was found in distal tubules of kidney, glandular epithelial cells of stomach and hepatocytes, implicating a co-expression of SIP1 and E-cadherin. Squamous epithelium of the esophagus and surface epithelium of colon and rectum were stained with moderate to weak intensity. Normal uterus, breast and lung tissues remained completely negative. By comparison with their normal tissues, we observed SIP1 overexpression in cancers of the kidney, breast, lung and uterus. However, SIP1 expression was found to be downregulated in tumors from colon, rectum, esophagus, liver and stomach tissues. Finally we did nuclear/cytoplasmic fractionation in 3 carcinoma cell lines and detected SIP1 in both fractions, nucleus being the dominant one. To our best knowledge, this is the first comprehensive immunohistochemical study of the expression of SIP1 in a series of human cancers. Our finding that SIP1 is not exclusively localized to nucleus suggests that the subcellular localization of SIP1 is regulated in normal and tumor tissues. These novel monoclonal antibodies may help elucidate the role of SIP1 in tumor development.

A top-down view of the tumor microenvironment: structure, cells and signaling

It is well established that the tumor microenvironment (TME) contributes to cancer progression. Stromal cells can be divided into mesenchymal, vascular, and immune. Signaling molecules secreted by the tumor corrupts these cells to create “activated” stroma. Equally, the extracellular matrix (ECM) contributes to tumor development and invasion by forming a biologically active scaffold. In this review we describe the key structural, cellular and signaling components of the TME with a perspective on stromal soluble factors and microRNAs (miRNAs).

NAPO as a novel marker for apoptosis

Apoptosis or programmed cell death plays a pivotal role in embryonic development and maintenance of homeostasis. It is also involved in the etiology of pathophysiological conditions such as cancer, neurodegenerative, autoimmune, infectious, and heart diseases. Consequently, the study of apoptosis is now at center of both basic and clinical research applications. Therefore, sensitive and simple apoptosis detection techniques are required. Here we describe a monoclonal antibody–defined novel antigen, namely NAPO (negative in apoptosis), which is specifically lost during apoptosis. The anti-NAPO antibody recognizes two nuclear polypeptides of 60 and 70 kD. The antigen is maintained in quiescent and senescent cells, as well as in different phases of the cell cycle, including mitosis. Thus, immunodetection of NAPO antigen provides a specific, sensitive, and easy method for differential identification of apoptotic and nonapoptotic cells.

Generation of ΔTAp73 Proteins by Translation from a Putative Internal Ribosome Entry Site

Abstract:  p73 belongs to a family of transcription factors, including p53 and p63, that mediate response to DNA damage and cellular stress by inducing DNA repair, cell cycle arrest, and apoptosis. TP73 gene contains two promotors and several splice variants resulting in up to 24 possible permutations of p73 proteins which underlies the complexity of the family and its regulatory mechanisms. p73 variants lacking the N‐terminal, denoted as ΔTAp73, are not transcriptionally competent and they act in a dominant negative fashion over TAp73. ΔTAp73 isoforms can be generated by alternative promotor usage, giving rise to ΔNp73, or alternative splicing of exons 2, 3 or 2, and 3 together. Such transcript isoforms potentially produce oncogenic proteins and they were shown to be present in primary tumors and tumor‐derived cell lines. We investigated the possibility of additional mechanisms by which p73 protein could be regulated and discovered a putative internal ribosome entry site (IRES) in exon 2. Translation initiation of TAp73 mRNA results in a ΔNp73‐like peptide, thus demonstrating an additional mechanism whereby a ΔTA p73 protein is produced from a transcript originally generated from the P1 promotor of the p73 gene.

Suppression of Hedgehog signalling promotes pro-tumourigenic integrin expression and function

Aberrant Hedgehog (Hh) signalling has been reported in a number of malignancies, particularly basal cell carcinoma (BCC) of the skin. Clinical trials of Hh inhibitors are underway in many cancers, and these have produced significant clinical benefit in BCC patients, although regrowth of new, or clinically aggressive, variants, as well as development of secondary malignancies, has been reported. αvβ6 integrin is expressed in many cancers, where it has been shown to correlate with an aggressive tumour phenotype and poor prognosis. We have previously reported αvβ6 up‐regulation in aggressive, morphoeic BCC variants, where it modulates the stromal response and induces invasion. To examine a possible link between Hh and αvβ6 function, we generated BCC models, overexpressing Gli1 in immortalized keratinocytes (NTert1, HaCaT). Unexpectedly, we found that suppressing Gli1 significantly increased αvβ6 expression. This promoted tumour cell motility and also stromal myofibroblast differentiation through integrin‐dependent TGF‐β1 activation. Gli1 inhibited αvβ6 expression by suppressing TGF‐β1‐induced Smad2/3 activation, blocking a positive feedback loop maintaining high αvβ6 levels. A similar mechanism was observed in AsPC1 pancreatic cancer cells expressing endogenous Gli1, suggesting a common mechanism across tumour types. In vitro findings were supported using human clinical samples, where we showed an inverse correlation between αvβ6 and Gli1 expression in different BCC subtypes and pancreatic cancers. In summary, we show that expression of Gli1 and αvβ6 inversely correlates in tumours in vivo, and Hh targeting up‐regulates TGF‐β1/Smad2/3‐dependent αvβ6 expression, promoting pro‐tumourigenic cell functions in vitro. These results have potential clinical significance, given the reported recurrence of BCC variants and secondary malignancies in patients treated by Hh targeting. Copyright

Exosomal microRNAs (exomiRs): Small molecules with a big role in cancer

Exosomes are secreted vesicles which can transmit molecular cargo between cells. Exosomal microRNAs (exomiRs) have drawn much attention in recent years because there is increasing evidence to suggest that loading of microRNAs into exosomes is not a random process. Preclinical studies have identified functional roles for exomiRs in influencing many hallmarks of cancer. Mechanisms underpinning their actions, such as exomiR receptors (“miRceptors”), are now becoming apparent. Even more exciting is the fact that exomiRs are highly suitable candidates for use as non-invasive biomarkers in an era of personalized cancer medicine.