Targeting extracellular matrix remodeling sensitizes glioblastoma to ionizing radiation.

Background: The median survival of Glioblastoma multiforme (GBM) patients is 14+ months due to poor responses to surgery and chemoradiation. Means to counteract radiation resistance are therefore highly desirable. We demonstrate the membrane bound matrix metalloproteinase MT1-MMP promotes resistance of GBM to radiation, and that using a selective and brain permeable MT1-MMP inhibitor, (R)-ND336, improved tumor control can be achieved in preclinical studies. Methods: Public microarray and RNA-sequencing data were used to determine MT1-MMP relevance in GBM patient survival. Glioma stem-like neurospheres (GSCs) were used for both in vitro and in vivo assays. An affinity resin coupled with proteomics was used to quantify active MT1-MMP in brain tissue of GBM patients. Short hairpin RNA (shRNA)-mediated knockdown of MT1-MMP and inhibition via the MT1-MMP inhibitor (R)-ND336, were used to assess the role of MT1-MMP in radio-resistance. Results: MT1-MMP expression inversely correlated with patient survival. Active MT1-MMP was present in brain tissue of GBM patients but not in normal brain. shRNA- or (R)-ND336-mediated inhibition of MT1-MMP sensitized GSCs to radiation leading to a significant increase in survival of tumor-bearing animals. MT1-MMP depletion reduced invasion via the effector protease MMP2; and increased the cytotoxic response to radiation via induction of replication fork stress and accumulation of double strand breaks (DSBs), making cells more susceptible to genotoxic insult. Conclusions: MT1-MMP is pivotal in maintaining replication fork stability. Disruption of MT1-MMP sensitizes cells to radiation and can counteract invasion. (R)-ND336, which efficiently penetrates the brain, is therefore a novel radio-sensitizer in GBM.

MiRNA-200C expression in Fanconi anemia pathway functionally deficient lung cancers.

The Fanconi Anemia (FA) pathway is essential for human cells to maintain genomic integrity following DNA damage. This pathway is involved in repairing damaged DNA through homologous recombination. Cancers with a defective FA pathway are expected to be more sensitive to cross-link based therapy or PARP inhibitors. To evaluate downstream effectors of the FA pathway, we studied the expression of 734 different micro RNAs (miRNA) using NanoString nCounter miRNA array in two FA defective lung cancer cells and matched control cells, along with two lung tumors and matched non-tumor tissue samples that were deficient in the FA pathway. Selected miRNA expression was validated with real-time PCR analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibits cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. miRNA-200C was increased in the FA defective lung cancers as compared to controls. AmpliSeq analysis showed significant reduction in ZEB1 and ZEB2 mRNA expression. Our findings indicate the miRNA-200C potentially play a very important role in FA pathway downstream regulation.

Spermidine/spermine N1-acetyltransferase 1 is a gene-specific transcriptional regulator that drives brain tumor aggressiveness.

Spermidine/spermine N1-acetyltransferase 1 (SAT1), the rate-limiting enzyme in polyamine catabolism, has broad regulatory roles due to near ubiquitous polyamine binding. We describe a novel function of SAT1 as a gene-specific transcriptional regulator through local polyamine acetylation. SAT1 expression is elevated in aggressive brain tumors and promotes resistance to radiotherapy. Expression profiling in glioma cells identified SAT1 target genes that distinguish high- and low-grade tumors, in support of the prognostic utility of SAT1 expression. We further discovered mechanisms of SAT1-driven tumor aggressiveness through promotion of expression of both DNA damage response pathways as well as cell cycle regulatory genes. Mechanistically, SAT1 associates specifically with the promoter of the MELK gene, which functionally controls other SAT1 targets, and leads biologically to maintenance of neurosphere stemness in conjunction with FOXM1 and EZH2. CRISPR knockin mutants demonstrate the essentiality of the polyamine acetyltransferase activity of SAT1 for its function as a transcriptional regulator. Together, the data demonstrate that gene-specific polyamine removal is a major transcriptional regulatory mechanism active in high-grade gliomas that drives poor outcomes.

The Ig superfamily protein PTGFRN coordinates survival signaling in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor with a median survival of approximately 14 months. Despite aggressive treatment of surgical resection, chemotherapy and radiation therapy, only 3-5% of GBM patients survive more than 3 years. Contributing to this poor therapeutic response, it is believed that GBM contains both intrinsic and acquired mechanisms of resistance, including resistance to radiation therapy. In order to define novel mediators of radiation resistance, we conducted a functional knockdown screen, and identified the immunoglobulin superfamily protein, PTGFRN. In GBM, PTGFRN is found to be overexpressed and to correlate with poor survival. Reducing PTGFRN expression radiosensitizes GBM cells and potently decreases the rate of cell proliferation and tumor growth. Further, PTGFRN inhibition results in significant reduction of PI3K p110ß and phosphorylated AKT, due to instability of p110ß. Additionally, PTGFRN inhibition decreases nuclear p110ß leading to decreased DNA damage sensing and DNA damage repair. Therefore overexpression of PTGFRN in glioblastoma promotes AKT-driven survival signaling and tumor growth, as well as increased DNA repair signaling. These findings suggest PTGFRN is a potential signaling hub for aggressiveness in GBM.

Type of TP53 mutation influences oncogenic potential and spectrum of associated K-ras mutations in lung-specific transgenic mice.

TP53 and K-ras mutations are two of the major genetic alterations in human nonsmall cell lung cancers. The association between these two genes during lung tumorigenesis is unknown. We evaluated the potential of two common Type I (273H, contact) and Type II (175H, conformational) TP53 mutations to induce lung tumors in transgenic mice, as well as K-ras status, and other driver mutations in these tumors. Among 516 (138 nontransgenic, 207 SPC-TP53-273H, 171 SPC-TP53-175H) mice analyzed, 91 tumors, all adenocarcinomas, were observed. Type II mutants developed tumors more frequently (as compared to nontransgenics, p = 0.0003; and Type I, p = 0.010), and had an earlier tumor onset compared to Type I (p = 0.012). K-ras mutations occurred in 21 of 50 (42%) of murine lung tumors sequenced. For both the nontransgenic and the SPC-TP53-273H transgenics, tumor K-ras codon 12-13 mutations occurred after 13 months with a peak incidence at 16-18 months. However, for the SPC-TP53-175H transgenics, K-ras codon 12-13 mutations were observed as early as 6 months, with a peak incidence between the ages of 10-12 months. Codons 12-13 transversion mutations were the predominant changes in the SPC-TP53-175H transgenics, whereas codon 61 transition mutations were more common in the SPC-TP53-273H transgenics. The observation of accelerated tumor onset, early appearance and high frequency of K-ras codon 12-13 mutations in the Type II TP53-175H mice suggests an enhanced oncogenic function of conformational TP53 mutations, and gains in early genetic instability for tumors containing these mutations compared to contact mutations.

The membrane tethered matrix metalloproteinase MT1-MMP triggers an outside-in DNA damage response that impacts chemo- and radiotherapy responses of breast cancer.

Breast cancer is the second leading cause of death among women in the US. Targeted therapies exist, however resistance is common and patients resort to chemotherapy. Chemotherapy is also a main treatment for triple negative breast cancer (TNBC) patients; while radiation is delivered to patients with advanced disease to counteract metastasis. Yet, resistance to both chemo- and radiotherapy is still frequent, highlighting a need to provide novel sensitizers. We discovered that MT1-MMP modulates DNA damage responses (DDR) in breast cancer. MT1-MMP expression inversely correlates to chemotherapy response of breast cancer patients. Inhibition of MT1-MMP sensitizes TNBC cells to IR and doxorubicin in vitro, and in vivo in an orthotopic breast cancer model. Specifically, depletion of MT1-MMP causes stalling of replication forks and Double Strand Breaks (DBSs), leading to increased sensitivity to additional genotoxic stresses. These effects are mediated by integrinß1, as a constitutive active integrinß1 reverts replication defects and protects cells depleted of MT1-MMP from IR and chemotherapy. These data highlight a novel DNA damage response triggered by MT1-MMP-integrinß1 and provide a new point of therapeutic targeting that may improve breast cancer patient outcomes.

MMR Deficiency Does Not Sensitize or Compromise the Function of Hematopoietic Stem Cells to Low and High LET Radiation.

One of the major health concerns on long-duration space missions will be radiation exposure to the astronauts. Outside the earth's magnetosphere, astronauts will be exposed to galactic cosmic rays (GCR) and solar particle events that are principally composed of protons and He, Ca, O, Ne, Si, Ca, and Fe nuclei. Protons are by far the most common species, but the higher atomic number particles are thought to be more damaging to biological systems. Evaluation and amelioration of risks from GCR exposure will be important for deep space travel. The hematopoietic system is one of the most radiation-sensitive organ systems, and is highly dependent on functional DNA repair pathways for survival. Recent results from our group have demonstrated an acquired deficiency in mismatch repair (MMR) in human hematopoietic stem cells (HSCs) with age due to functional loss of the MLH1 protein, suggesting an additional risk to astronauts who may have significant numbers of MMR deficient HSCs at the time of space travel. In the present study, we investigated the effects gamma radiation, proton radiation, and 56 Fe radiation on HSC function in Mlh1+/+ and Mlh1-/- marrow from mice in a variety of assays and have determined that while cosmic radiation is a major risk to the hematopoietic system, there is no dependence on MMR capacity. Stem Cells Translational Medicine 2018;7:513-520.

HIF drives lipid deposition and cancer in ccRCC via repression of fatty acid metabolism.

Clear cell renal cell carcinoma (ccRCC) is histologically defined by its lipid and glycogen-rich cytoplasmic deposits. Alterations in the VHL tumor suppressor stabilizing the hypoxia-inducible factors (HIFs) are the most prevalent molecular features of clear cell tumors. The significance of lipid deposition remains undefined. We describe the mechanism of lipid deposition in ccRCC by identifying the rate-limiting component of mitochondrial fatty acid transport, carnitine palmitoyltransferase 1A (CPT1A), as a direct HIF target gene. CPT1A is repressed by HIF1 and HIF2, reducing fatty acid transport into the mitochondria, and forcing fatty acids to lipid droplets for storage. Droplet formation occurs independent of lipid source, but only when CPT1A is repressed. Functionally, repression of CPT1A is critical for tumor formation, as elevated CPT1A expression limits tumor growth. In human tumors, CPT1A expression and activity are decreased versus normal kidney; and poor patient outcome associates with lower expression of CPT1A in tumors in TCGA. Together, our studies identify HIF control of fatty acid metabolism as essential for ccRCC tumorigenesis.

Rad51C-ATXN7 fusion gene expression in colorectal tumors.

BACKGROUND: Fusion proteins have unique oncogenic properties and their identification can be useful either as diagnostic or therapeutic targets. Next generation sequencing data have previously shown a fusion gene formed between Rad51C and ATXN7 genes in the MCF7 breast cancer cell line. However, the existence of this fusion gene in colorectal patient tumor tissues is largely still unknown. METHODS: We evaluated for the presence of Rad51C-ATXN7 fusion gene in colorectal tumors and cells by RT-PCR, PCR, Topo TA cloning, Real time PCR, immunoprecipitation and immunoblotting techniques. RESULTS: We identified two forms of fusion mRNAs between Rad51C and ATXN7 in the colorectal tumors, including a Variant 1 (fusion transcript between Rad51C exons 1-7 and ATXN7 exons 6-13), and a Variant 2 (between Rad51C exons 1-6 and ATXN7 exons 6-13). In silico analysis showed that the Variant 1 produces a truncated protein, whereas the Variant 2 was predicted to produce a fusion protein with molecular weight of 110 KDa. Immunoprecipitation and Western blot analysis further showed a 110 KDa protein in colorectal tumors. 5-Azacytidine treatment of LS-174 T cells caused a 3.51-fold increase in expression of the fusion gene (Variant 2) as compared to no treatment controls evaluated by real time PCR. CONCLUSION: In conclusion we found a fusion gene between DNA repair gene Rad51C and neuro-cerebral ataxia Ataxin-7 gene in colorectal tumors. The in-frame fusion transcript of Variant 2 results in a fusion protein with molecular weight of 110 KDa. In addition, we found that expression of fusion gene is associated with functional impairment of Fanconi Anemia (FA) DNA repair pathway in colorectal tumors. The expression of Rad51C-ATXN7 in tumors warrants further investigation, as it suggests the potential of the fusion gene in treatment and predictive value in colorectal cancers.

Overexpression of Rad51C splice variants in colorectal tumors.

Functional alterations in Rad51C are the cause of the Fanconi anemia complementation group O (FANCO) gene disorder. We have identified novel splice variants of Rad51C mRNA in colorectal tumors and cells. The alternatively spliced transcript variants are formed either without exon-7 (variant 1), without exon 6 and 7 (variant 2) or without exon 7 and 8 (variant 3). Real time PCR analysis of nine pair-matched colorectal tumors and non-tumors showed that variant 1 was overexpressed in tumors compared to matched non-tumors. Among 38 colorectal tumor RNA samples analyzed, 18 contained variant 1, 12 contained variant 2, 14 contained variant 3, and eight expressed full length Rad51C exclusively. Bisulfite DNA sequencing showed promoter methylation of Rad51C in tumor cells. 5-azacytidine treatment of LS-174T cells caused a 14 fold increase in variant 1, a 4.8 fold increase for variant 3 and 3.4 fold for variant 2 compared to 2.5 fold increase in WT. Expression of Rad51C variants is associated with FANCD2 foci positive colorectal tumors and is associated with microsatellite stability in those tumors. Further investigation is needed to elucidate differential function of the Rad51C variants to evaluate potential effects in drug resistance and DNA repair.

Fanconi anemia repair pathway dysfunction, a potential therapeutic target in lung cancer.

The Fanconi anemia (FA) pathway is a major mechanism of homologous recombination DNA repair. The functional readout of the pathway is activation through mono-ubiquitination of FANCD2 leading to nuclear foci of repair. We have recently developed an FA triple-staining immunofluorescence based method (FATSI) to evaluate FANCD2 foci formation in formalin fixed paraffin-embedded (FFPE) tumor samples. DNA-repair deficiencies have been considered of interest in lung cancer prevention, given the persistence of damage produced by cigarette smoke in this setting, as well as in treatment, given potential increased efficacy of DNA-damaging drugs. We screened 139 non-small cell lung cancer (NSCLC) FFPE tumors for FANCD2 foci formation by FATSI analysis. Among 104 evaluable tumors, 23 (22%) were FANCD2 foci negative, thus repair deficient. To evaluate and compare novel-targeted agents in the background of FA deficiency, we utilized RNAi technology to render several lung cancer cell lines FANCD2 deficient. Successful FANCD2 knockdown was confirmed by reduction in the FANCD2 protein. Subsequently, we treated the FA defective H1299D2-down and A549D2-down NSCLC cells and their FA competent counterparts (empty vector controls) with the PARP inhibitors veliparib (ABT-888) (5 µM) and BMN673 (0.5 µM), as well as the CHK1 inhibitor Arry-575 at a dose of 0.5 µM. We also treated the FA defective small cell lung cancer cell lines H719D2-down and H792D2-down and their controls with the BCL-2/XL inhibitor ABT-263 at a dose of 2 µM. The treated cells were harvested at 24, 48, and 72 h post treatment. MTT cell viability analysis showed that each agent was more cytotoxic to the FANCD2 knock-down cells. In all tests, the FA defective lung cancer cells had less viable cells as comparing to controls 72 h post treatment. Both MTT and clonogenic analyses comparing the two PARP inhibitors, showed that BMN673 was more potent compared to veliparib. Given that FA pathway plays essential roles in response to DNA damage, our results suggest that a subset of lung cancer patients are likely to be more susceptible to DNA cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. These subjects can be identified through FATSI analysis. Clinical trials to evaluate this therapeutic concept are needed.