The histone deacetylase SIRT6 is a tumor suppressor that controls cancer metabolism.

Reprogramming of cellular metabolism is a key event during tumorigenesis. Despite being known for decades (Warburg effect), the molecular mechanisms regulating this switch remained unexplored. Here, we identify SIRT6 as a tumor suppressor that regulates aerobic glycolysis in cancer cells. Importantly, loss of SIRT6 leads to tumor formation without activation of known oncogenes, whereas transformed SIRT6-deficient cells display increased glycolysis and tumor growth, suggesting that SIRT6 plays a role in both establishment and maintenance of cancer. By using a conditional SIRT6 allele, we show that SIRT6 deletion in vivo increases the number, size, and aggressiveness of tumors. SIRT6 also functions as a regulator of ribosome metabolism by corepressing MYC transcriptional activity. Lastly, Sirt6 is selectively downregulated in several human cancers, and expression levels of SIRT6 predict prognosis and tumor-free survival rates, highlighting SIRT6 as a critical modulator of cancer metabolism. Our studies reveal SIRT6 to be a potent tumor suppressor acting to suppress cancer metabolism.

The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes.

Gap junction channels, composed of connexins, allow direct cell-to-cell communication. Connexin 43 (Cx43; also known as GJA1) is widely expressed in tissues, including the epidermis. In a previous study of human papillomavirus-positive cervical epithelial tumour cells, we identified Cx43 as a binding partner of the human homologue of Drosophila Discs large (Dlg1; also known as SAP97). Dlg1 is a member of the membrane associated-guanylate kinase (MAGUK) scaffolding protein family, which is known to control cell shape and polarity. Here, we show that Cx43 also interacts with Dlg1 in uninfected keratinocytes in vitro and in keratinocytes, dermal cells and adipocytes in normal human epidermis in vivo. Depletion of Dlg1 in keratinocytes did not alter Cx43 transcription but was associated with a reduction in Cx43 protein levels. Reduced Dlg1 levels in keratinocytes resulted in a reduction in Cx43 at the plasma membrane with a concomitant reduction in gap junctional intercellular communication and relocation of Cx43 to the Golgi compartment. Our data suggest a key role for Dlg1 in maintaining Cx43 at the plasma membrane in keratinocytes.

Structural basis for topological regulation of Tn3 resolvase.

Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely related resolvases from the Tn3 and γδ transposons have historically served as paradigms for the regulation of recombinase activity by DNA topology. However, despite many proposals, models of the multi-subunit protein-DNA complex (termed the synaptosome) that enforces this regulation have been unsatisfying due to a lack of experimental constraints and incomplete concordance with experimental data. Here, we present new structural and biochemical data that lead to a new, detailed model of the Tn3 synaptosome, and discuss how it harnesses DNA topology to regulate the enzymatic activity of the recombinase.

Site-specific DNA recombinases alter the connectivity of DNA by recognizing specific DNA sequences, then cutting the DNA strands and pasting them together in a new configuration. Such enzymes play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful biotechnology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. However, others have evolved elaborate mechanisms to ensure that only one of multiple possible recombination products is produced. Tn3 resolvase has long been known to be regulated by DNA topology­that is, it will cut and reconnect two target sequences only if they lie on the same DNA molecule, and if they are in the proper relative orientation. This study presents new structural and biochemical data that lead to a new, detailed model of the large protein­DNA complex formed by Tn3 resolvase and its cognate sites. This 3D model illustrates how DNA topology can be harnessed to regulate the activity of a recombinase and provides a basis for engineering Tn3 resolvase and related recombination systems as genome editing tools.

Human Papillomavirus 16 Oncoprotein Expression Is Controlled by the Cellular Splicing Factor SRSF2 (SC35).

UNLABELLED: High-risk human papillomaviruses (HR-HPV) cause anogenital cancers, including cervical cancer, and head and neck cancers. Human papillomavirus 16 (HPV16) is the most prevalent HR-HPV. HPV oncogenesis is driven by two viral oncoproteins, E6 and E7, which are expressed through alternative splicing of a polycistronic RNA to yield four major splice isoforms (E6 full length, E6*I, E6*II, E6*X). The production of multiple mRNA isoforms from a single gene is controlled by serine/arginine-rich splicing factors (SRSFs), and HPV16 infection induces overexpression of a subset of these, SRSFs 1, 2, and 3. In this study, we examined whether these proteins could control HPV16 oncoprotein expression. Small interfering RNA (siRNA) depletion experiments revealed that SRSF1 did not affect oncoprotein RNA levels. While SRSF3 knockdown caused some reduction in E6E7 expression, depletion of SRSF2 resulted in a significant loss of E6E7 RNAs, resulting in reduced levels of the E6-regulated p53 proteins and E7 oncoprotein itself. SRSF2 contributed to the tumor phenotype of HPV16-positive cervical cancer cells, as its depletion resulted in decreased cell proliferation, reduced colony formation, and increased apoptosis. SRSF2 did not affect transcription from the P97 promoter that controls viral oncoprotein expression. Rather, RNA decay experiments showed that SRSF2 is required to maintain stability of E6E7 mRNAs. These data show that SRSF2 is a key regulator of HPV16 oncoprotein expression and cervical tumor maintenance. IMPORTANCE: Expression of the HPV16 oncoproteins E7 and E6 drives HPV-associated tumor formation. Although increased transcription may yield increased levels of E6E7 mRNAs, it is known that the RNAs can have increased stability upon integration into the host genome. SR splicing factors (SRSFs) control splicing but can also control other events in the RNA life cycle, including RNA stability. Previously, we demonstrated increased levels of SRSFs 1, 2, and 3 during cervical tumor progression. Now we show that SRSF2 is required for expression of E6E7 mRNAs in cervical tumor but not nontumor cells and may act by inhibiting their decay. SRSF2 depletion in W12 tumor cells resulted in increased apoptosis, decreased proliferation, and decreased colony formation, suggesting that SRSF2 has oncogenic functions in cervical tumor progression. SRSF function can be targeted by known drugs that inhibit SRSF phosphorylation, suggesting a possible new avenue in abrogating HPV oncoprotein activity.

Nuclear expression of Lyn, a Src family kinase member, is associated with poor prognosis in renal cancer patients.

BACKGROUND: 8000 cases of renal cancer are diagnosed each year in the UK, with a five-year survival rate of 50%. Treatment options are limited; a potential therapeutic target is the Src family kinases (SFKs). SFKs have roles in multiple oncogenic processes and promote metastases in solid tumours. The aim of this study was to investigate SFKs as potential therapeutic targets for clear cell renal cell carcinoma (ccRCC). METHODS: SFKs expression was assessed in a tissue microarray consisting of 192 ccRCC patients with full clinical follow-up. SFK inhibitors, dasatinib and saracatinib, were assessed in early ccRCC cell lines, 786-O and 769-P and a metastatic ccRCC cell line, ACHN (± Src) for effects on protein expression, apoptosis, proliferation and wound healing. RESULTS: High nuclear expression of Lyn and the downstream marker of activation, paxillin, were associated with decreased patient survival. Conversely, high cytoplasmic expression of other SFK members and downstream marker of activation, focal adhesion kinase (FAK) were associated with increased patient survival. Treatment of non-metastatic 786-O and 769-P cells with dasatinib, dose dependently reduced SFK activation, shown via SFK (Y(419)) and FAK (Y(861)) phosphorylation, with no effect in metastatic ACHN cells. Dasatinib also increased apoptosis, while decreasing proliferation and migration in 786-O and 769-P cell lines, both in the presence and absence of Src protein. CONCLUSIONS: Our data suggests that nuclear Lyn is a potential therapeutic target for ccRCC and dasatinib affects cellular functions associated with cancer progression via a Src kinase independent mechanism.

Variable orthogonality of RDF - large serine integrase interactions within the ϕC31 family.

Large serine integrases are phage- (or mobile element-) encoded enzymes that catalyse site-specific recombination reactions between a short DNA sequence on the phage genome (attP) and a corresponding host genome sequence (attB), thereby integrating the phage DNA into the host genome. Each integrase has its unique pair of attP and attB sites, a feature that allows them to be used as orthogonal tools for genome modification applications. In the presence of a second protein, the Recombination Directionality Factor (RDF), integrase catalyses the reverse, excisive reaction, generating new recombination sites, attR and attL. In addition to promoting attR x attL reaction, the RDF inhibits attP x attB recombination. This feature makes the directionality of integrase reactions programmable, allowing them to be useful for building synthetic biology devices. In this report, we describe the degree of orthogonality of both integrative and excisive reactions for three related integrases (ϕC31, ϕBT1, and TG1) and their RDFs. Among these, TG1 integrase is the most active, showing near complete recombination in both attP x attB and attR x attL reactions, and the most directional in the presence of its RDF. Our findings show that there is varying orthogonality among these three integrases - RDF pairs: ϕC31 integrase was the least selective, with all three RDFs activating it for attR x attL recombination. Similarly, ϕC31 RDF was the least effective among the three RDFs in promoting the excisive activities of the integrases, including its cognate ϕC31 integrase. ϕBT1 and TG1 RDFs were noticeably more effective than ϕC31 RDF at inhibiting attP x attB recombination by their respective integrases, making them more suitable for building reversible genetic switches. AlphaFold-Multimer predicts very similar structural interactions between each cognate integrase - RDF pair. The binding surface on RDF is much more conserved than the binding surface on integrase, an indication that specificity is determined more by the integrase than the RDF. Overall, the observed weak integrase/RDF orthogonality across the three enzymes emphasizes the need for identifying and characterizing more integrase - RDF pairs. Additionally, the ability of a particular integrase's preferred reaction direction to be controlled to varying degrees by non-cognate RDFs provides a path to tunable, non-binary genetic switches.

A functional interaction between the MAGUK protein hDlg and the gap junction protein connexin 43 in cervical tumour cells.

Gap junctions, composed of Cxs (connexins), allow direct intercellular communication. Gap junctions are often lost during the development of malignancy, although the processes behind this are not fully understood. Cx43 is a widely expressed Cx with a long cytoplasmic C-terminal tail that contains several potential protein-interaction domains. Previously, in a model of cervical carcinogenesis, we showed that the loss of gap junctional communication correlated with relocalization of Cx43 to the cytoplasm late in tumorigenesis. In the present study, we demonstrate a similar pattern of altered expression for the hDlg (human discs large) MAGUK (membrane-associated guanylate kinase) family tumour suppressor protein in cervical tumour cells, with partial co-localization of Cx43 and hDlg in an endosomal/lysosomal compartment. Relocalization of these proteins is not due to a general disruption of cell membrane integrity or Cx targeting. Cx43 (via its C-terminus) and hDlg interact directly in vitro and can form a complex in cells. This novel interaction requires the N- and C-termini of hDlg. hDlg is not required for Cx43 internalization in W12GPXY cells. Instead, hDlg appears to have a role in maintaining a cytoplasmic pool of Cx43. These results demonstrate that hDlg is a physiologically relevant regulator of Cx43 in transformed epithelial cells.

Gaps in detailed knowledge of human papillomavirus (HPV) and the HPV vaccine among medical students in Scotland.

BACKGROUND: A vaccination programme targeted against human papillomavirus (HPV) types 16 and 18 was introduced in the UK in 2008, with the aim of decreasing incidence of cervical disease. Vaccine roll out to 12-13 year old girls with a catch-up programme for girls aged up to 17 years and 364 days was accompanied by a very comprehensive public health information (PHI) campaign which described the role of HPV in the development of cervical cancer. METHODS: A brief questionnaire, designed to assess acquisition of knowledge of HPV infection and its association to cervical cancer, was administered to two different cohorts of male and female 1st year medical students (school leavers: 83% in age range 17-20) at a UK university. The study was timed so that the first survey in 2008 immediately followed a summer's intensive PHI campaign and very shortly after vaccine roll-out (150 students). The second survey was exactly one year later over which time there was a sustained PHI campaign (213 students). RESULTS: We addressed three research questions: knowledge about three specific details of HPV infection that could be acquired from PHI, whether length of the PHI campaign and/or vaccination of females had any bearing on HPV knowledge, and knowledge differences between men and women regarding HPV. No female student in the 2008 cohort had completed the three-dose vaccine schedule compared to 58.4% of female students in 2009. Overall, participants' knowledge regarding the sexually transmitted nature of HPV and its association with cervical cancer was high in both year groups. However, in both years, less than 50% of students correctly identified that HPV causes over 90% of cases of cervical cancer. Males gave fewer correct answers for these two details in 2009. In 2008 only around 50% of students recognised that the current vaccine protects against a limited subset of cervical cancer-causing HPV sub-types, although there was a significant increase in correct response among female students in the 2009 cohort compared to the 2008 cohort. CONCLUSIONS: This study highlights a lack of understanding regarding the extent of protection against cervical cancer conferred by the HPV vaccine, even among an educated population in the UK who could have a vested interest in acquiring such knowledge. The intensive PHI campaign accompanying the first year of HPV vaccination seemed to have little effect on knowledge over time. This is one of the first studies to assess detailed knowledge of HPV in both males and females. There is scope for continued improvements to PHI regarding the link between HPV infection and cervical cancer.

Microvesicles but Not Exosomes from Pathfinder Cells Stimulate Functional Recovery of the Pancreas in a Mouse Streptozotocin-Induced Diabetes Model.

Pathfinder cells (PCs), a novel cell type derived from the pancreas of adult rats, have been demonstrated to stimulate recovery of tissue structure and function in two animal models of acute tissue damage to date-streptozotocin (STZ)-induced diabetes and ischemia-reperfusion damage to the kidney. In repaired tissue, PCs and their progeny typically represent only 0.02% of the repaired tissue, suggesting that they act via a paracrine mechanism on native cells in the damaged area. Extracellular vesicles are strong candidates for mediating such a paracrine effect. Therefore, we studied the effects of two PC-derived extracellular vesicle fractions on tissue repair in the STZ diabetes model, one containing primarily microvesicles and the second containing predominantly exosomes. Treatment of STZ-induced diabetic mice with the microvesicles preparation led to blood glucose, insulin, glucagon, and C-peptide levels similar to those found with PC treatment. Furthermore, analysis of the histopathology of the pancreas indicated islet regeneration. In contrast, the exosome fraction demonstrated no repair activity, and STZ diabetic mice treated with exosome preparations had blood glucose values that were indistinguishable from those of vehicle-only treated controls. Therefore, we conclude that exosomes play no part in PC action as detected by this assay, whereas microvesicles provide all or a large component of the paracrine activity of PCs. Because they act to stimulate repair of multiple tissues, PC-derived microvesicles may similarly have the potential to stimulate repair of many damaged tissues, identifying a very significant cell-free therapeutic opportunity in regenerative medicine.

HPV16 E6 Controls the Gap Junction Protein Cx43 in Cervical Tumour Cells.

Human papillomavirus type 16 (HPV16) causes a range of cancers including cervical and head and neck cancers. HPV E6 oncoprotein binds the cell polarity regulator hDlg (human homologue of Drosophila Discs Large). Previously we showed in vitro, and now in vivo, that hDlg also binds Connexin 43 (Cx43), a major component of gap junctions that mediate intercellular transfer of small molecules. In HPV16-positive non-tumour cervical epithelial cells (W12G) Cx43 localised to the plasma membrane, while in W12T tumour cells derived from these, it relocated with hDlg into the cytoplasm. We now provide evidence that E6 regulates this cytoplasmic pool of Cx43. E6 siRNA depletion in W12T cells resulted in restoration of Cx43 and hDlg trafficking to the cell membrane. In C33a HPV-negative cervical tumour cells expressing HPV16 or 18 E6, Cx43 was located primarily in the cytoplasm, but mutation of the 18E6 C-terminal hDlg binding motif resulted in redistribution of Cx43 to the membrane. The data indicate for the first time that increased cytoplasmic E6 levels associated with malignant progression alter Cx43 trafficking and recycling to the membrane and the E6/hDlg interaction may be involved. This suggests a novel E6-associated mechanism for changes in Cx43 trafficking in cervical tumour cells.

SIRT3 & SIRT7: Potential Novel Biomarkers for Determining Outcome in Pancreatic Cancer Patients.

PURPOSE: The sirtuin gene family has been linked with tumourigenesis, in both a tumour promoter and suppressor capacity. Information regarding the function of sirtuins in pancreatic cancer is sparse and equivocal. We undertook a novel study investigating SIRT1-7 protein expression in a cohort of pancreatic tumours. The aim of this study was to establish a protein expression profile for SIRT1-7 in pancreatic ductal adenocarcinomas (PDAC) and to determine if there were associations between SIRT1-7 expression, clinico-pathological parameters and patient outcome. MATERIAL AND METHODS: Immunohistochemical analysis of SIRT1-7 protein levels was undertaken in a tissue micro-array comprising 77 resected PDACs. Statistical analyses determined if SIRT1-7 protein expression was associated with clinical parameters or outcome. RESULTS: Two sirtuin family members demonstrated significant associations with clinico-pathological parameters and patient outcome. Low level SIRT3 expression in the tumour cytoplasm correlated with more aggressive tumours, and a shorter time to relapse and death, in the absence of chemotherapeutic intervention. Low levels of nuclear SIRT7 expression were also associated with an aggressive tumour phenotype and poorer outcome, as measured by disease-free and disease-specific survival time, 12 months post-diagnosis. CONCLUSIONS: Our data suggests that SIRT3 and SIRT7 possess tumour suppressor properties in the context of pancreatic cancer. SIRT3 may also represent a novel predictive biomarker to determine which patients may or may not respond to chemotherapy. This study opens up an interesting avenue of investigation to potentially identify predictive biomarkers and novel therapeutic targets for pancreatic cancer, a disease that has seen no significant improvement in survival over the past 40 years.

SIRT2: tumour suppressor or tumour promoter in operable breast cancer?

PURPOSE: Sirtuins comprise a family of genes involved in cellular stress, survival and damage responses. They have been implicated in a range of diseases including cancer, with most information pertaining to their function in tumourigenesis being derived from in vitro studies, or model organisms. Their putative roles as tumour suppressors or tumour promoters remain to be validated in vivo. Little is known about their role in breast tumourigenesis. We sought to evaluate the seven sirtuin family members (SIRT1-7) in a human breast cancer cohort, in relation to clinico-pathological features and outcome of the disease. MATERIALS AND METHODS: Immunohistochemical analysis of SIRT1-7 protein levels was undertaken in 392 oestrogen receptor (ER+ve) and 153 ER-ve breast tumour samples. SIRT1-7 transcriptional levels were assessed in normal (n=25), non-malignant (n=73) and malignant (n=70) breast tissue using Relative Quantitative Real Time PCR. Statistical analyses determined if SIRT1-7 transcription or protein expression was associated with clinical parameters or outcome. RESULTS: In ER-ve tumours, high protein levels of nuclear SIRT2 were associated with reduced time to recurrence and disease-specific death. This association was only observed in Grade 3 tumours. In the ER+ve cohort, high SIRT2 nuclear levels were associated with shorter disease-free survival and time to recurrence whilst on Tamoxifen, in patients with Grade 3 tumours. Conversely, in Grade 2 tumours, high SIRT2 levels were associated with increased time to recurrence. CONCLUSIONS: Our data suggest that SIRT2 is the sirtuin predominantly involved in breast tumourigenesis and prognosis. It indicates that SIRT2 acts as a tumour suppressor or tumour promoter dependent upon breast tumour grade.

Pathfinder cells provide a novel therapeutic intervention for acute kidney injury.

Pathfinder cells (PCs) are a novel class of adult-derived cells that facilitate functional repair of host tissue. We used rat PCs to demonstrate that they enable the functional mitigation of ischemia reperfusion (I/R) injury in a mouse model of renal damage. Female C57BL/6 mice were subjected to 30 min of renal ischemia and treated with intravenous (i.v.) injection of saline (control) or male rat pancreas-derived PCs in blinded experimentation. Kidney function was assessed 14 days after treatment by measuring serum creatinine (SC) levels. Kidney tissue was assessed by immunohistochemistry (IHC) for markers of cellular damage, proliferation, and senescence (TUNEL, Ki67, p16(ink4a), p21). Fluorescence in situ hybridization (FISH) was performed to determine the presence of any rat (i.e., pathfinder) cells in the mouse tissue. PC-treated animals demonstrated superior renal function at day 14 post-I/R, in comparison to saline-treated controls, as measured by SC levels (0.13 mg/dL vs. 0.23 mg/dL, p<0.001). PC-treated kidney tissue expressed significantly lower levels of p16(ink4a) in comparison to the control group (p=0.009). FISH analysis demonstrated that the overwhelming majority of repaired kidney tissue was mouse in origin. Rat PCs were only detected at a frequency of 0.02%. These data confirm that PCs have the ability to mitigate functional damage to kidney tissue following I/R injury. Kidneys of PC-treated animals showed evidence of improved function and reduced expression of damage markers. The PCs appear to act in a paracrine fashion, stimulating the host tissue to recover functionally, rather than by differentiating into renal cells. This study demonstrates that pancreatic-derived PCs from the adult rat can enable functional repair of renal damage in mice. It validates the use of PCs to regenerate damaged tissues and also offers a novel therapeutic intervention for repair of solid organ damage in situ.

PepA and ArgR do not regulate Cre recombination at the bacteriophage P1 loxP site.

In the lysogenic state, bacteriophage P1 is maintained as a low copy-number circular plasmid. Site-specific recombination at loxP by the phage-encoded Cre protein keeps P1 monomeric, thus helping to ensure stable plasmid inheritance. Two Escherichia coli DNA-binding proteins, PepA and ArgR, were recently reported to be necessary for maintenance or establishment of P1 lysogeny. PepA and ArgR bind to regulatory DNA sequences upstream of the ColE1 cer recombination site to regulate site-specific recombination by the XerCD recombinases. This recombination keeps ColE1 in a monomeric state and helps to ensure stable plasmid maintenance. It has been suggested that ArgR and PepA play a similar role in P1 maintenance, regulating Cre recombination by binding to DNA sequences upstream of loxP. Here, we show that ArgR does not bind to its proposed binding site upstream of loxP, and that Cre recombination at loxP in its natural P1 context is not affected by PepA and ArgR in vitro. When sequences upstream of loxP were mutated to allow ArgR binding, PepA and ArgR still had no effect on Cre recombination. Our results demonstrate that PepA requires specific DNA sequences for binding, and that PepA and ArgR have no direct role in Cre recombination at P1 loxP.

Proteasomes begin ornithine decarboxylase digestion at the C terminus.

Proteasomes denature folded protein substrates and thread them through a narrow pore that leads to the sequestered sites of proteolysis. Whether a protein substrate initiates insertion from its N or C terminus or in a random orientation has not been determined for any natural substrate. We used the labile enzyme ornithine decarboxylase (ODC), which is recognized by the proteasome via a 37-residue C-terminal tag, to answer this question. Three independent approaches were used to assess orientation as follows. 1) The 461-residue ODC protein chain was interrupted at position 305. The C-terminal fragment was degraded by purified proteasomes, but because processivity requires continuity of the polypeptide chain, the N-terminal fragment was spared. 2) A proteasome-inhibitory viral sequence prevented degradation when introduced near the C terminus but not when inserted elsewhere in ODC. 3) A bulky tightly folded protein obstructed in vivo degradation most effectively when positioned near the C terminus. These data demonstrate that the proteasome initiates degradation of this native substrate at the C terminus. The co-localization of entry site and degradation tag to the ODC C terminus suggests that recognition tags determine the site for initiating entry. Flexibility of a polypeptide terminus may promote the initiation of degradation.