Lingtao Jin

Glutamate Dehydrogenase 1 Signals through Antioxidant Glutathione Peroxidase 1 to Regulate Redox Homeostasis and Tumor Growth

How mitochondrial glutaminolysis contributes to redox homeostasis in cancer cells remains unclear. Here we report that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1) is commonly upregulated in human cancers. GDH1 is important for redox homeostasis in cancer cells by controlling the intracellular levels of its product alpha-ketoglutarate and subsequent metabolite fumarate. Mechanistically, fumarate binds to and activates a reactive oxygen species scavenging enzyme glutathione peroxidase 1. Targeting GDH1 by shRNA or a small molecule inhibitor R162 resulted in imbalanced redox homeostasis, leading to attenuated cancer cell proliferation and tumor growth.

Glutaminolysis as a target for cancer therapy.

Cancer cells display an altered metabolic circuitry that is directly regulated by oncogenic mutations and loss of tumor suppressors. Mounting evidence indicates that altered glutamine metabolism in cancer cells has critical roles in supporting macromolecule biosynthesis, regulating signaling pathways, and maintaining redox homeostasis, all of which contribute to cancer cell proliferation and survival. Thus, intervention in these metabolic processes could provide novel approaches to improve cancer treatment. This review summarizes current findings on the role of glutaminolytic enzymes in human cancers and provides an update on the development of small molecule inhibitors to target glutaminolysis for cancer therapy.

Lysine acetylation activates 6-phosphogluconate dehydrogenase to promote tumor growth

Although the oxidative pentose phosphate pathway is important for tumor growth, how 6-phosphogluconate dehydrogenase (6PGD) in this pathway is upregulated in human cancers is unknown. We found that 6PGD is commonly activated in EGF-stimulated cells and human cancer cells by lysine acetylation. Acetylation at K76 and K294 of 6PGD promotes NADP(+) binding to 6PGD and formation of active 6PGD dimers, respectively. Moreover, we identified DLAT and ACAT2 as upstream acetyltransferases of K76 and K294, respectively, and HDAC4 as the deacetylase of both sites. Expressing acetyl-deficient mutants of 6PGD in cancer cells significantly attenuated cell proliferation and tumor growth. This is due in part to reduced levels of 6PGD products ribulose-5-phosphate and NADPH, which led to reduced RNA and lipid biosynthesis as well as elevated ROS. Furthermore, 6PGD activity is upregulated with increased lysine acetylation in primary leukemia cells from human patients, providing mechanistic insights into 6PGD upregulation in cancer cells.

The Prometastatic Ribosomal S6 Kinase 2-cAMP Response Element-binding Protein (RSK2-CREB) Signaling Pathway Up-regulates the Actin-binding Protein Fascin-1 to Promote Tumor Metastasis

Background: RSK2-mediated prometastatic signaling mechanism remains unclear. Results: RSK2-CREB pathway up-regulates Fascin-1 to promote filopodia formation, cancer cell invasion, and tumor metastasis. Conclusion: RSK2-CREB-Fascin-1 pathway could be a promising therapeutic target in metastatic cancers. Significance: These data will advance our understanding of signaling pathways that mediates RSK2-dependent prometastatic signals. Metastasis is the leading cause of death in patients with breast, lung, and head and neck cancers. However, the molecular mechanisms underlying metastases in these cancers remain unclear. We found that the p90 ribosomal S6 kinase 2 (RSK2)-cAMP response element-binding protein (CREB) pathway is commonly activated in diverse metastatic human cancer cells, leading to up-regulation of a CREB transcription target Fascin-1. We also observed that the protein expression patterns of RSK2 and Fascin-1 correlate in primary human tumor tissue samples from head and neck squamous cell carcinoma patients. Moreover, knockdown of RSK2 disrupts filopodia formation and bundling in highly invasive cancer cells, leading to attenuated cancer cell invasion in vitro and tumor metastasis in vivo, whereas expression of Fascin-1 significantly rescues these phenotypes. Furthermore, targeting RSK2 with the small molecule RSK inhibitor FMK-MEA effectively attenuated the invasive and metastatic potential of cancer cells in vitro and in vivo, respectively. Taken together, our findings for the first time link RSK2-CREB signaling to filopodia formation and bundling through the up-regulation of Fascin-1, providing a proinvasive and prometastatic advantage to human cancers. Therefore, protein effectors of the RSK2-CREB-Fascin-1 pathway represent promising biomarkers and therapeutic targets in the clinical prognosis and treatment of metastatic human cancers.

Phosphorylation-mediated activation of LDHA promotes cancer cell invasion and tumour metastasis

Metastases remain the major cause of death from cancer. Recent molecular advances have highlighted the importance of metabolic alterations in cancer cells, including the Warburg effect that describes an increased glycolysis in cancer cells. However, how this altered metabolism contributes to tumour metastasis remains elusive. Here, we report that phosphorylation-induced activation of lactate dehydrogenase A (LDHA), an enzyme that catalyses the interconversion of pyruvate and lactate, promotes cancer cell invasion, anoikis resistance and tumour metastasis. We demonstrate that LDHA is phosphorylated at tyrosine 10 by upstream kinases, HER2 and Src. Targeting HER2 or Src attenuated LDH activity as well as invasive potential in head and neck cancer and breast cancer cells. Inhibition of LDH activity by small hairpin ribonucleic acid or expression of phospho-deficient LDHA Y10F sensitized the cancer cells to anoikis induction and resulted in attenuated cell invasion and elevated reactive oxygen species, whereas such phenotypes were reversed by its product lactate or antioxidant N-acetylcysteine, suggesting that Y10 phosphorylation-mediated LDHA activity promotes cancer cell invasion and anoikis resistance through redox homeostasis. In addition, LDHA knockdown or LDHA Y10F rescue expression in human cancer cells resulted in decreased tumour metastasis in xenograft mice. Furthermore, LDHA phosphorylation at Y10 positively correlated with progression of metastatic breast cancer in clinical patient tumour samples. Our findings demonstrate that LDHA phosphorylation and activation provide pro-invasive, anti-anoikis and pro-metastatic advantages to cancer cells, suggesting that Y10 phosphorylation of LDHA may represent a promising therapeutic target and a prognostic marker for metastatic human cancers.

p90RSK2 is essential for FLT3-ITD- but dispensable for BCR-ABL-induced myeloid leukemia.

p90 ribosomal S6 kinase 2 (p90RSK2) is important in diverse cellular processes including gene expression, cell proliferation, and survival. We found that p90RSK2 is commonly activated in diverse leukemia cell lines expressing different leukemogenic tyrosine kinases, including BCR-ABL and FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD). Interestingly, in a murine BM transplantation (BMT) model, genetic deficiency of RSK2 did not affect the pathogenesis or disease progression of BCR-ABL-induced myeloproliferative neoplasm (PN). In contrast, FLT3-ITD induced a T-cell acute lymphoblastic leukemia in BMT mice receiving RSK2 knockout (KO) BM cells, phenotypically distinct from the myeloproliferative neoplasm induced by FLT3-ITD using wild-type BM cells. In consonance with these results, inhibition of RSK2 by an RSK inhibitor, fmk, did not effectively induce apoptosis in BCR-ABL-expressing murine Ba/F3 cells, human K562 cells or primary tissue samples from CML patients, whereas fmk treatment induced significant apoptotic cell death not only in FLT3-ITD-positive Ba/F3 cells, human Molm14 and Mv(4;11) leukemia cells, but also in primary tissue samples from AML patients. These results suggest that RSK2 is dispensable for BCR-ABL-induced myeloid leukemia, but may be required for pathogenesis and lineage determination in FLT3-ITD-induced hematopoietic transformation. RSK2 may thus represent an alternative therapeutic target in the treatment of FLT3-ITD-positive leukemia.

p90 RSK2 mediates antianoikis signals by both transcription-dependent and -independent mechanisms.

ABSTRACT How invasive and metastatic tumor cells evade anoikis induction remains unclear. We found that knockdown of RSK2 sensitizes diverse cancer cells to anoikis induction, which is mediated through phosphorylation targets including apoptosis signal-regulating kinase 1 (ASK1) and cyclic AMP (cAMP) response element-binding protein (CREB). We provide evidence to show that RSK2 inhibits ASK1 by phosphorylating S83, T1109, and T1326 through a novel mechanism in which phospho-T1109/T1326 inhibits ATP binding to ASK1, while phospho-S83 attenuates ASK1 substrate MKK6 binding. Moreover, the RSK2→CREB signaling pathway provides antianoikis protection by regulating gene expression of protein effectors that are involved in cell death regulation, including the antiapoptotic factor protein tyrosine kinase 6 (PTK6) and the proapoptotic factor inhibitor-of-growth protein 3 (ING3). PTK6 overexpression or ING3 knockdown in addition to ASK1 knockdown further rescued the increased sensitivity to anoikis induction in RSK2 knockdown cells. These data together suggest that RSK2 functions as a signal integrator to provide antianoikis protection to cancer cells in both transcription-independent and -dependent manners, in part by signaling through ASK1 and CREB, and contributes to cancer cell invasion and tumor metastasis.

RSK2 signals through stathmin to promote microtubule dynamics and tumor metastasis

Metastasis is responsible for >90% of cancer-related deaths. Complex signaling in cancer cells orchestrates the progression from a primary to a metastatic cancer. However, the mechanisms of these cellular changes remain elusive. We previously demonstrated that p90 ribosomal S6 kinase 2 (RSK2) promotes tumor metastasis. Here we investigated the role of RSK2 in the regulation of microtubule dynamics and its potential implication in cancer cell invasion and tumor metastasis. Stable knockdown of RSK2 disrupted microtubule stability and decreased phosphorylation of stathmin, a microtubule-destabilizing protein, at serine 16 in metastatic human cancer cells. We found that RSK2 directly binds and phosphorylates stathmin at the leading edge of cancer cells. Phosphorylation of stathmin by RSK2 reduced stathmin-mediated microtubule depolymerization. Moreover, overexpression of phospho-mimetic mutant stathmin S16D significantly rescued the decreased invasive and metastatic potential mediated by RSK2 knockdown in vitro and in vivo. Furthermore, stathmin phosphorylation positively correlated with RSK2 expression and metastatic cancer progression in primary patient tumor samples. Our finding demonstrates that RSK2 directly phosphorylates stathmin and regulates microtubule polymerization to provide a pro-invasive and pro-metastatic advantage to cancer cells. Therefore, the RSK2–stathmin pathway represents a promising therapeutic target and a prognostic marker for metastatic human cancers.

MAST1 Drives Cisplatin Resistance in Human Cancers by Rewiring cRaf-Independent MEK Activation

Platinum-based chemotherapeutics represent a mainstay of cancer therapy, but resistance limits their curative potential. Through a kinome RNAi screen, we identified microtubule-associated serine/threonine kinase 1 (MAST1) as a main driver of cisplatin resistance in human cancers. Mechanistically, cisplatin but no other DNA-damaging agents inhibit the MAPK pathway by dissociating cRaf from MEK1, while MAST1 replaces cRaf to reactivate the MAPK pathway in a cRaf-independent manner. We show clinical evidence that expression of MAST1, both initial and cisplatin-induced, contributes to platinum resistance and worse clinical outcome. Targeting MAST1 with lestaurtinib, a recently identified MAST1 inhibitor, restores cisplatin sensitivity, leading to the synergistic attenuation of cancer cell proliferation and tumor growth in human cancer cells and patient-derived xenograft models.

Abstract 3154: RSK2-mediated phosphorylation of stathmin promotes microtubule polymerization, providing a pro-invasive advantage to metastatic cancer cells

Metastasis is responsible for over 90% of cancer related deaths. Breast cancer, lung cancer, and head and neck cancer commonly metastasize to vital organs resulting in poor clinical outcomes. However, the molecular processes underlying such cellular changes remain elusive. Therefore, identifying genes that induce cancer cell signaling changes and understanding how these changes control pro-metastatic mechanisms is necessary to develop molecular therapies for metastasis and to improve clinical prognosis for late-stage cancer patients. Protein kinases have been implicated in mediating pro-metastatic signaling in human cancers. We previously demonstrated that a serine/threonine kinase, p90 ribosomal S6 kinase 2 (RSK2) promotes cancer cell invasion and migration, anoikis resistance, and tumor metastasis in human cancers, including head and neck cancer, breast cancer, and lung cancer. Here, we investigated the role of RSK2 in the regulation of microtubule dynamics and its potential implication in cancer cell invasion and tumor metastasis. SiRNA-mediated knockdown of RSK2 disrupted microtubules in diverse metastatic human cancer cell lines including lung cancer A549, breast cancer SKBR3, and head and neck cancer 212LN cells. Stathmin (STMN) is a microtubule destabilizing protein, which is an essential regulator of microtubule polymerization. We found that stable knockdown of RSK2 resulted in decreased phosphorylation of STMN at Serine 16 in the aforementioned metastatic cancer cell lines. Moreover, we found that RSK2 directly phosphorylated STMN at Ser16 in an in vitro RSK2 kinase assay using recombinant RSK2 and STMN proteins. We observed that phosphorylation of STMN by RSK2 resulted in reduced STMN-mediated microtubule depolymerization in vitro, while stable expression of phospho-deficient STMN S16A mutant but not STMN wild type (WT) led to decreased invasive potential of metastatic cancer cells. These data together suggest STMN as a potential novel RSK2 substrate/effector, which may contribute to RSK2-mediated pro-metastatic signaling. Our finding demonstrates that RSK2 signals through STMN to regulate microtubule polymerization and cancer cell invasion via direct phosphorylation of STMN at Serine 16, providing a pro-invasive and metastatic advantage to cancer cells. Therefore, the RSK2-STMN pathway represents a promising therapeutic target in the clinical prognosis and treatment of metastatic human cancers. Citation Format: Gina Alesi, Dan Li, Lingtao Jin, Georgia Z. Chen, Dong M. Shin, Fadlo Khuri, Sumin Kang. RSK2-mediated phosphorylation of stathmin promotes microtubule polymerization, providing a pro-invasive advantage to metastatic cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3154. doi:10.1158/1538-7445.AM2014-3154