Preclinical Studies of OBI-999: A Novel Globo H-Targeting Antibody-Drug Conjugate.

Globo H (GH), a hexasaccharide, is expressed at low levels in normal tissues but is highly expressed in multiple cancer types, rendering it a promising target for cancer immunotherapy. OBI-999, a novel antibody-drug conjugate, is derived from a conjugation of a GH-specific mAb with a monomethyl auristatin E (MMAE) payload through a site-specific ThioBridge and a cleavable linker. OBI-999 high homogeneity with a drug-to-antibody ratio of 4 (>95%) was achieved using ThioBridge. OBI-999 displayed GH-dependent cellular internalization and trafficked to endosome and lysosome within 1 and 5 hours, respectively. Furthermore, OBI-999 showed low nanomolar cytotoxicity in the assay with high GH expression on tumor cells and exhibited a bystander killing effect on tumor cells with minimal GH expression. Tissue distribution indicated that OBI-999 and free MMAE gradually accumulated in the tumor, reaching maximum level at 168 hours after treatment, whereas OBI-999 and free MMAE decreased quickly at 4 hours after treatment in normal organs. Maximum MMAE level in the tumor was 16-fold higher than in serum, suggesting that OBI-999 is stable during circulation and MMAE is selectively released in the tumor. Excellent tumor growth inhibition of OBI-999 was demonstrated in breast, gastric, and pancreatic cancer xenograft or lung patient-derived xenograft models in a dose-dependent manner. The highest nonseverely toxic dose in cynomolgus monkeys is 10 mg/kg determined by a 3-week repeated-dose toxicology study demonstrating an acceptable safety margin. Taken together, these results support further clinical development of OBI-999, which is currently in a phase I/II clinical study in multiple solid tumors (NCT04084366). OBI-999, the first GH-targeting ADC, displayed excellent tumor inhibition in animal models across multiple cancer types, including breast, gastric, pancreatic, and lung cancers, warranting further investigation in the treatment of solid tumors.

p21-Activated kinase 3 promotes cancer stem cell phenotypes through activating the Akt-GSK3ß-ß-catenin signaling pathway in pancreatic cancer cells.

Relative to several other p21-activated kinase (PAK) family members, the role of PAK3 in regulating cancer cell functions remains unclear. Our study obtained evidence that PAK3 regulates the Akt-GSK3ß-ß-catenin signaling by acting as Ser473-Akt kinase in several pancreatic cancer cell lines. Specifically, knockdown of PAK3 or overexpression of dominant-negative PAK3 inhibited the phosphorylation of Ser473-Akt and GSK3ß, resulting in the proteasomal degradation of ß-catenin. Conversely, overexpression of PAK3 led to activation of Akt signaling and increased ß-catenin expression. These changes, however, were not noted with the silencing and/or overexpression of PAK1, PAK2, or PAK4, which underlies the impetus of PAK3 as a key effector in governing malignant phenotypes in these pancreatic cancer cells, including cancer stem cell (CSC) expansion. Accordingly, PAK3 depletion effectively suppresses tumorsphere formation, ALDH activity, and the expression of CSC surface markers. Moreover, we used a stable knockdown clone of AsPC-1 cells to demonstrate the in vivo efficacy of PAK3 inhibition in suppressing tumorigenesis and xenograft tumor growth. Together, these findings suggest the potential role of PAK3 as a target for pancreatic cancer therapy, which warrants further investigations.

Inhibition of Jak/STAT signaling reduces the activation of pancreatic stellate cells in vitro and limits caerulein-induced chronic pancreatitis in vivo.

Chronic pancreatitis (CP) is a fibro-inflammatory disease leading to pain, maldigestion, and pancreatic insufficiency. No therapeutic options exist due to a limited understanding of the biology of CP pathology. Recent findings implicate pancreatic stellate cells (PSC) as prominent mediators of inflammatory and fibrotic processes during CP. Here, we utilized primary and immortalized PSC obtained from mice and patients with CP or pancreatic cancer to examine the effect of Jak/STAT and MAPK pathway inhibition in vitro. The well-characterized caerulein model of CP was used to assess the therapeutic efficacy of Jak1/2 inhibition in vivo. Treatment of cultured PSC with the Jak1/2 inhibitor ruxolitinib reduced STAT3 phosphorylation, cell proliferation, and expression of alpha-smooth muscle actin (α-SMA), a marker of PSC activation. Treatment with the MAPK inhibitor, MEK162, had less consistent effects on PSC proliferation and no impact on activation. In the caerulein-induced murine model of CP, administration of ruxolitinib for one week significantly reduced biomarkers of inflammation and fibrosis. These data suggest that the Jak/STAT pathway plays a prominent role in PSC proliferation and activation. In vivo treatment with the Jak1/2 inhibitor ruxolitinib reduced the severity of experimental CP, suggesting that targeting Jak/STAT signaling may represent a promising therapeutic strategy for CP.

Integrin-linked kinase as a novel molecular switch of the IL-6-NF-κB signaling loop in breast cancer.

Substantial evidence has clearly demonstrated the role of the IL-6-NF-κB signaling loop in promoting aggressive phenotypes in breast cancer. However, the exact mechanism by which this inflammatory loop is regulated remains to be defined. Here, we report that integrin-linked kinase (ILK) acts as a molecular switch for this feedback loop. Specifically, we show that IL-6 induces ILK expression via E2F1 upregulation, which, in turn, activates NF-κB signaling to facilitate IL-6 production. shRNA-mediated knockdown or pharmacological inhibition of ILK disrupted this IL-6-NF-κB signaling loop, and blocked IL-6-induced cancer stem cells in vitro and estrogen-independent tumor growth in vivo Together, these findings establish ILK as an intermediary effector of the IL-6-NF-κB feedback loop and a promising therapeutic target for breast cancer.

Integrin-linked kinase affects signaling pathways and migration in thyroid cancer cells and is a potential therapeutic target.

BACKGROUND: Integrin-linked kinase (ILK) is a serine-threonine kinase that regulates interactions between the cell and the extracellular matrix. In many cancers, overexpression of ILK leads to increased cell proliferation, motility, and invasion. We hypothesized that ILK functions as a regulator of viability and migration in thyroid cancer cells. METHODS: Eleven human thyroid cancer cell lines were screened for ILK protein expression. The cell lines with the greatest expression were treated with either ILK small interfering RNA (siRNA) or a novel ILK inhibitor, T315, and the effects were evaluated via Western blot and migration assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assays were performed to assess cell viability. RESULTS: siRNA against ILK decreased phosphorylation of downstream effectors Akt and MLC, as well as decreased migration. Treatment with T315 showed a dose-related decrease in both Akt and MLC phosphorylation, as well as decreased migration. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assays showed T315 to have an half maximal inhibitory concentration of less than 1 µM in cell lines with high ILK expression. CONCLUSION: ILK is expressed differentially in thyroid cancer cell lines. Both ILK siRNA and T315 inhibit motility of thyroid cancer cell lines, and T315 is shown to be cytotoxic at low concentrations. Altogether, our study suggests that ILK may represent an important kinase in aggressive thyroid cancers.

Blockade of autophagy reduces pancreatic cancer stem cell activity and potentiates the tumoricidal effect of gemcitabine.

BACKGROUND: Cancer stem cells (CSCs) are considered responsible for the recurrence and chemoresistance of cancer. Dysregulated autophagy is highly prevalent in many types of cancer including pancreatic cancer and has been implicated in cytoprotection and tumor promotion. This study aimed to investigate the role of autophagy in regulating cancer stemness and chemoresistance of pancreatic cancer. METHODS: The correlation between autophagy and CSCs and its clinical significance were analyzed using pancreatic cancer tissue microarrays. Genetic and pharmacological approaches were applied to explore the function of autophagy on CSC activity and gemcitabine resistance of pancreatic cancer cells in vitro and in vivo. RESULTS: LC3 expression positively correlated with the expression of CSC markers aldehyde dehydrogenase 1 (ALDH1), CD44, and CD133 in pancreatic cancer tissues. High coexpression of LC3/ALDH1 was associated with both poor overall survival and progression-free survival. In pancreatic cancer cell lines, higher LC3-II expression was observed in the sphere-forming cells than in the bulk cells. Blockade of autophagy by silencing ATG5, ATG7, and BECN1 or the administration of autophagy inhibitor chloroquine markedly reduced the CSC populations, ALDH1 activity, sphere formation, and resistance to gemcitabine in vitro and in vivo. Furthermore, osteopontin (OPN) was found to stimulate LC3-II, ALDH1, CD44, and CD133 expression in PANC-1 cells, whereas this effect could be prevented by OPN knockdown and autophagy blockade. After treatment with various inhibitors against the major signaling pathways downstream of OPN, only the inhibitor of NF-κB activation, BAY 1170-82, could effectively counteract OPN-induced autophagy and CSC activity. According to the histochemical results, pancreatic cancer patients manifesting high levels of OPN/LC3/ALDH1 and OPN/CD44/CD133 had poor survival. CONCLUSIONS: Induction of autophagy mediated by OPN/NF-κB signaling is required for maintenance of pancreatic CSC activity. Combination of gemcitabine with pharmacological autophagy inhibitors is a promising therapeutic strategy for pancreatic cancer.

Hepatic stellate cells secretes type I collagen to trigger epithelial mesenchymal transition of hepatoma cells.

Liver fibrosis is a risk factor for hepatoma. Activated hepatic stellate cells (HSCs) play a critical role in progression of hepatoma. Resected hepatoma patients with high α-SMA+HSCs infiltration had worse survival, OR: 2.2 and p=0.0434. We hypothesized that HSCs could increase the epithelial-mesenchymal transition (EMT) ability of hepatoma cells. In murine model of liver fibrosis with injection of ML1 mice HCC cell line, E-cadherin was lost at the margin of tumor nodule around α-SMA+HSC sites. In subcutaneous tumor model, HSCs could increase the metastatic nodules in the lung, and the expression of E-cadherin was decreased and the Slug was induced. To elucidate the effect of HSCs on hepatoma cells, HSC-T6 was co-cultured with ML1 and the condition medium of HSC-T6 can trigger ML1 cell morphological change, down-expression of E-cadherin, induction of Slug expression, and cell migration. Proteomic analysis of the condition medium showed that collagen I was the target molecule. Collagen type I alone also induced EMT of ML1 cells. Knockdown of collagen type I in HSC-T6 could decrease its induction of EMT on ML1 cells. In conclusion, HSC can secrete collagen type I to trigger hepatoma cells to undergo EMT for metastasis.

Concanavalin A/IFN-gamma triggers autophagy-related necrotic hepatocyte death through IRGM1-mediated lysosomal membrane disruption.

Interferon-gamma (IFN-γ), a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A) can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP) is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1) translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ(-/-) mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.

Induction of liver fibrosis in a murine hepatoma model by thioacetamide is associated with enhanced tumor growth and suppressed antitumor immunity.

Liver cirrhosis and hepatocellular carcinomas are two major causes of morbidity and mortality worldwide, and can synergistically interact to expedite the tumor progression. How fibrosis promotes the hepatoma growth remains completely unexplained. Using an in situ murine hepatoma model together with fibrosis induction by thioacetamide (TAA), the hepatoma growth and the immune factors in the fibrotic liver were analyzed. We found that TAA-fibrosis induction enhanced hepatoma cell growth in the liver and increased the mortality of hepatoma-bearing mice. The tumor-infiltrating CD4(+) or CD8(+) T cells are downregulated by fibrosis induction. The Foxp3(+) regulatory T cells (Treg) cells were induced. We conclude that fibrosis induction causes further immunosuppression, in which Treg cells exert a downregulation effect on the antitumor immunity.

Dihydrolipoic acid inhibits tetrachlorohydroquinone-induced tumor promotion through prevention of oxidative damage.

alpha-Lipoic acid (LA) has been intensely investigated as a therapeutic agent for several diseases, including hepatic disorder and diabetic polyneuropathy. However, the effects of LA or its reduced form, dihydrolipoic acid (DHLA), on cancer chemoprevention has seldom been studied. Tetrachlorohydroquinone (TCHQ) is a toxic metabolite of pentachlorophenol (PCP) that was proven to be a tumor promoter in our previous study. We recently reported that DHLA can inhibit DMBA/TPA-induced skin tumor formation through its anti-inflammatory and anti-oxidizing functions. In the present study, we further examined the effects of DHLA on DMBA/TCHQ-induced skin tumor formation and the possible mechanisms. We found that DHLA significantly inhibited tumor incidence and tumor multiplicity in DMBA/TCHQ-induced skin tumor formation. Administration of DHLA prevented ROS generation, cytotoxicity, genotoxicity and apoptotic cell death in cells treated with TCHQ. In addition, activation of JNK and p38 MAPK may be involved in TCHQ-mediated apoptosis. Nonetheless, the detailed mechanisms of DHLA in attenuating TCHQ-induced skin tumor promotion are still unclear and need to be further investigated. We conclude that DHLA may be a useful protective agent against TCHQ-induced toxicity in epithelial cells, and for reversing TCHQ-induced damage in mouse skin.

High frequency of frameshift mutation on p53 gene in Taiwanese with non small cell lung cancer.

Extensive researches have found that the mutation of p53 tumor suppressor gene is the most frequent event in many human cancers and associated with a poor clinical outcome in lung cancer patients. Because the p53 molecular mutation involved in tumorigenesis of patients with lung cancer in Taiwan remains poorly defined, the aim of this study was to assess the p53 mutation spectrum and possible etiological factors of Taiwan's patients with Non-Small Cell Lung Cancer (NSCLC). Cancer specimens were obtained surgically from 61 patients with pathologically proven NSCLC. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing were used to study p53 mutations in exon 4-8. We also performed immunohistochemistry (IHC) to detect p53 protein expression. Our results provided that 34 mutations of p53 gene were found in 27 cases with a mutation rate of 44% (27/61). There were six cases having more than two p53 mutations. Among the 34 mutations, 19 were point mutations (56%, 19/34) consisted of a majority of missense mutations including transversion (13/19, 68%) and transitions (6/19, 32%) with four cases (4/6, 67%) occurring in the CpG sequence. One of the most important finding in our study was the high frequency of frameshift (44%, 15/34) which included 11 insertions and 4 deletions of p53 in NSCLC in Taiwan. Surprisingly, our results disclosed distinct novel mutations at codon 181, 185, 208 (Exon 5-6) of p53. Especially, 4 cases with mutation at codon181 and codon 185 seemed to have more advanced clinical outcome with survival time less than 6 months. In addition, there were two recurring mutations at codon 168 and three at condon193. The different mutation spectrum in our series, including a high frequency of frameshift mutations and distinctly novel hot spots suggested the heterogenous entity of exogenous mutagens in NSCLC in Taiwan.

Bcl-2 overexpression inhibits tetrachlorohydroquinone-induced apoptosis in NIH3T3 cells: a possible mechanism for tumor promotion.

TCHQ is a major carcinogenic metabolite of the widely used wood preservative PCP. Recently, we found that TCHQ was a promoter in a mouse skin carcinogenesis model. However, the mechanism is still not clear. In this study, we showed that overexpression of Bcl-2 effectively suppressed TCHQ-induced apoptosis in NIH3T3 cells, as evidenced by morphological changes and DNA fragmentation. Although production of ROS contributes to TCHQ-induced apoptosis, Bcl-2 failed to attenuate TCHQ-elicited increase of intracellular ROS level. In addition, overexpressed Bcl-2 provides only partial protection against TCHQ-induced cellular DNA damage. We also found that TCHQ induced a change in mitochondrial transmembrane potential, and that caspase-9 and subsequent caspase-3 can be activated during TCHQ-induced acute apoptosis. Interestingly, TCHQ induced a significant upregulation of Bcl-2 expression, and over-expressed Bcl-2 can dramatically inhibit the change of mitochondria membrane potential and activation of both caspase-9 and -3. Thus, our results suggest TCHQ-induced tumor promotion may be through a mechanism of upregulation of Bcl-2 protein and subsequent apoptosis inhibition.