Hsp72 mediates TAp73α anti-apoptotic effects in small cell lung carcinoma cells.

The transcription factor p73, a member of the p53 family of proteins, is involved in the regulation of cell cycle progression and apoptosis. Due to alternative promoters and carboxy-terminal splicing, the P73 gene gives rise to a range of different isoforms. Interestingly, a particular increase in expression of the TAp73α isoform has been reported in various tumours. In addition, TAp73α has been shown to inhibit Bax activation and mitochondrial dysfunctions and thereby to confer small cell lung carcinoma (SCLC) cells resistance to drug-induced apoptosis. However, the precise mechanism by which TAp73α exerts its pro-survival effect is yet unclear. Here we report that TAp73α, but not TAp73ß, regulates the expression of inducible Hsp72/HSPA1A. Hsp72 proved to be required for the survival effects of TAp73α as antisense knockdown of Hsp72 resulted in an abolishment of the anti-apoptotic effect of TAp73α in SCLC cells upon Etoposide treatment. Importantly, depletion of Hsp72 allowed activation of Bax, loss of mitochondrial membrane potential and lysosomal membrane permeabilization in SCLC cells even in the presence of TAp73α. Finally, we revealed that TAp73ß counteracts the anti-apoptotic effect of TAp73α by preventing Hsp72 induction. Our results thus provide additional evidence for the potential oncogenic role of TAp73α, and extend the understanding of the mechanism for its anti-apoptotic effect.

A fine-needle aspiration-based protein signature discriminates benign from malignant breast lesions.

There are increasing demands for informative cancer biomarkers, accessible via minimally invasive procedures, both for initial diagnostics and to follow-up personalized cancer therapy. Fine-needle aspiration (FNA) biopsy provides ready access to relevant tissues; however, the minute sample amounts require sensitive multiplex molecular analysis to achieve clinical utility. We have applied proximity extension assays (PEA) and NanoString (NS) technology for analyses of proteins and of RNA, respectively, in FNA samples. Using samples from patients with breast cancer (BC, n = 25) or benign lesions (n = 33), we demonstrate that these FNA-based molecular analyses (a) can offer high sensitivity and reproducibility, (b) may provide correct diagnosis in shorter time and at a lower cost than current practice, (c) correlate with results from routine analysis (i.e., benchmarking against immunohistochemistry tests for ER, PR, HER2, and Ki67), and (d) may also help identify new markers related to immunotherapy. A specific 11-protein signature, including FGF binding protein 1, decorin, and furin, distinguished all cancer patient samples from all benign lesions in our main cohort and in smaller replication cohort. Due to the minimally traumatic sampling and rich molecular information, this combined proteomics and transcriptomic methodology is promising for diagnostics and evaluation of treatment efficacy in BC.

PLD-Specific Small-Molecule Inhibitors Decrease Tumor-Associated Macrophages and Neutrophils Infiltration in Breast Tumors and Lung and Liver Metastases.

Phospholipase D-2 (PLD2) has a key role in breast cancer formation and metastasis formation with PLD small inhibitors reducing primary tumor growth. This study aimed to evaluate the importance of targeting PLD on the tumor microenvironment. We provide evidence about the beneficial effect of PLD inhibitors [FIPI (dual PLD1/PLD2) or VU0155072-2 (PLD2 inhibitor)] on avoiding infiltration of tumor-helping macrophages and neutrophils. Tumor growth and metastasis within the primary tumors had low (<20% over controls) PLD enzyme activity. Unexpectedly, we found that the inhibitors also affected PLD2 gene expression and protein albeit at a lesser extent. The later could indicate that targeting both the actual PLD enzyme and its activity could be beneficial for potential cancer treatments in vivo. F4/80 and Ly6G staining of macrophages and neutrophils, respectively, and Arg1 staining data were consistent with M2 and N2 polarization. NOS2 staining increased in xenotransplants upon treatment with PLD2 inhibitors suggesting the novel observation that an increased recruitment of M1 macrophages occurred in primary tumors. PLD inhibitor-treated primary tumors had large, fragile, necrotic areas that were Arg1+ for M2 macrophages. The xenotransplants also caused the formation of large F4/80+ and Ly6G+ (>100 µm) clusters in lungs. However, PLD inhibitors, particularly FIPI, were able to diminish leukocyte presence. Ex vivo chemotaxis and PLD activity of peripheral blood neutrophils (PMN) and peritoneal macrophages was also determined. Whereas PMN had impaired functionality, macrophages did not. This significantly increased ("emboldened") macrophage function was due to PLD inhibition. Since tumor-associated leukocytes in primary tumors and metastases were targeted via PLD inhibition, we posit that these inhibitors have a key role in cancer regression, while still affording an appropriate inflammatory response at least from off-site innate immunity macrophages.

ERK3 regulates TDP2-mediated DNA damage response and chemoresistance in lung cancer cells.

Posttranslational modifications (PTMs), such as phosphorylation and ubiquitination, play critical regulatory roles in the assembly of DNA damage response proteins on the DNA damage site and their activities in DNA damage repair. Tyrosyl DNA phosphodiesterase 2 (TDP2) repairs Topoisomerase 2 (Top2)-linked DNA damage, thereby protecting cancer cells against Top2 inhibitors-induced growth inhibition and cell death. The regulation of TDP2 activity by post-translational modifications in DNA repair, however, remains unclear. In the current study, we have found that ERK3, an atypical MAPK, phosphorylates TDP2 at S60 and regulates TDP2's phosphodiesterase activity, thereby cooperatively protecting lung cancer cells against Top2 inhibitors-induced DNA damage and growth inhibition. As such, our study revealed a post-translational regulation of TDP2 activity and discovered a new role of ERK3 in increasing cancer cells' DNA damage response and chemoresistance to Top2 inhibitors.

TAp73alpha protects small cell lung carcinoma cells from caspase-2 induced mitochondrial mediated apoptotic cell death.

Caspase-2 is ubiquitously expressed and the most evolutionarily conserved mammalian caspase. It can be activated by a range of death stimuli prior to Bax activation and the occurrence of apoptotic mitochondrial dysfunctions. Caspase-2 has also been reported to exert tumour suppressor function in vivo. The full length TAp73alpha isoform is found up-regulated in various tumour types, and is reported in a cell-type specific manner to repress drug-induced apoptosis. Here, we report that TAp73alpha represses caspase-2 enzymatic activity and by this means reduce caspase-2 induced Bax activation, loss of mitochondrial transmembrane potential and resulting apoptosis. The inhibitory effect on caspase-2 requires the presence of the DNA binding domain and SAM domain region of TAp73alpha. In conclusion, the ability of TAp73alpha to act as an inhibitor of caspase-2-induced cell death together with its up-regulation in certain tumour types strengthen the potential oncogenic activities for this protein.