Shepherd, T. | bioRxiv Science

bioRxiv2019

AMPK-independent LKB1 activity is required for efficient epithelial ovarian cancer metastasis

Epithelial ovarian cancer (EOC) spreads by direct dissemination of malignant cells and multicellular clusters, known as spheroids, into the peritoneum followed by implantation and growth on abdominal surfaces. Using a spheroid model system of EOC metastasis, we discovered that Liver kinase B1 (LKB1), encoded by the STK11 gene, and its canonical substrate AMP-activated protein kinase (AMPK) are activated in EOC spheroids, yet only LKB1 is required for cell survival. We have now generated STK11-knockout cell lines using normal human FT190 cells and three EOC cell lines, OVCAR8, HeyA8, and iOvCa147. STK11KO did not affect growth and viability in adherent culture, but it decreased anchorage-independent growth of EOC cells. EOC spheroids lacking LKB1 had markedly impaired growth and viability, whereas there was no difference in normal FT190 spheroids. To test whether LKB1 loss affects EOC metastasis, we performed intraperitoneal injections of OVCAR8-, HeyA8-, and iOvCa147-STK11KO cells, and respective controls. LKB1 loss exhibited a dramatic reduction on tumour burden and metastatic potential; in particular, OVCAR8-STK11KO tumours had evidence of extensive necrosis, apoptosis and hypoxia. Interestingly, LKB1 loss did not affect AMPK phosphorylation in EOC spheroids and tumour xenografts, indicating that LKB1 signaling to support EOC cell survival in spheroids and metastatic tumour growth occurs via other downstream mediators. We identified the dual-specificity phosphatase DUSP4 as a commonly upregulated protein due to LKB1 loss; indeed, DUSP4 knockdown in HeyA8-STK11KO cells restored spheroid formation and viability. Our results strongly indicate that intact LKB1 activity independent of downstream AMPK signaling is required during EOC metastasis.

cancer biology↗

bioRxiv2019

Relating growth potential and biofilm formation of Shigatoxigenic Escherichia coli to in planta colonisation and the metabolome of ready- to-eat crops

Contamination of fresh produce with pathogenic Escherichia coli, including Shigatoxigenic E. coli (STEC), represents a serious risk to human health. Colonisation is governed by multiple bacterial and plant factors that can impact on the probability and suitability of bacterial growth. Thus, we aimed to determine whether the growth potential of STEC for plants associated with foodborne outbreaks (two leafy vegetables and two sprouted seed species), is predictive for colonisation of living plants as assessed from growth kinetics and biofilm formation in plant extracts. Fitness of STEC was compared to environmental E. coli, at temperatures relevant to plant growth. Growth kinetics in plant extracts varied in a plant-dependent and isolate-dependent manner for all isolates, with spinach leaf lysates supporting the fastest rates of growth. Spinach extracts also supported the highest levels of biofilm formation. Saccharides were identified as the major driver of bacterial growth, although no single metabolite could be correlated with growth kinetics. The highest level of in planta colonisation occurred on alfalfa sprouts, though internalisation was 10-times more prevalent in the leafy vegetables than in sprouted seeds. Marked differences in in planta growth meant that growth potential could only be inferred for STEC for sprouted seeds. In contrast, biofilm formation in extracts related to spinach colonisation. Overall, the capacity of E. coli to colonise, grow and internalise within plants or plant-derived matrices were influenced by the isolate type, plant species, plant tissue type and temperature, complicating any straight-forward relationship between in vitro and in planta behaviours.\n\nImportanceFresh produce is an important vehicle for STEC transmission and experimental evidence shows that STEC can colonise plants as secondary hosts, but differences in the capacity to colonise occur between different plant species and tissues. Therefore, an understanding of the impact of these plant factors have on the ability of STEC to grow and establish is required for food safety considerations and risk assessment. Here, we determined whether growth and the ability of STEC to form biofilms in plants extracts could be related to specific plant metabolites or could predict the ability of the bacteria to colonise living plants. Growth rates for sprouted seeds (alfalfa and fenugreek) exhibited a positive relationship between plant extracts and living plants, but not for leafy vegetables (lettuce and spinach). Therefore, the detailed variations at the level of the bacterial isolate, plant species and tissue type all need to be considered in risk assessment.

microbiology↗