Michael Blank

PROGRAMS FOR CELL DEATH: APOPTOSIS IS ONLY ONE WAY TO GO

Cell death programs are major players in tissue homeostasis, development and cellular stress responses. A prominent cause of malignant transformation is the cumulative genetic alterations in pathways that regulate cellular growth and death. The processes that govern cell death following genotoxic stress are a major focus of basic research and are also very relevant to translational research in clinical oncology: understanding cell death following cancer therapy is essential for designing new treatment modalities. Cell death is usually, and sometimes automatically, linked with one of its major programs, apoptosis. Recent advances have led, however, to the emergence of additional, non-apoptotic cell death pathways, each with its triggers and readouts. Genotoxic stress appears to induce several cell death pathways, only part of which fall within the classical definition of apoptosis. Accordingly, solid tumor cells that are refractive to apoptosis were shown to die via non-apoptotic mechanisms. Recently we demonstrated that mitotic cell death induced by DNA damage in cells with defective G2/M checkpoint is mechanistically distinct from apoptosis. This review outlines recent advances in the understanding of molecular networks operative in apoptotic and non-apoptotic cell death mechanisms and their cross-talks.

A tumor suppressor function of Smurf2 associated with controlling chromatin landscape and genome stability through RNF20

In addition to allelic mutations, cancers are known to harbor alterations in their chromatin landscape. Here we show that genomic ablation of Smad ubiquitin regulatory factor 2 (Smurf2), a HECT-domain E3 ubiquitin ligase, results in dysregulation of both the DNA damage response and genomic stability, culminating in increased susceptibility to various types of cancers in aged mice. We show that Smurf2 regulates the monoubiquitination of histone H2B as well as the trimethylation of histone H3 at Lys4 and Lys79 by targeting ring finger protein 20 (RNF20) for proteasomal degradation in both mouse and human cells. We also show that Smurf2 and RNF20 are colocalized at the γ-H2AX foci of double-stranded DNA breaks in the nucleus. Thus, Smurf2 has a tumor suppression function that normally maintains genomic stability by controlling the epigenetic landscape of histone modifications through RNF20.

ANTI-cancer Activities of Hypericin in the Dark¶

Abstract The potent photodynamic properties of hypericin (HY) elicit a range of light-dependent virucidal and tumoricidal activities. Yet, a relatively low reduction/oxidation potential endows HY with electron accepting and donating properties enabling it to act as both an oxidizing and a reducing agent. HY can thus compete as an electron acceptor from bioenergized reduction/oxidation reactions generating its excitation energy for biological activities from physiological reduction/oxidation reactions in the absence of light. Our studies show that HY can inhibit the growth of highly metastatic murine breast adenocarcinoma and squamous cell carcinoma tumors in culture. Furthermore, we show that HY can interfere with the growth of these tumors in mice reducing tumor size and prolonging animal survival in complete absence of light. While there is no evidence that HY induces apoptosis in these cells in the dark, 3H-thymidine incorporation into DNA was significantly reduced indicating effects that are apparently cytostatic in nature compared to the cytocidal effects of HY with light.

Antimetastatic activity of the photodynamic agent hypericin in the dark.

A unique property of the photodynamic signal transduction inhibitor hypericin (HY) is high functionality in the dark, which has been shown to result in portfolio of anticancer activities both in vitro and in vivo. Here we show that treatment with HY significantly reduces growth rate of metastases in 2 murine models: breast adenocarcinoma (DA3) and squamous cell carcinoma (SQ2). Focus on metastases was achieved by resection of primary tumors at stages in which micrometastases exist in lungs. Long‐term animal survival in DA3 tumor‐excised groups increased from 15.6% in controls to 34.5% following supplementary treatment with HY. In mice bearing SQ2 tumor metastases, therapy with HY increased animal survival from 17.7% in controls to 46.1%. Using Laser‐induced fluorescence and multipixel spectral image analyses, we demonstrate that HY has a high tendency to accumulate in primary and metastatic tumors; HY content in lungs bearing metastases was approximately 2‐fold higher than in the lungs of healthy animals. The tendency of HY to preferentially concentrate in lung metastases, combined with its potent antiproliferative activities, may render HY as a useful supplementary modality in the treatment of metastatic cancer irrespective of photoactivation.

Molecular functions of NEDD4 E3 ubiquitin ligases in cancer

The initiation, progression and cure of cancer rely heavily on altered gene expression and posttranslational functions. Protein ubiquitination is a major mechanism for posttranslational reorganization of the genome. This evolutionary conserved cascade, through regulation of protein stability, distribution, and function, governs nearly every biological process in the cell. E3 ubiquitin ligases are pivotal components of the ubiquitination pathway. Genetic alterations, abnormal expression, and dysfunctions of E3s have been implicated in the pathogenesis of a wide spectrum of human malignancies. In this review, we summarize and discuss recent discoveries on the roles of NEDD4 E3s in cancer. Over the past decade, members of this family have increasingly surfaced as fundamental components and critical regulators of molecular pathways central to the pathogenesis and cure of the disease.

Condensin I recruitment and uneven chromatin condensation precede mitotic cell death in response to DNA damage

Mitotic cell death (MCD) is a prominent but poorly defined form of death that stems from aberrant mitosis. One of the early steps in MCD is premature mitosis and uneven chromatin condensation (UCC). The mechanism underlying this phenomenon is currently unknown. In this study, we show that DNA damage in cells with a compromised p53-mediated G2/M checkpoint triggers the unscheduled activation of cyclin-dependent kinase 1 (Cdk1), activation and chromatin loading of the condensin I complex, and UCC followed by the appearance of multimicronucleated cells, which is evidence of MCD. We demonstrate that these processes engage some of the players of normal mitotic chromatin packaging but not those that drive the apoptotic chromatin condensation. Our findings establish a link between the induction of DNA damage and mitotic abnormalities (UCC) through the unscheduled activation of Cdk1 and recruitment of condensin I. These results demonstrate a clear distinction between the mechanisms that drive MCD-associated and apoptosis-related chromatin condensation and provide mechanistic insights and new readouts for a major cell death process in treated tumors.

Wavelength-dependent Properties of Photodynamic Therapy Using Hypericin in vitro and in an Animal Model ¶

Abstract Wavelength effects in photodynamic therapy (PDT) with hypericin (HY) were examined in a C26 colon carcinoma model both in vitro and in vivo. Irradiation of HY-sensitized cells in vitro with either 550 or 590 nm caused the loss of cell viability in a drug- and light-dose–dependent manner. The calculated ratio of HY-based PDT (HY-PDT) efficiencies at these two wavelengths was found to correlate with the numerical ratio of absorbed photons at each wavelength. In vivo irradiation of C26-derived tumors, 6 h after intraperitoneal administration of HY (5 mg/kg), caused extensive vascular damage and tumor necrosis. The depth of tumor necrosis (d) was more pronounced at 590 than at 550 nm and increased when the light dose was raised from 60 to 120 J/cm2. The maximal depths of tumor necrosis (at 120 J/cm2) were 7.5 ± 1.5 mm at 550 nm and 9.9 ± 0.8 mm at 590 nm. Both values are rather high in view of the limited penetration of green-yellow light into the tissue. Moreover, the depth ratio, d590/d550 = 1.3 (P < 0.001), is smaller than expected considering the 2.2-fold lower HY absorbance and the 1.7-fold lower tissue penetration of radiation at 550 than at 590 nm. This finding indicates that in vivo the depth at which HY-PDT elicits tumor necrosis is not only determined by photophysical considerations (light penetration, number of absorbed photons) but is also influenced significantly by other mechanisms such as vascular effects. Therefore, despite the relatively short-wavelength peaks of absorption, our observations suggest that HY is an effective photodynamic agent that can be useful in the treatment of tumors with depths in the range of 1 cm.

Effects of photodynamic therapy with hypericin in mice bearing highly invasive solid tumors.

The tumoricidal properties of photodynamic therapy (PDT) with hypericin (HY) were evaluated in a highly metastatic adenocarcinoma (DA3Hi) and anaplastic squamous cell carcinoma (SQ2) tumors in vivo. Photosensitization of the tumor site with hypericin (HY-PDT) reduced primary tumor development and significantly prolonged the survival of tumor-bearing (TB) mice. Of these two tumors the squamous cell carcinoma emerged as more sensitive to HY-PDT compared with DA3Hi adenocarcinoma both in vitro and in vivo. HY-PDT caused extensive tumor necrosis that was followed by local, intratumoral, and systemic inflammatory reactions. Analyses of cytokine mRNA profiles reveal increases in mRNA levels of expression confined to inflammation-related cytokines both within the tumor and also systemically (measured in spleens). However, there was no evidence for any HY-PDT-induced antitumoral immune reactions. Our results suggest that PDT with hypericin can be considered as a supplementary treatment in the management of some invasive and metastatic cancers such as squamous carcinoma and similar tumors.

Integrative genomics identifies YY1AP1 as an oncogenic driver in EpCAM(+) AFP(+) hepatocellular carcinoma.

Identification of key drivers and new therapeutic targets is important given the poor prognosis for hepatocellular carcinoma (HCC) patients, particularly those ineligible for surgical resection or liver transplant. However, the approach to identify such driver genes is facing significant challenges due to the genomically heterogenous nature of HCC. Here we tested whether the integrative genomic profiling of a well-defined HCC subset that is classified by an extreme EpCAM+ AFP+ gene expression signature and associated with poor prognosis, all attributes of a stem cell-like phenotype, could uncover survival-related driver genes in HCC. Following transcriptomic analysis of the well-defined HCC cases, a Gene Set Enrichment Analysis coupled with genomic copy number alteration assessment revealed that YY1-associated protein 1 (YY1AP1) is a critical oncoprotein specifically activated in EpCAM+ AFP+ HCC. YY1AP1 silencing eliminates oncogene addiction by altering the chromatin landscape and triggering massive apoptosis in vitro and tumor suppression in vivo. YY1AP1 expression promotes HCC proliferation and is required for the maintenance of stem cell features. We revealed that YY1AP1 cooperates with YY1 to alter the chromatin landscape and activate transcription of stemness regulators. Thus YY1AP1 may serve as a key molecular target for EpCAM+ AFP+ HCC subtype. Our results demonstrate the feasibility and power of a new strategy by utilizing well-defined patient samples and integrative genomics to uncover critical pathways linked to HCC subtypes with prognostic impact.

The COP9 signalosome is vital for timely repair of DNA double-strand breaks

The DNA damage response is vigorously activated by DNA double-strand breaks (DSBs). The chief mobilizer of the DSB response is the ATM protein kinase. We discovered that the COP9 signalosome (CSN) is a crucial player in the DSB response and an ATM target. CSN is a protein complex that regulates the activity of cullin ring ubiquitin ligase (CRL) complexes by removing the ubiquitin-like protein, NEDD8, from their cullin scaffold. We find that the CSN is physically recruited to DSB sites in a neddylation-dependent manner, and is required for timely repair of DSBs, affecting the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair (HRR). The CSN is essential for the processivity of deep end-resection—the initial step in HRR. Cullin 4a (CUL4A) is recruited to DSB sites in a CSN- and neddylation-dependent manner, suggesting that CSN partners with CRL4 in this pathway. Furthermore, we found that ATM-mediated phosphorylation of CSN subunit 3 on S410 is critical for proper DSB repair, and that loss of this phosphorylation site alone is sufficient to cause a DDR deficiency phenotype in the mouse. This novel branch of the DSB response thus significantly affects genome stability.