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Summary The density of nerves in the tumor microenvironment is increasingly reported to be associated with worse clinical outcome in various cancers. Therefore, it is time to consider the assessment of nerve density in clinical cancer pathology, and interestingly, the development of artificial intelligence may facilitate this clinical translation. See related article by Perez-Pacheco et al., p. xxxx

Shell-isolated nanoparticle-enhanced Raman spectroscopy has been successfully applied to probe electroreductive dehalogenation of aryl halides at an Ag electrode.

Supplementary Figure S1. AUY922 does not significantly affect viability of normal colon epithelial cells; Supplementary Figure S2. Sensitivity of colon cancer cells to AUY922 is not associated with the expression levels of HSP90; Supplementary Figure S3. The general caspase inhibitor z-VAD-fmk efficiently inhibits AUY922-mediated inhibition of mutant KRAS colon cancer cell survival; Supplementary Figure S4. AUY922 kills colon cancer cells by induction of apoptosis; Supplementary Figure S5. AUY922 triggers reduction in the mitochondrial membrane potential, release of mitochondrial apoptogenic proteins in sensitive but not resistant colon cells; Supplementary Figure S6. Inhibition of Bim does not alter sensitivity of Caco-2 cells to AUY922; Supplementary Figure S7. Knockdown of PUMA or p53 does not inhibit cell death induced by AUY922; Supplementary Figure S8. Upregulation of Bim by AUY922 in mutant KRAS colon cancer cells is due to a transcriptional increase; Supplementary Figure S9. Knockdown of CHOP inhibits cell death induced by AUY922; Supplementary Figure S10. BGP-15 and 4-PBA attenuate XBP-1 splicing induced by AUY922; Supplementary Figure S11. Overexpression of GRP78 reduces AUY922-induced upregulation of CHOP and Bim; Supplementary Figure S12. A schematic model comparing different responses of mutant and wild-type KRAS colon cancer cells to AUY922; Supplementary Figure S13. AUY922 does not cause any significant change in the expression of STK33 in mutant KRAS colon cancer cells; Supplementary Table 1. List of Antibodies; Supplementary Table 2. List of reagents; Supplementary Table 3. siRNAs

Past studies have shown that activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK is a common cause for resistance of melanoma cells to death receptor–mediated or mitochondria-mediated apoptosis. We report in this study that inhibition of the MEK/ERK pathway also sensitizes melanoma cells to endoplasmic reticulum (ER) stress-induced apoptosis, and this is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperon glucose-regulated protein 78 (GRP78) expression. Treatment with the ER stress inducer tunicamycin or thapsigargin did not induce significant apoptosis in the majority of melanoma cell lines, but resistance to these agents was reversed by the MEK inhibitor U0126 or MEK1 small interfering RNA (siRNA). Induction of apoptosis by ER stress when MEK was inhibited was caspase dependent with caspase-4, caspase-9, and caspase-3 being involved. Caspase-4 seemed to be the apical caspase in that caspase-4 activation occurred before activation of caspase-9 and caspase-3 and that inhibition of caspase-4 by a specific inhibitor or siRNA blocked activation of caspase-9 and caspase-3, whereas inhibition of caspase-9 or caspase-3 did not inhibit caspase-4 activation. Moreover, overexpression of Bcl-2 inhibited activation of caspase-9 and caspase-3 but had minimal effect on caspase-4 activation. Inhibition of MEK/ERK also resulted in down-regulation of GRP78, which was physically associated with caspase-4, before and after treatment with tunicamycin or thapsigargin. In addition, siRNA knockdown of GRP78 increased ER stress-induced caspase-4 activation and apoptosis. Taken together, these results seem to have important implications for new treatment strategies in melanoma by combinations of agents that induce ER stress and inhibitors of the MEK/ERK pathway. [Cancer Res 2007;67(20):9750–61]

We have previously shown that most melanoma cell lines are insensitive to endoplasmic reticulum (ER) stress–induced apoptosis, and this involves activation of the mitogen-activated protein/extracellular signal-regulated kinase (MEK)/ERK signaling pathway and expression of the apoptosis repressor with caspase recruitment domain (ARC) protein in the cells. In the present study, we show that up-regulation of the antiapoptotic Bcl-2 family member Mcl-1 is another mechanism critical for protection of melanoma cells against ER stress–induced apoptosis. Inhibition of Mcl-1 by small interference RNA (siRNA) rendered melanoma cells sensitive to apoptosis induced by the ER stress inducers thapsigargin and tunicamycin, but this sensitization was partially reversed by siRNA knockdown of PUMA or Noxa, as shown in Mcl-1–deficient melanoma cells. Both PUMA and Noxa were increased by ER stress through transcriptional up-regulation, but only up-regulation of Noxa was dependent on p53, whereas up-regulation of PUMA seemed to be mediated by a p53-independent mechanism(s). Up-regulation of Mcl-1 was also due to increased transcription that involved the IRE1α and activating transcription factor 6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway seemed to be necessary for optimal up-regulation of Mcl-1. Taken together, these results reveal the mechanisms of resistance of melanoma cells to apoptosis induction mediated by BH3-only proteins upon ER stress, and identify Mcl-1 as a target for the treatment of melanoma in combination with therapeutics that induce ER stress. [Cancer Res 2008;68(16):6708–17]

Supplementary Experimental Procedures

Supplementary Figure 2 from Inhibition of Endoplasmic Reticulum Stress–Induced Apoptosis of Melanoma Cells by the ARC Protein

Supplementary Figs. S1-S8 and Table S1 Supplementary Materials and Methods - The primers used for qPCR. Supplementary Fig. S1. Quantitation of RIP1 expression in melanocytic tumors. Supplementary Fig. S2. Melanocytes of different origins express lower levels of RIP1 than melanoma cells. Supplementary Fig. S3. Overexpression of RIP1 triggers cell death independently of its kinase domain. Supplementary Fig. S4. NF-kB is constitutively activated in melanoma cells. Supplementary Fig. S5. RIP1 overexpression triggers moderate reduction in melanocyte viability independently of caspases. Supplementary Fig. S6. RIP1 mRNA expression and its gene copy number variations in fresh melanoma isolates. Supplementary Fig. S7. The turnover rates of RIP1 mRNA are comparable between melanoma cells and melanocytes. Supplementary Fig. S8. Overexpression of RIP1 reverses reduction in NF-kB activation and melanoma cell proliferation caused by knockdown of TNFR1. Supplementary Table S1. Summary of melanocytic tumors and their expression levels of RIP1.

Supplementary Figure 4 from Inhibition of MEK Sensitizes Human Melanoma Cells to Endoplasmic Reticulum Stress-Induced Apoptosis

Supplementary Figure 4 from Inhibition of Endoplasmic Reticulum Stress–Induced Apoptosis of Melanoma Cells by the ARC Protein

Supplementary Figure 2 from Inhibition of MEK Sensitizes Human Melanoma Cells to Endoplasmic Reticulum Stress-Induced Apoptosis

Supplementary Figure 1 from Inhibition of Endoplasmic Reticulum Stress–Induced Apoptosis of Melanoma Cells by the ARC Protein

Supplementary Figure 1 from Inhibition of MEK Sensitizes Human Melanoma Cells to Endoplasmic Reticulum Stress-Induced Apoptosis

Supplementary Figures 1-9 from Up-regulation of Mcl-1 Is Critical for Survival of Human Melanoma Cells upon Endoplasmic Reticulum Stress

Supplementary Figure 3 from Inhibition of MEK Sensitizes Human Melanoma Cells to Endoplasmic Reticulum Stress-Induced Apoptosis

Supplementary Figure 3 from Inhibition of Endoplasmic Reticulum Stress–Induced Apoptosis of Melanoma Cells by the ARC Protein

We have shown previously that most melanoma cell lines are insensitive to endoplasmic reticulum (ER) stress–induced apoptosis, but resistance can be reversed through activation of caspase-4 by inhibition of the MEK/ERK pathway. We report in this study that apoptosis was induced by the ER stress inducer thapsigargin or tunicamycin via a caspase-8–mediated pathway in the melanoma cell line Me1007, although the MEK/ERK pathway was activated in this cell line. The high sensitivity of Me1007 to ER stress–induced apoptosis was associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein that was expressed at relatively high levels in the resistant melanoma cell lines. Transfection of cDNA encoding ARC into Me1007 cells inhibited both caspase-8 activation and apoptosis induced by thapsigargin or tunicamycin. In contrast, inhibition of ARC by small interfering RNA knockdown sensitized the resistant melanoma cell lines to ER stress–induced apoptosis, which was inhibitable by blockage of caspase-8 activation. Both exogenous and endogenous ARC seemed to predominantly locate to the cytoplasm and mitochondria and could be coimmunoprecipitated with caspase-8. Taken together, ER stress can potentially activate multiple apoptosis signaling pathways in melanoma cells in a context-dependent manner. Whereas the MEK/ERK signaling pathway plays an important role in inhibiting ER stress–induced caspase-4 activation, ARC seems to be critical in blocking activation of casapse-8 in melanoma cells subjected to ER stress. [Cancer Res 2008;68(3):834–42]

In situ shell-isolated nanoparticle-enhanced Raman spectroscopy provides molecular-level insights into the electroreductive dehalogenation mechanism of aryl halides.

The comorbidity of autism spectrum disorder and anxiety is common, but the underlying circuitry is poorly understood. Here, Tmem74-/- mice showed autism- and anxiety-like behaviors along with increased excitability of pyramidal neurons (PNs) in the prelimbic cortex (PL), which were reversed by Tmem74 re-expression and chemogenetic inhibition in PNs of the PL. To determine the underlying circuitry, we performed conditional deletion of Tmem74 in the PNs of PL of mice, and we found that alterations in the PL projections to fast-spiking interneurons (FSIs) in the dorsal striatum (dSTR) (PLPNs–dSTRFSIs) mediated the hyperexcitability of FSIs and autism-like behaviors and that alterations in the PL projections to the PNs of the basolateral amygdaloid nucleus (BLA) (PLPNs–BLAPNs) mediated the hyperexcitability of PNs and anxiety-like behaviors. However, the two populations of PNs in the PL had different spatial locations, optogenetic manipulations revealed that alterations in the activity in the PL–dSTR or PL–BLA circuits led to autism- or anxiety-like behaviors, respectively. Collectively, these findings highlight that the hyperactivity of the two populations of PNs in the PL mediates autism and anxiety comorbidity through the PL–dSTR and PL–BLA circuits, which may lead to the development of new therapeutics for the autism and anxiety comorbidity.

This paper explored the protective effect of total flavonoids of Rhododendron simsii(TFR) on focal cerebral ischemia-reperfusion injury(CIRI) in rats and its relationship with the store-operated calcium entry(SOCE) pathway regulated by stromal intera-ction molecule(STIM) and calcium release-activated calcium modulator(Orai).Rats were randomly assigned into the sham group, model(middle cerebral artery occlusion, MCAO) group, TFR(60 mg·kg~(-1)) group, TFR(60 mg·kg~(-1))+SOCE pathway inhibitor 2-aminoethoxydiphenyl borate(2-APB, 2.5 mg·kg~(-1)) group, and 2-APB(2.5 mg·kg~(-1)) group.The rats in the sham group and MCAO group were administrated with normal saline, and those in the TFR group and TFR+2-APB group were administrated with TFR(60 mg·kg~(-1)) by gavage for 14 days until sampling.The rats in the 2-APB group and TFR+2-APB group were intraperitoneally injected with 2-APB(2.5 mg·kg~(-1)) after operation.The levels of interleukin-1(IL-1), interleukin-6(IL-6), and tumor necrosis factor-alpha(TNF-α) in serum were measured by ELISA.The cerebral infarction and the pathological status of ischemic brain tissue were detected via TTC staining and HE staining, respectively.The protein and mRNA levels of STIM1, STIM2, Orai1, cysteinyl aspartate specific proteinase 3(caspase-3), and protein kinase B(PKB) in brain tissue were respectively determined by Western blot and RT-qPCR.The growth of brain neurons in each group was observed via immunofluorescence method.The results showed that compared with the MCAO group, TFR lowered the levels of IL-1, IL-6 and TNF-α in serum and the score of neurological function, ameliorated the pathological injury of brain tissue, and decreased the infarct size.Moreover, TFR up-regulated the mRNA and protein levels of STIM1, STIM2, Orai1, and PKB, down-regulated those of caspase-3 in brain tissue, and increased the double-labeled positive cells under fluorescence microscope.However, the above effects were significantly weakened by the addition of 2-APB, a SOCE inhibitor.The results suggested that TFR may play a protective role against focal cerebral ischemia-reperfusion injury by up-regulating the expression of SOCE-related signal molecules, promoting neurogenesis around the ischemic area, improving the survival state of neurons, and redu-cing the activity of inflammatory mediators.

Pancreatic cancer is a highly aggressive malignancy characterized by poor survival, recurrence after surgery and resistance to therapy. Nerves infiltrate the microenvironment of pancreatic cancers and contribute to tumor progression, however the clinicopathological significance of tumor innervation is unclear. In this study, the presence of nerves and their cross-sectional size were quantified by immunohistochemistry for the neuronal markers S-100, PGP9.5 and GAP-43 in a series of 99 pancreatic cancer cases versus 71 normal adjacent pancreatic tissues. A trend was observed between the presence of nerves in the tumor microenvironment of pancreatic cancer and worse overall patient survival (HR = 1.8, 95% CI 0.77–4.28, p = 0.08). The size of nerves, as measured by cross-sectional area, were significantly higher in pancreatic cancer than in the normal adjacent tissue (p = 0.002) and larger nerves were directly associated with worse patient survival (HR = 0.41, 95% CI 0.19–0.87, p = 0.04). In conclusion, this study suggests that the presence and size of nerves within the pancreatic cancer microenvironment are associated with tumor aggressiveness.

Shikonin is the main component of the traditional Chinese medicine comfrey, which can inhibit the activity of PKM2 by regulating glycolysis and ATP production. Rheumatoid arthritis synovial cells (RA-FLSs) have been reported to increase glycolytic activity and have other similar hallmarks of metabolic activity. In this study, we investigated the effects of shikonin on glycolysis, mitochondrial function, and cell death in RA-FLSs. The results showed that shikonin induced apoptosis and autophagy in RA-FLSs by activating the production of reactive oxygen species (ROS) and inhibiting intracellular ATP levels, glycolysis-related proteins, and the PI3K-AKT-mTOR signaling pathway. Shikonin can significantly reduce the expression of apoptosis-related proteins, paw swelling in rat arthritic tissues, and the levels of inflammatory factors in peripheral blood, such as TNF-α, IL-6, IL-8, IL-10, IL-17A, and IL-1β while showing less toxicity to the liver and kidney.

Tumor metastasis is the ultimate stage of cancer, and the primary cause of mortality in patients. Tumor cells breaking through the natural barrier consisting of the basement membrane (BM) and extracellular matrix (ECM) is the a crucial step in tumor invasion and metastasis. Thus, protecting this barrier is the key to reducing mortality. Heparanase is a mammalian endo-β-glucuronidase which has been found to promote the cleavage of heparan sulfate (HS), and plays a significant role in tumor cell invasion and metastasis. Although chemotherapeutic reagents have a strong antitumor activity, they may promote the invasion and migration of cancer cells, as has been observed during clinical treatment. Chemotherapeutic reagents can induce endoplasmic reticulum (ER) stress; in this study, we used adriamycin (ADM) and a classical ER stress inducer, tunicamycin (TM). We report that the activation of ER stress is involved in the enhanced invasion and migration ability of breast cancer cells and we hypothesized that this effect is associated with the activation of heparanase. In support of this, we used the heparanase inhibitor, OGT2115, and low molecular weight heparin (LMWH) to inhibit the expression and activity of heparanase, and we found that the invasion and migration ability of the cells was suppressed. Our findings demonstrate that heparanase inhibitors suppress breast cancer cell invasion and migration induced by ER stress, and provide a strong rationale for the development of heparanase-based therapeutics for the prevention of metastasis induced by chemotherapeutic reagents.

Current experimental results indicate that endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which rebuilds ER homeostasis, through which tumor cells can become resistant chemotherapeutic agents. Autophagy is a form of programmed cell death, but it can also play a cytoprotective role in tumor cells, indicating that it has an inverse function. The aim of the present study was to investigate whether tunicamycin (TM) induces autophagy, as well as whether the inhibition of autophagy enhances the apoptosis ofbreast cancer cells induced by TM. In addition, we wished to investigate the mechanisms through which specific UPR targets control autophagy. We found that MCF-7 and MDA-MB‑231 breast cancer cells were insensitive to TM at a relatively low concentration. As shown by western blot analysis, treatment with TM increased the expression of 78 kDa glucose-regulated protein (GRP78), inositol requiring enzyme 1 (IRE1), beclin-1, IRE1α, p-JNK and microtubule-associated protein 1 light chain 3 (LC3); the expression of p62 increased at an early time point during treatment and subsequently decreased. We also used the specific inhibitor of autophagy, 3-methyladenine (3-MA), to elucidate the role of autophagy in ER stress in the breast cancer cells treated with TM. The transformation of LC3-I to LC3-II which was induced by TM, was reversed following treatment with 3-MA. The inhibition of autophagy by 3-MA treatment enhanced the inhibitory and apoptotic rates of TM in the breast cancer cells, as shown by confocal microscopy and flow cytometry. TM increased the misfolded proteins that lead to the activation of ER stress-mediated protection and induced apoptosis paralleled by autophagy in breast cancer cells which was regulated by IRE1/JNK/beclin-1. Autophagy attenuates ER stress by clearing ubiquitinated proteins and decreasing apoptosis, which plays a protective role. The inhibition of autophagy or the promotion of ER stress may be used as therapeutic targets to improve the efficacy of chemotherapeutic drugs.

Nerve infiltration in the tumor microenvironment is emerging as a promoter of cancer progression that could be targeted in therapies, but the mechanisms initiating tumor innervation remain to be elucidated. Here we report that endoplasmic reticulum (ER) stress in cancer cells is transmitted to neuronal cells, resulting in neurite outgrowth and tumor innervation. In vitro, the induction of ER stress in various human cancer cells resulted in the synthesis and release of the precursor for brain-derived neurotrophic factor (proBDNF) through a mechanism dependent on the transcription factor X-box binding protein 1 (XBP1). Cancer cell-released proBDNF was found to mediate the transmission of ER stress to neurons, resulting in the stimulation of neurite outgrowth. Next-generation sequencing indicated the increased expression of the Egl-9 family hypoxia inducible factor 3 (EGLN3) that was mediated by c-MYC and necessary to neurite outgrowth induced by proBDNF. In orthotopic tumor xenograft, ER stress stimulated XBP1 and proBDNF expression as well as tumor innervation. Anti-proBDNF antibody inhibited both tumor innervation and cancer progression induced by ER stress. Interestingly, the chemotherapeutic drug 5-Fluorouracil (5-FU) was found to induce ER stress and tumor innervation, and this effect was inhibited by anti-proBDNF antibody. Finally, in human tumors, cancer tissues with nerve infiltration expressed high XBP1 and proBDNF while EGLN3 was upregulated in infiltrated nerves. This study reveals that ER stress participates in tumor innervation through the release of proBDNF and that targeting this pathway could be used in future therapies.

Microchromosome maintenance protein 7 (MCM7), a DNA replication permitting factor, plays an essential role in initiating DNA replication. MCM7 is reported to be involved in tumor formation and progression, whereas the expression profile and molecular function of MCM7 in colorectal cancer (CRC) remain unknown. In this study, we aimed to evaluate the clinical significance and biological function of MCM7 in CRC and investigated whether MCM7 can be used for a differential diagnosis in CRC and whether it may serve as a more sensitive proliferation marker for CRC evaluation. Moreover, immunohistochemical analysis of MCM7 was performed in a total of 89 specimens, and high MCM7 expression levels were associated with worse overall survival (OS) in CRC patients. Furthermore, the cell functional test suggested that lentivirus-mediated silencing of MCM7 with shRNA in CRC cells significantly inhibited cellular proliferation and promoted apoptosis in vitro and inhibited tumor growth in vivo. Additionally, mechanistic studies further demonstrated that P21-activated protein kinase 2 (PAK2) was regulated by MCM7 via microarray analysis and cell functional recovery tests, and miR-107 played a role in regulating expression MCM7 via miRNA microarray analysis and 3’UTR reporter assays. Taken together, our results suggest that the miR-107/MCM7/PAK2 pathway may participate in cancer progression and that MCM7 may serve as a prognostic biomarker in CRC

Bronsted acid and base interactions are a cornerstone of chemistry describing a wide range of chemical phenomena. However, probing such interaction at the solid-liquid interface to extract the elementary and intrinsic information at a single-molecule level remains a big challenge. Herein, we employ an STM break junction (STM-BJ) technique to investigate the acid-base chemistry of carboxylic acid-based molecules at a Au (111) model surface and propose a prototype of a single-molecule pH sensor for the first time. The single-molecule measurements in different environmental conditions verify that the formation probability of molecular junctions is determined by the populations of deprotonated -COO- form in a self-assembled monolayer. Furthermore, the variation of the intensity of the conductance peaks (i.e., junction-forming probability) with the pH of the bulk solution fits well to the Henderson-Hasselbalch type equation. From the equation, a good linear relation is found between the degree of dissociation of the immobilized -COOH group and the environmental pH, providing a feasible way to design chemicals and biosensors and a detector at the single-molecule scale.

Pancreatic cancer is a lethal malignancy and no screening biomarker or targeted therapy is currently available. Here, we performed a shotgun proteomic label-free quantification (LFQ) to define protein changes in the cellular proteome and secretome of four pancreatic cancer cell lines (PANC1, Paca44, Paca2, and BXPC3) versus normal human pancreatic ductal epithelial cells (HPDE). In the cellular proteome and secretome, 149 and 43 proteins were dysregulated in the most cancer cell lines, respectively. Using Ingenuity Pathway Analysis (IPA), the most dysregulated signaling pathways in pancreatic cancer cells included the activation of epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), extracellular regulated kinase (ERK), and the deactivation of type-I interferon (IFN) pathways, which could promote cancer cell progression and decrease antitumor immunity. Parallel reaction monitoring (PRM) mass spectrometry was used to confirm the changes of seven regulated proteins quantified by LFQ: EGFR, growth/differentiation factor 15 (GDF15), protein-glutamine gamma-glutamyltransferase 2 (TGM2), leukemia inhibitory factor (LIF), interferon-induced GTP-binding protein Mx1 (MX1), signal transducer and activator of transcription 1 (STAT1), and serpin B5 (SERPINB5). Together, this proteomic analysis highlights protein changes associated with pancreatic cancer cells that should be further investigated as potential biomarkers or therapeutic targets.

Pancreatic cancer has a dismal prognosis, and there is no targeted therapy against this malignancy. The neuronal membrane protein sortilin is emerging as a regulator of cancer cell development, but its expression and impact in pancreatic cancer are unknown. This study found that sortilin expression was higher in pancreatic cell lines versus normal pancreatic ductal epithelial cells, as shown by Western blot analysis and mass spectrometry. The increased sortilin level in pancreatic cancer cells was confirmed by immunohistochemistry in a series of 99 human pancreatic adenocarcinomas versus 48 normal pancreatic tissues (P = 0.0014). Sortilin inhibition by siRNA and the pharmacologic inhibitor AF38469 strongly reduced the adhesion and invasion of pancreatic cancer cells without affecting cell survival and viability. Sortilin inhibition also decreased the phosphorylation of the focal adhesion kinase in Tyr925. Together, these data show that sortilin contributes to pancreatic cancer invasion and could eventually be targeted in therapy.

Although the expression of programmed death-ligand 1 (PD-L1) is an important mechanism by which cancer cells evade the immune system, PD-L1 expression in cancer cells is commonly associated with patients' responses to treatment with anti-programmed death 1/PD-L1 antibodies. However, how PD-L1 expression is regulated in melanoma cells remains to be fully elucidated. Here we report that the class I histone deacetylase (HDAC) HDAC8 controls transcriptional activation of PD-L1 by a transcription complex consisting of transcription factors homeobox A5 and signal transducer and activator of transcription 3. Inhibition of HDAC8 upregulated PD-L1 in melanoma cells. This was due to an increase in the activity of a fragment of the PD-L1 gene promoter that is enriched with binding sites for both homeobox A5 and signal transducer and activator of transcription 3. Indeed, knockdown of homeobox A5 or signal transducer and activator of transcription 3 abolished upregulation of PD-L1 by HDAC8 inhibition. Moreover, homeobox A5 and signal transducer and activator of transcription 3 were physically associated and appeared interdependent in activating PD-L1 transcription. Functional studies showed that HDAC8-mediated regulation of PD-L1 expression participated in modulating anti-melanoma T-cell responses. Collectively, these results identify HDAC8 as an important epigenetic regulator of PD-L1 expression, with implications for better understanding of the interaction between melanoma cells and the immune system.

Neurotrophin receptors are emerging targets in oncology, but their clinicopathologic significance in thyroid cancer is unclear. In this study, the neurotrophin tyrosine receptor kinase TrkA (also called NTRK1), the common neurotrophin receptor p75 NTR , and the proneurotrophin receptor sortilin were analyzed with immunohistochemistry in a cohort of thyroid cancers ( n = 128) and compared with adenomas and normal thyroid tissues ( n = 62). TrkA was detected in 20% of thyroid cancers, compared with none of the benign samples ( P = 0.0007). TrkA expression was independent of histologic subtypes but associated with lymph node metastasis ( P = 0.0148), suggesting the involvement of TrkA in tumor invasiveness. Nerves in the tumor microenvironment were positive for TrkA. p75 NTR was overexpressed in anaplastic thyroid cancers compared with papillary and follicular subtypes ( P P NTR , and sortilin decreased cell survival and cell migration through decreased SRC and ERK activation. Together, these data reveal TrkA, p75 NTR , and sortilin as potential therapeutic targets in thyroid cancer.

Past studies have shown that mutant KRAS colon cancer cells are susceptible to apoptosis induced by the HSP90 inhibitor AUY922. Nevertheless, intrinsic and acquired resistance remains an obstacle for the potential application of the inhibitor in the treatment of the disease. Here we report that Mcl-1 is important for survival of colon cancer cells in the presence of AUY922. Mcl-1 was upregulated in mutant KRAS colon cancer cells selected for resistance to AUY922-induced apoptosis. This was due to its increased stability mediated by Bcl-2-associated athanogene domain 3 (BAG3), which was also increased in resistant colon cancer cells by heat shock factor 1 (HSF1) as a result of chronic endoplasmic reticulum (ER) stress. Functional investigations demonstrated that inhibition of Mcl-1, BAG3, or HSF1 triggered apoptosis in resistant colon cancer cells, and rendered AUY922-naïve colon cancer cells more sensitive to the inhibitor. Together, these results identify that the HSF1-BAG3-Mcl-1 signal axis is critical for protection of mutant KRAS colon cancer cells from AUY922-induced apoptosis, with potential implications for targeting HSF1/BAG3/Mcl-1 to improve the efficacy of AUY922 in the treatment of colon cancer.

// Fen Liu 1, 2, * , Chen Chen Jiang 2, * , Xu Guang Yan 3 , Hsin-Yi Tseng 3 , Chun Yan Wang 3 , Yuan Yuan Zhang 2 , Hamed Yari 3 , Ting La 3 , Margaret Farrelly 3 , Su Tang Guo 3 , Rick F. Thorne 4 , Lei Jin 2 , Qi Wang 1 and Xu Dong Zhang 3 1 Department of Respiratory Medicine, The Second Hospital, Dalian Medical University, Dalian, China 2 School of Medicine and Public Health, The University of Newcastle, NSW, Australia 3 School of Biomedical Sciences and Pharmacy, The University of Newcastle, NSW, Australia 4 School of Environmental and Life Sciences, University of Newcastle, NSW, Australia * These authors contributed equally to this work Correspondence to: Qi Wang, email: wqdlmu@163.com Xu Dong Zhang, email: Xu.Zhang@newcastle.edu.au Keywords: CD47, BRAF/MEK inhibitors, NRF-1, melanoma, phagocytosis Received: March 04, 2017 Accepted: April 19, 2017 Published: May 09, 2017 ABSTRACT The expression of CD47 on the cancer cell surface transmits “don’t eat me” signalling that not only inhibits phagocytosis of cancer cells by phagocytes but also impairs anti-cancer T cell responses. Here we report that oncogenic activation of ERK plays an important role in transcriptional activation of CD47 through nuclear respiratory factor 1 (NRF-1) in melanoma cells. Treatment with BRAF/MEK inhibitors upregulated CD47 in cultured melanoma cells and fresh melanoma isolates. Similarly, melanoma cells selected for resistance to the BRAF inhibitor vemurafenib expressed higher levels of CD47. The increase in CD47 expression was mediated by ERK signalling, as it was associated with rebound activation of ERK and co-knockdown of ERK1/2 by siRNA diminished upregulation of CD47 in melanoma cells after exposure to BRAF/MEK inhibitors. Furthermore, ERK1/2 knockdown also reduced the constitutive expression of CD47 in melanoma cells. We identified a DNA fragment that was enriched with the consensus binding sites for NRF-1 and was transcriptionally responsive to BRAF/MEK inhibitor treatment. Knockdown of NRF-1 inhibited the increase in CD47, indicating that NRF-1 has a critical role in transcriptional activation of CD47 by ERK signalling. Functional studies showed that melanoma cells resistant to vemurafenib were more susceptible to macrophage phagocytosis when CD47 was blocked. So these results suggest that NRF-1-mediated regulation of CD47 expression is a novel mechanism by which ERK signalling promotes the pathogenesis of melanoma, and that the combination of CD47 blockade and BRAF/MEK inhibitors may be a useful approach for improving their therapeutic efficacy.

The effect of MTH1 inhibition on cancer cell survival has been elusive. Here we report that although silencing of MTH1 does not affect survival of melanoma cells, TH588, one of the first-in-class MTH1 inhibitors, kills melanoma cells through apoptosis independently of its inhibitory effect on MTH1. Induction of apoptosis by TH588 was not alleviated by MTH1 overexpression or introduction of the bacterial homolog of MTH1 that has 8-oxodGTPase activity but cannot be inhibited by TH588, indicating that MTH1 inhibition is not the cause of TH588-induced killing of melanoma cells. Although knockdown of MTH1 did not impinge on the viability of melanoma cells, it rendered melanoma cells sensitive to apoptosis induced by the oxidative stress inducer elesclomol. Of note, treatment with elesclomol also enhanced TH588-induced apoptosis, whereas a reactive oxygen species scavenger or an antioxidant attenuated the apoptosis triggered by TH588. Indeed, the sensitivity of melanoma cells to TH588 was correlated with endogenous levels of reactive oxygen species. Collectively, these results indicate that the cytotoxicity of TH588 toward melanoma cells is not associated with its inhibitory effect on MTH1, although it is mediated by cellular production of ROS.

Pancreatic cancer has a dismal prognosis and to date there are no targeted therapies for this malignancy. Using shotgun proteomics, the mRNA binding protein cold shock domain containing E1 (CSDE1), also called upstream-of-N-Ras, is detected in pancreatic cancer cell lines but not in normal pancreatic epithelial cells. The expression of CSDE1 in pancreatic cancer cells is confirmed by Western blotting and immunohistochemistry of human pancreatic tumors. In vitro functional assays show that siRNA downregulation of CSDE1 or gene knockout using CRISPR-Cas9 significantly reduce the invasiveness of pancreatic cancer cells. Together, this study reveals that CSDE1 is overexpressed in pancreatic cancer and is a potential therapeutic target to inhibit pancreatic cancer cell invasion.

Nerves are emerging regulators of cancer progression. Cancer cells induce the outgrowth of nerves in the tumor microenvironment through the release of neurotrophic factors, and in return nerves liberate neurotransmitters that activate cancer growth and dissemination. Although sympathetic nerves drive tumor angiogenesis via the liberation of noradrenaline, sensory and parasympathetic nerves stimulate cancer stem cells. Interestingly, recent evidence indicates that parasympathetic nerves can eventually inhibit tumor progression, suggesting a yin–yang type of regulation of cancer by nerves. From a broader perspective, the question of a higher level of control of cancer development by the central nervous system should be raised. Significance: Nerves are emerging regulators of cancer initiation, progression, and metastasis. Here, we review the evidence to date and explore the basic and clinical ramifications of these findings.

Radiotherapy and adjuvant cisplatin chemotherapy are the mainstream approaches in the treatment of nasopharyngeal carcinoma (NPC). These have been shown to effectively improve the outcome and reduce tumor recurrence. However, radiotherapy and chemotherapy resistance during the course of treatment has become more common recently, resulting in the failure of NPC therapy. Therefore, new therapeutic strategies or adjuvant drugs are urgently needed. The current study was designed to look for new treatment strategies or auxiliary drugs in the treatment of NPC. Two human NPC cell lines, HNE1 and HNE1/DDP, were used to examine the relationship between endoplasmic reticulum stress and cell resistance to ionizing radiation (IR) and cisplatin (DDP). Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Meanwhile, propidium iodide (PI) staining and PI/Annexin V staining were used to observe cell apoptosis. Finally, western blot was used to detect the endogenous expression of glucose-regulated protein 78 (GRP78) and other apoptosis-related proteins. GRP78 small interference RNA was transfected using Lipofectamine 2000. Compared with HNE1/DDP, IR and DDP increased the cell apoptosis and inhibited the cell proliferation of HNE1. Inhibition of GRP78 can reverse IR and DDP resistance in NPC cells by PI/Annexin V staining. Knockdown of GRP78 upregulates the expression of pro-apoptotic proteins and downregulates the expression of antiapoptotic proteins. These results indicate that HNE1 is more sensitive to DDP and IR than HNE1/DDP. Knockdown GRP78 can reverse IR and DDP resistance in NPC cells. Inhibition of GRP78 gives us a new target to overcome resistance to radiotherapy and chemotherapy of NPC cells. Thus, this study should be further explored in vivo and assessed for possible clinical applications.

// Chun Yan Wang 1, 2 , Su Tang Guo 1, 2 , Jia Yu Wang 1 , Xu Guang Yan 1 , Margaret Farrelly 1 , Yuan Yuan Zhang 3 , Fen Liu 1 , Hamed Yari 1 , Ting La 1 , Fu Xi Lei 1 , Lei Jin 3 , Xu Dong Zhang 1 , Chen Chen Jiang 3 1 School of Biomedical Sciences and Pharmacy, The University of Newcastle, NSW, Australia 2 Department of Molecular Biology, Shanxi Cancer Hospital and Institute, Taiyuan, Shanxi, China 3 School of Medicine and Public Health, The University of Newcastle, NSW, Australia Correspondence to: Chen Chen Jiang, email: Chenchen.Jiang@newcastle.edu.au Xu Dong Zhang, email: Xu.Zhang@newcastle.edu.au Keywords: colon cancer, mutant BRAF, heat shock protein 90, AUY922, CDC37 Received: May 20, 2016 Accepted: June 29, 2016 Published: July 06, 2016 ABSTRACT Oncogenic mutations of BRAF occur in approximately 10% of colon cancers and are associated with their resistance to clinically available therapeutic drugs and poor prognosis of the patients. Here we report that colon cancer cells with mutant BRAF are also resistant to the heat shock protein 90 (HSP90) inhibitor AUY922, and that this is caused by rebound activation of ERK and Akt. Although AUY922 triggered rapid reduction in ERK and Akt activation in both wild-type and mutant BRAF colon cancer cells, activation of ERK and Akt rebounded shortly in the latter leading to resistance of the cells to AUY922-induced apoptosis. Reactivation of ERK was associated with the persistent expression of mutant BRAF, which, despite being a client of HSP90, was only partially degraded by AUY922, whereas reactivation of Akt was related to the activity of the HSP90 co-chaperone, cell division cycle 37 (CDC37), in that knockdown of CDC37 inhibited Akt reactivation in mutant colon cancer cells treated with AUY922. In support, as a HSP90 client protein, Akt was only diminished by AUY922 in wild-type but not mutant BRAF colon cancer cells. Collectively, these results reveal that reactivation of ERK and Akt associated respectively with the activity of mutant BRAF and CDC37 renders mutant BRAF colon cancer cells resistant to AUY922, with implications of co-targeting mutant BRAF and/or CDC37 and HSP90 in the treatment of mutant BRAF colon cancers.