AMPK-Independent LKB1 Activity Is Required for Efficient Epithelial Ovarian Cancer Metastasis

Abstract

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 AMPKα 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.

Activated CAMKKβ-AMPK signaling promotes autophagy in a spheroid model of ovarian tumour metastasis

“…Furthermore, work in our laboratory identified recently that LKB1-deficient EOC spheroids still retain the capacity to induce p-AMPK through CAMKKβ activity [27]. This finding together with our results herein suggest a crucial role for CAMKKβ in regulating p-AMPK levels in this disease.…”

“…LKB1 is a highly-conserved serine-threonine kinase that typically functions as a regulator of cellular metabolism within the AMPK signaling axis [26]. Surprisingly, recent work from our laboratory identified that EOC spheroids lacking LKB1 expression by CRISPR-mediated STK11 knockout sustain elevated p-AMPK suggesting alternative kinase(s) target AMPK in our system [27].…”

A Novel Role for NUAK1 in Promoting Ovarian Cancer Metastasis through Regulation of Fibronectin Production in Spheroids

“…The elucidation of key intracellular signaling pathways in ovarian cancer spheroids would allow for an improved understanding of metastatic processes and would aid in the identification of novel therapeutic targets. Our group previously showed that Liver kinase B1 (LKB1) is critical for ovarian cancer metastasis [15,16]. LKB1, which is encoded by STK11, is a serine-threonine kinase that is known best as having tumour suppressive-like activity in cancers [17].…”

Age-related copy number variations and expression levels of F-box protein FBXL20 predict ovarian cancer prognosis

“…The F-box proteins are categorized into sub-families, FBXL, FBXW, and FBXO based on their substrate-binding motifs, including Leucine Rich Repeats, WD40 motifs, and other domains, respectively [ 35 ]. Relatively well studied F-box proteins that are related with OvCa progression including FBXW1/11 [ 27 , [36] , [37] , [38] , [39] , [40] ], FBXW7 [ [41] , [42] , [43] , [44] , [45] , [46] ], FBXW8 [ [47] , [48] , [49] ], FBXL1 (SKP2) [ [50] , [51] , [52] ], FBXL3 [ 53 , 54 ], FBXL10 [ 55 , 56 ], FBXL20 [ 26 ], FBXO4 [ 57 , 58 ], FBXO7 [ [59] , [60] , [61] ] and FBXO22 [ [62] , [63] , [64] ]. Abbreviations: AEBP2, AE binding protein 2; Cdc25A, cell division cycle 25 A; cIAP1, cellular inhibitor of apoptosis protein 1; DEPTOR, DEP domain containing MTOR interacting protein; FMRP, fragile X mental retardation protein; KDM4B, lysine demethylase 4B; LKB1, liver kinase B1; Mdm2, murine double minute 2 homolog; TLK1/2, tousled-like kinases 1/2; TRAF1/2, TNF receptor associated factor 1/2; TRF1, telomeric repeat factor 1; Vps34, vacuolar protein sorting 34.…”