Shuang Huang

Poly(ADP-ribose) polymerase-1 signaling to mitochondria in necrotic cell death requires RIP1/TRAF2-mediated JNK1 activation

Poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation-induced necrosis has been implicated in several pathophysiological conditions. Although mitochondrial dysfunction and apoptosis-inducing factor translocation from the mitochondria to the nucleus have been suggested to play very important roles in PARP-1-mediated cell death, the signaling events downstream of PARP-1 activation in initiating mitochondria dysfunction are not clear. Here we used the DNA alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine, a potent PARP-1 activator, to study PARP-1 activation-mediated cell death. We found, based on genetic knockouts and pharmacological inhibition, that c-Jun N-terminal kinase (JNK), especially JNK1, but not the other groups of mitogen-activated protein kinase, is required for PARP-1-induced mitochondrial dysfunction, apoptosis-inducing factor translocation, and subsequent cell death. We reveal that receptor-interacting protein 1 (RIP1) and tumor necrosis factor receptor-associated factor 2 (TRAF2), are upstream of JNK in PARP-1 hyperactivated cells, because PARP-1-induced JNK activation was attenuated in RIP1-/- and TRAF2-/- mouse embryonic fibroblast cells. Consistently, knockouts of RIP1 and TRAF2 caused a resistance to PARP-1-induced cell death. Therefore, our study uncovers that RIP1, TRAF2, and JNK comprise a pathway to mediate the signaling from PARP-1 overactivation to mitochondrial dysfunction.

ATR-Chk2 Signaling in p53 Activation and DNA Damage Response during Cisplatin-induced Apoptosis

Cisplatin is one of the most effective anti-cancer drugs; however, the use of cisplatin is limited by its toxicity in normal tissues, particularly injury of the kidneys. The mechanisms underlying the therapeutic effects of cisplatin in cancers and side effects in normal tissues are largely unclear. Recent work has suggested a role for p53 in cisplatin-induced renal cell apoptosis and kidney injury; however, the signaling pathway leading to p53 activation and renal apoptosis is unknown. Here we demonstrate an early DNA damage response during cisplatin treatment of renal cells and tissues. Importantly, in the DNA damage response, we demonstrate a critical role for ATR, but not ATM (ataxia telangiectasia mutated) or DNA-PK (DNA-dependent protein kinase), in cisplatin-induced p53 activation and apoptosis. We show that ATR is specifically activated during cisplatin treatment and co-localizes with H2AX, forming nuclear foci at the site of DNA damage. Blockade of ATR with a dominant-negative mutant inhibits cisplatin-induced p53 activation and renal cell apoptosis. Consistently, cisplatin-induced p53 activation and apoptosis are suppressed in ATR-deficient fibroblasts. Downstream of ATR, both Chk1 and Chk2 are phosphorylated during cisplatin treatment in an ATR-dependent manner. Interestingly, following phosphorylation, Chk1 is degraded via the proteosomal pathway, whereas Chk2 is activated. Inhibition of Chk2 by a dominant-negative mutant or gene deficiency attenuates cisplatin-induced p53 activation and apoptosis. In vivo in C57BL/6 mice, ATR and Chk2 are activated in renal tissues following cisplatin treatment. Together, the results suggest an important role for the DNA damage response mediated by ATR-Chk2 in p53 activation and renal cell apoptosis during cisplatin nephrotoxicity.

Extracellular Signal–Regulated Kinase Signaling Pathway Regulates Breast Cancer Cell Migration by Maintaining slug Expression

Cell migration is a critical step in cancer cell invasion. Recent studies have implicated the importance of the extracellular signal-regulated kinase (ERK) signaling pathway in cancer cell migration. However, the mechanism associated with ERK-regulated cell migration is poorly understood. Using a panel of breast cancer cell lines, we detected an excellent correlation between ERK activity and cell migration. Interestingly, we noticed that a 48-hour treatment with U0126 [specific mitogen-activated protein/ERK kinase (MEK)-1/2 inhibitor] was needed to significantly inhibit breast cancer cell migration, whereas this inhibitor blocked ERK activity within 1 hour. This observation suggests that ERK-dependent gene expression, rather than direct ERK signaling, is essential for cell migration. With further study, we found that ERK activity promoted the expression of the activator protein-1 (AP1) components Fra-1 and c-Jun, both of which were necessary for cell migration. Combination of U0126 treatment and Fra-1/c-Jun knockdown did not yield further reduction in cell migration than either alone, indicating that ERKs and Fra-1/c-Jun act by the same mechanism to facilitate cell migration. In an attempt to investigate the role of Fra-1/c-Jun in cell migration, we found that the ERK-Fra-1/c-Jun axis regulated slug expression in an AP1-dependent manner. Moreover, the occurrence of U0126-induced migratory inhibition coincided with slug reduction, and silencing slug expression abrogated breast cancer cell migration. These results suggest an association between ERK-regulated cell migration and slug expression. Indeed, cell migration was not significantly inhibited by U0126 treatment or Fra-1/c-Jun silencing in cells expressing slug transgene. Our study suggests that the ERK pathway regulates breast cancer cell migration by maintaining slug expression.

Ratio of miR-196s to HOXC8 messenger RNA correlates with breast cancer cell migration and metastasis.

Expression profiling has identified metastasis-associated microRNAs (miRNA) but technical limitations hinder the discovery of metastasis-suppressing miRNAs. In this study, we sought metastasis-suppressing miRNAs by functional screening. Individual miRNAs were lentivirally introduced into metastatic MDA-MB-231 breast cancer cells and analyzed for effects on cell migration, a critical step in cancer metastasis. Among 486 miRNAs screened, 14 were identified that included all of the members of the miRNA-196 family (miR-196a1, miR-196a2, and miR-196b). Enforced expression of miR-196a1/2 or miR-196b abrogated in vitro invasion and in vivo spontaneous metastasis of breast cancer cells, indicating that members of the miR-196 family are potent metastasis suppressors. We found that miR-196 inhibited the expression of transcription factor HOXC8. Functional linkage was implied by small interfering RNA-mediated knockdown of HOXC8, which suppressed cell migration and metastasis, and by ectopic expression of HOXC8, which prevented the effects of miR-196 on cell migration and metastasis. Unlike other metastasis-associated miRNAs that have been described, the expressions of miR-196 were not correlated with breast cancer cell migration or the metastatic status of clinical breast tumor specimens. Instead, we detected an excellent correlation between the ratio of miR-196 to HOXC8 messages and the migratory behavior of breast cancer cell lines as well as the metastatic status of clinical samples. Our findings identify miRNA-196s as potent metastasis suppressors and reveal that the ratio of miR-196s to HOXC8 mRNA might be an indicator of the metastatic capability of breast tumors.

Lysophosphatidic Acid Stimulates Ovarian Cancer Cell Migration via a Ras-MEK Kinase 1 Pathway

Lysophosphatidic acid (LPA) is present at high concentrations in ascites and plasma of ovarian cancer patients. Studies conducted in experimental models demonstrate that LPA promotes ovarian cancer invasion/metastasis by up-regulating protease expression, elevating protease activity, and enhancing angiogenic factor expression. In this study, we investigated the effect of LPA on ovarian cancer migration, an essential component of cancer cell invasion. LPA stimulates both chemotaxis and chemokinesis of ovarian cancer cells and LPA-stimulated cell migration is GI dependent. Moreover, constitutively active H-Ras enhances ovarian cancer cell migration, whereas dominant negative H-Ras blocks LPA-stimulated cell migration, suggesting that Ras works downstream of Gi to mediate LPA-stimulated cell migration. Interestingly, H-Ras mutants that specifically activate Raf-1, Ral-GDS, or phosphatidylinositol 3′-kinase are unable to significantly enhance ovarian cancer cell migration, suggesting that a Ras downstream effector distinct from Raf-1, Ral-GDS, and phosphatidylinositol 3′-kinase is responsible for LPA-stimulated cell migration. In this article, we demonstrate that LPA activates mitogen-activated protein kinase kinase 1 (MEKK1) in a Gi-Ras-dependent manner and that MEKK1 activity is essential for LPA-stimulated ovarian cancer cell migration. Inhibitors that block MEKK1 downstream pathways, including MEK1/2, MKK4/7, and nuclear factor-κB pathways, do not significantly alter LPA-stimulated cell migration. Instead, LPA induces the redistribution of focal adhesion kinase to focal contact regions of the cytoplasm membrane, and this event is abolished by pertussis toxin, dominant negative H-Ras, or dominant negative MEKK1. Our studies thus suggest that the Gi-Ras-MEKK1 signaling pathway mediates LPA-stimulated ovarian cancer cell migration by facilitating focal adhesion kinase redistribution to focal contacts.

Inhibition of PKCδ reduces cisplatin-induced nephrotoxicity without blocking chemotherapeutic efficacy in mouse models of cancer

Cisplatin is a widely used cancer therapy drug that unfortunately has major side effects in normal tissues, notably nephrotoxicity in kidneys. Despite intensive research, the mechanism of cisplatin-induced nephrotoxicity remains unclear, and renoprotective approaches during cisplatin-based chemotherapy are lacking. Here we have identified PKCδ as a critical regulator of cisplatin nephrotoxicity, which can be effectively targeted for renoprotection during chemotherapy. We showed that early during cisplatin nephrotoxicity, Src interacted with, phosphorylated, and activated PKCδ in mouse kidney lysates. After activation, PKCδ regulated MAPKs, but not p53, to induce renal cell apoptosis. Thus, inhibition of PKCδ pharmacologically or genetically attenuated kidney cell apoptosis and tissue damage, preserving renal function during cisplatin treatment. Conversely, inhibition of PKCδ enhanced cisplatin-induced cell death in multiple cancer cell lines and, remarkably, enhanced the chemotherapeutic effects of cisplatin in several xenograft and syngeneic mouse tumor models while protecting kidneys from nephrotoxicity. Together these results demonstrate a role of PKCδ in cisplatin nephrotoxicity and support targeting PKCδ as an effective strategy for renoprotection during cisplatin-based cancer therapy.

The Histone H3 Methyltransferase G9A Epigenetically Activates the Serine-Glycine Synthesis Pathway to Sustain Cancer Cell Survival and Proliferation

Increased activation of the serine-glycine biosynthetic pathway is an integral part of cancer metabolism that drives macromolecule synthesis needed for cell proliferation. Whether this pathway is under epigenetic control is unknown. Here we show that the histone H3 lysine 9 (H3K9) methyltransferase G9A is required for maintaining the pathway enzyme genes in an active state marked by H3K9 monomethylation and for the transcriptional activation of this pathway in response to serine deprivation. G9A inactivation depletes serine and its downstream metabolites, triggering cell death with autophagy in cancer cell lines of different tissue origins. Higher G9A expression, which is observed in various cancers and is associated with greater mortality in cancer patients, increases serine production and enhances the proliferation and tumorigenicity of cancer cells. These findings identify a G9A-dependent epigenetic program in the control of cancer metabolism, providing a rationale for G9A inhibition as a therapeutic strategy for cancer.

MicroRNA-34a is induced via p53 during cisplatin nephrotoxicity and contributes to cell survival.

MicroRNAs are small noncoding RNAs that are produced endogenously and have emerged as important regulators in pathophysiological conditions such as development and tumorigenesis. Very little is known about the regulation of microRNAs in renal diseases, including acute kidney injury (AKI). In this study, we examined the regulation of microRNA-34a (miR-34a) in experimental models of cisplatin-induced AKI and nephrotoxicity. By Northern blot and real-time polymerase chain reaction analyses, we detected an induction of miR-34a in vitro during cisplatin treatment of mouse proximal tubular cells and also in vivo during cisplatin nephrotoxicity in C57BL/6 mice. In cultured cells, miR-34a was induced within a few hours. In mice, miR-34a induction was detectable in renal tissues after 1 d of cisplatin treatment and increased to approximately four-fold of control at d 3. During cisplatin treatment, p53 was activated. Inhibition of p53 with pifithrin-α abrogated the induction of miR-34a during cisplatin treatment of proximal tubular cells. In vivo, miR-34a induction by cisplatin was abrogated in p53-deficient mice, a result that further confirms a role for p53 in miR-34a induction during cisplatin nephrotoxicity. Functionally, antagonism of miR-34a with specific antisense oligonucleotides increased cell death during cisplatin treatment. Collectively, the results suggest that miR-34a is induced via p53 during cisplatin nephrotoxicity and may play a cytoprotective role for cell survival.

Integrity of SOS1/EPS8/ABI1 Tri-Complex Determines Ovarian Cancer Metastasis

Ovarian cancer is mainly confined in peritoneal cavity and its metastasis is often associated with the formation of malignant ascites. As lysophosphatidic acid (LPA) is present at high levels in ascites of ovarian cancer patients and potently stimulates cell migration, we reason that LPA-stimulated cell migration may play an important role in ovarian cancer metastasis. Here, we show that only ovarian cancer cell lines with LPA migratory response undergo peritoneal metastatic colonization. LPA-stimulated cell migration is required for metastatic colonization because knockdown of LPA receptor subtype 1 (LPAR(1)) abolishes this event. However, the difference in metastatic potentials is not caused by the absence of LPAR(1) because both metastatic and nonmetastatic lines express similar levels of LPAR(1). Instead, we find that LPA can activate Rac only in metastatic cells and that metastatic colonization of ovarian cancer cells necessitates Rac activity. These results thus suggest that LPA-induced Rac activation is a prerequisite for ovarian cancer metastasis. In metastatic cells, Rac activation is facilitated by SOS1/EPS8/ABI1 tri-complex and the integrity of this tri-complex is essential for LPA-stimulated cell migration and metastatic colonization. We show that at least 1 member of SOS1/EPS8/ABI1 tri-complex is absent in nonmetastatic ovarian cancer cells and reexpressing the missing one conferred them with metastatic capability. Importantly, coexpression of SOS1, EPS8, and ABI1, but not of any individual member of SOS1/EPS8/ABI1 tri-complex, correlates with advanced stages and shorter survival of ovarian cancer patients. Our study implicates that the integrity of SOS1/EPS8/ABI1 tri-complex is a determinant of ovarian cancer metastasis.

Protein kinase Cα-CARMA3 signaling axis links Ras to NF-κB for lysophosphatidic acid-induced urokinase plasminogen activator expression in ovarian cancer cells

We reported previously that a signaling pathway consisting of Gi-Ras-NF-κB mediates lysophosphatidic acid (LPA)-induced urokinase plasminogen activator (uPA) upregulation in ovarian cancer cells. However, it is not clear what signaling components link Ras to nuclear factor (NF)-κB for this LPA-induced event. In the present study, we found that treatment of protein kinase C (PKC) inhibitors including conventional PKC (cPKC) inhibitor Gö6976 abolished LPA-induced uPA upregulation in ovarian cancer cell lines tested, indicating the importance of cPKC activity in this LPA-induced event. Indeed, LPA stimulation led to the activation of PKCα and Ras–PKCα interaction. Although constitutively active mutants of PKCα (a cPKC), PKCθ (a novel PKC (nPKC)) and PKCζ (an atypical PKC (aPKC)) were all able to activate NF-κB and upregulate uPA expression, only dominant-negative PKCα mutant attenuated LPA-induced NF-κB activation and uPA upregulation. These results suggest that PKCα, rather than PKC isoforms in other PKC classes, participates in LPA-induced NF-κB activation and uPA upregulation in ovarian cancer cells. To determine the signaling components downstream of PKCα mediating LPA-induced uPA upregulation, we showed that forced expression of dominant-negative CARMA3 or silencing CARMA3, Bcl10 and MALT1 with specific siRNAs diminished these LPA-induced events. Furthermore, we demonstrated that PKCα/CARMA3 signaling axis is important in LPA-induced ovarian cancer cell in vitro invasion.