GRK2 negatively regulates IGF‐1R signaling pathway and cyclins' expression in HepG2 cells

G protein coupled receptor kinase 2 (GRK2) plays a central role in the regulation of a variety of important signaling pathways. Alternation of GRK2 protein level and activity casts profound effects on cell physiological functions and causes diseases such as heart failure, rheumatoid arthritis, and obesity. We have previously reported that overexpression of GRK2 has an inhibitory role in cancer cell growth. To further examine the role of GRK2 in cancer, in this study, we investigated the effects of reduced protein level of GRK2 on insulin‐like growth factor 1 receptor (IGF‐1R) signaling pathway in human hepatocellular carcinoma (HCC) HepG2 cells. We created a GRK2 knockdown cell line using a lentiviral vector mediated expression of GRK2 specific short hairpin RNA (shRNA). Under IGF‐1 stimulation, HepG2 cells with reduced level of GRK2 showed elevated total IGF‐1R protein expression as well as tyrosine phosphorylation of receptor. In addition, HepG2 cells with reduced level of GRK2 also demonstrated increased tyrosine phosphorylation of IRS1 at the residue 612 and increased phosphorylation of Akt, indicating a stronger activation of IGF‐1R signaling pathway. However, HepG2 cells with reduced level of GRK2 did not display any growth advantage in culture as compared with the scramble control cells. We further detected that reduced level of GRK2 induced a small cell cycle arrest at G2/M phase by enhancing the expression of cyclin A, B1, and E. Our results indicate that GRK2 has contrasting roles on HepG2 cell growth by negatively regulating the IGF‐1R signaling pathway and cyclins' expression. J. Cell. Physiol. 228: 1897–1901, 2013. © 2013 Wiley Periodicals, Inc.     

The RNF26/CBX7 axis modulates the TNF pathway to promote cell proliferation and regulate sensitivity to TKIs in ccRCC

Clear cell renal cell carcinoma (ccRCC) accounts for 85% of all malignant renal tumors. Currently, the pathogenesis of ccRCC is not fully understood. Chromobox (CBX) family proteins are the major subunits of PcG complexes and are implicated in regulating mammalian development. The CBX family consists of eight members, namely, CBX1-8. Numerous studies have highlighted that each CBX protein exhibits distinct functions and prognostic roles in specific cancer types. In this study, in silico analysis indicated that CBX7 was downregulated in ccRCC and correlated with favorable prognosis in a ccRCC cohort. Subsequent studies showed that CBX7 inhibited cancer cell proliferation and invasion. Then, we showed that CBX7 downregulated ETS1 to inactivate the tumor necrosis factor (TNF) signaling pathway, which inhibited tumor proliferation and enhanced the sensitivity of ccRCC cells to tyrosine kinase inhibitors (TKIs). Moreover, we found that CBX7 was a bona fide substrate of RNF26. RNF26 promoted the degradation of CBX7 and enhanced ccRCC tumor growth. Therefore, our results revealed a novel RNF26/CBX7 axis that modulates the TNF signaling pathway in ccRCC.     

Nimbolide inhibits IGF‐I‐mediated PI3K/Akt and MAPK signalling in human breast cancer cell lines (MCF‐7 and MDA‐MB‐231)

The insulin‐like growth factor I (IGF‐I) signalling pathway contributes a major role on various cancer cell proliferation, survival and cell cycle. The present study was aimed to investigate the effect of nimbolide on IGF signalling and cell cycle arrest in MCF‐7 and MDA‐MB‐231 breast cancer cell lines. The protein expression of IGF signalling molecules and cell cycle protein levels was assessed by western blot analysis. In order to study the interaction of nimbolide on IGF‐1 signalling pathway, IGF‐I and phosphoinositide 3‐kinase (PI3K) inhibitor (LY294002) were used to treat MCF‐7 and MDA‐MB‐231 cells. Further, the cell cycle arrest was analysed by flow cytometry. The protein expression of IGF signalling molecules was significantly decreased in nimbolide‐treated breast cancer cells. PI3K inhibitor and IGF‐I with nimbolide treatment notably inhibited phosphorylated Akt. The cell cycle arrest was observed at the G0/G1 phase, and accumulation of apoptotic cells was observed in nimbolide‐treated breast cancer cell lines. Nimbolide also increased the protein expression of p21 and decreased the cyclins in both the cell lines. Nimbolide decreases the proliferation of breast cancer cells by modulating the IGF signalling molecules, which could be very useful for the breast cancer treatment. Copyright © 2014 John Wiley & Sons, Ltd.     

Aggravated Cardiac Remodeling post Aortocaval Fistula in Unilateral Nephrectomized Rats

Background

Aortocaval fistula (AV) in rat is a unique model of volume-overload congestive heart failure and cardiac hypertrophy. Living donor kidney transplantation is regarded as beneficial to allograft recipients and not particularly detrimental to the donors. Impact of AV on animals with mild renal dysfunction is not fully understood. In this study, we explored the effects of AV in unilateral nephrectomized (UNX) rats.

Methods

Adult male Sprague-Dawley (SD) rats were divided into Sham (n = 10), UNX (right kidney remove, n = 10), AV (AV established between the levels of renal arteries and iliac bifurcation, n = 18) and UNX+AV (AV at one week after UNX, n = 22), respectively. Renal outcome was measured by glomerular filtration rate, effective renal plasma flow, fractional excretion of sodium, albuminuria, plasma creatinine, and cystatin C. Focal glomerulosclerosis (FGS) incidence was evaluated by renal histology. Cardiac function was measured by echocardiography and hemodynamic measurements.

Results

UNX alone induced compensatory left kidney enlargement, increased plasma creatinine and cystatin C levels, and slightly reduced glomerular filtration rate and increased FGS. AV induced significant cardiac enlargement and hypertrophy and reduced cardiac function and increased FGS, these changes were aggravated in UNX+AV rats.

Conclusions

Although UNX only induces minor renal dysfunction, additional chronic volume overload placement during the adaptation phase of the remaining kidney is associated with aggravated cardiac dysfunction and remodeling in UNX rats, suggesting special medical care is required for UNX or congenital monokidney subjects in case of chronic volume overload as in the case of pregnancy and hyperthyroidism to prevent further adverse cardiorenal events in these individuals.     

Formononetin-induced apoptosis of human prostate cancer cells through ERK1/2 mitogen-activated protein kinase inactivation

Formononetin is a main active component of red clover plants (Trifolium pratense L.), and is considered as a phytoestrogen. Our previous studies demonstrated that formononetin caused cell cycle arrest at the G0/G1 phase by inactivating insulin-like growth factor 1(IGF1)/IGF1R-phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in MCF-7 cells. In the present study, we investigated the molecular mechanisms involved in the effect of formononetin on prostate cancer cells. Our results suggested that higher concentrations of formononetin inhibited the proliferation of prostate cancer cells (LNCaP and PC-3), while the most striking effect was observed in LNCaP cells. We further found that formononetin inactivated extracellular signal-regulated kinase1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway in a dose-dependent manner, which resulted in increased the expression levels of BCL2-associated X (Bax) mRNA and protein, and induced apoptosis in LNCaP cells. Thus, we concluded that the induced apoptosis effect of formononetin on human prostate cancer cells was related to ERK1/2 MAPK-Bax pathway. Considering that red clover plants were widely used clinically, our results provided the foundation for future development of different concentrations formononetin for treatment of prostate cancer.     

A complete map of the Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) signaling pathway

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine-protein kinase belonging to the Ca²⁺/calmodulin-dependent protein kinase subfamily. CAMKK2 has an autocatalytic site, which gets exposed when Ca²⁺/calmodulin (CAM) binds to it. This results in autophosphorylation and complete activation of CAMKK2. The three major known downstream targets of CAMKK2 are 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPKα), calcium/calmodulin-dependent protein kinase 1 (CAMK1) and calcium/calmodulin-dependent protein kinase 4 (CAMK4). Activation of these targets by CAMKK2 is important for the maintenance of different cellular and physiological processes within the cell. CAMKK2 is found to be important in neuronal development, bone remodeling, adipogenesis, and systemic glucose homeostasis, osteoclastgensis and postnatal myogensis. CAMKK2 is reported to be involved in pathologies like Duchenne muscular dystrophy, inflammation, osteoporosis and bone remodeling and is also reported to be overexpressed in prostate cancer, hepatic cancer, ovarian and gastric cancer. CAMKK2 is involved in increased cell proliferation and migration through CAMKK2/AMPK pathway in prostate cancer and activation of AKT in ovarian cancer. Although CAMKK2 is a molecule of great importance, a public resource of the CAMKK2 signaling pathway is currently lacking. Therefore, we carried out detailed data mining and documentation of the signaling events associated with CAMKK2 from published literature and developed an integrated reaction map of CAMKK2 signaling. This resulted in the cataloging of 285 reactions belonging to the CAMKK2 signaling pathway, which includes 33 protein–protein interactions, 74 post-translational modifications, 7 protein translocation events, and 22 activation/inhibition events. Besides, 124 gene regulation events and 25 activator/inhibitors involved in CAMKK2 activation were also cataloged. The CAMKK2 signaling pathway map data is made freely accessible through WikiPathway database (https://www.wikipathways.org/index.php/Pathway:WP4874). We expect that data on a signaling map of CAMKK2 will provide the scientific community with an improved platform to facilitate further molecular as well as biomedical investigations on CAMKK2 and its utility in the development of biomarkers and therapeutic targets.Electronic supplementary materialThe online version of this article (10.1007/s12079-020-00592-1) contains supplementary material, which is available to authorized users.     

Interstitial Fluid Flow Increases Hepatocellular Carcinoma Cell Invasion through CXCR4/CXCL12 and MEK/ERK Signaling

Hepatocellular carcinoma (HCC) is the most common form of liver cancer (~80%), and it is one of the few cancer types with rising incidence in the United States. This highly invasive cancer is very difficult to detect until its later stages, resulting in limited treatment options and low survival rates. There is a dearth of knowledge regarding the mechanisms associated with the effects of biomechanical forces such as interstitial fluid flow (IFF) on hepatocellular carcinoma invasion. We hypothesized that interstitial fluid flow enhanced hepatocellular carcinoma cell invasion through chemokine-mediated autologous chemotaxis. Utilizing a 3D in vitro invasion assay, we demonstrated that interstitial fluid flow promoted invasion of hepatocellular carcinoma derived cell lines. Furthermore, we showed that autologous chemotaxis influences this interstitial fluid flow-induced invasion of hepatocellular carcinoma derived cell lines via the C-X-C chemokine receptor type 4 (CXCR4)/C-X-C motif chemokine 12 (CXCL12) signaling axis. We also demonstrated that mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling affects interstitial fluid flow-induced invasion; however, this pathway was separate from CXCR4/CXCL12 signaling. This study demonstrates, for the first time, the potential role of interstitial fluid flow in hepatocellular carcinoma invasion. Uncovering the mechanisms that control hepatocellular carcinoma invasion will aid in enhancing current liver cancer therapies and provide better treatment options for patients.     

Inhibition of RPS6K reveals context-dependent Akt activity in luminal breast cancer cells

Aberrant signaling through insulin (Ins) and insulin-like growth factor I (IGF1) receptors contribute to the risk and advancement of many cancer types by activating cell survival cascades. Similarities between these pathways have thus far prevented the development of pharmacological interventions that specifically target either Ins or IGF1 signaling. To identify differences in early Ins and IGF1 signaling mechanisms, we developed a dual receptor (IGF1R & InsR) computational response model. The model suggested that ribosomal protein S6 kinase (RPS6K) plays a critical role in regulating MAPK and Akt activation levels in response to Ins and IGF1 stimulation. As predicted, perturbing RPS6K kinase activity led to an increased Akt activation with Ins stimulation compared to IGF1 stimulation. Being able to discern differential downstream signaling, we can explore improved anti-IGF1R cancer therapies by eliminating the emergence of compensation mechanisms without disrupting InsR signaling.     

Protein Secretion Is Required for Pregnancy-Associated Plasma Protein-A to Promote Lung Cancer Growth In Vivo

Pregnancy-associated plasma protein-A (PAPPA) has been reported to regulate the activity of insulin-like growth factor (IGF) signal pathway through proteolytic degradation of IGF binding proteins (IGFBPs) thereby increasing the local concentration of free IGFs available to receptors. In this study we found that PAPPA is secreted from two out of seven lung cancer cell lines examined. None of immortalized normal bronchial epithelial cells (HBE) tested secrets PAPPA. There is no correlation between expression level and secretion of PAPPA in these cells. A cell line over-expressing PAPPA accompanied with secretion shows no notable changes in proliferation under cell culture conditions in vitro, but displays significantly augmentation of tumor growth in vivo in a xenograft model. In contrast, a cell line over-expressing PAPPA without secretion exhibits reduction of tumor growth both in vitro and in vivo. Down-regulation of PAPPA expression and secretion by RNAi knockdown decreases tumor growth after implanted in vivo. The tumor promoting activity of PAPPA appears to be mediated mainly through augmentation of the IGF signaling pathway as indicated by notable increases in downstream Akt kinase phosphorylation in tumor samples. Our results indicate that PAPPA secretion may play an important role in lung cancer growth and progression.     

Opposing effects of 15-lipoxygenase-1 and -2 metabolites on MAPK signaling in prostate. Alteration in peroxisome proliferator-activated receptor gamma

Human prostate tumors have elevated levels of 15-lipoxygenase-1 (15-LOX-1) and data suggest that 15-LOX-1 may play a role in the development of prostate cancer. In contrast, 15-LOX-2 expression is higher in normal rather than in tumor prostate tissue and appears to suppress cancer development. We recently reported that 13-(S)-HODE, the 15-LOX-1 metabolite, up-regulates the MAP kinase signaling pathway and subsequently down-regulates PPARgamma in human colorectal carcinoma cells. To determine whether this mechanism is applicable to prostate cancer and what the effects of 15-LOX-2 are, we investigated the effect of 15-LOX-1, 15-LOX-2, and their metabolites on epidermal growth factor (EGF)- and insulin-like growth factor (IGF)-1 signaling in prostate carcinoma cells. In PC3 cells, 13-(S)-HODE, a 15-LOX-1 metabolite, up-regulated MAP kinase while in contrast 15-(S)-HETE, a 15-LOX-2 metabolite, down-regulated MAP kinase. As a result, 13-(S)-HODE increased PPARgamma phosphorylation while a subsequent decrease in PPARgamma phosphorylation was observed with 15-(S)-HETE. Thus, 15-LOX metabolites have opposing effects on the regulation of the MAP kinase signaling pathway and a downstream target of MAP kinase signaling like PPARgamma. In addition to the EGF signaling pathway, the IGF signaling pathway appears to be linked to prostate cancer. 13-(S)-HODE and 15-(S)-HETE up-regulate or down-regulate, respectively, both the MAPK and Akt pathways after activation with IGF-1. Thus, the effect of these lipid metabolites is not solely restricted to EGF signaling and not solely restricted to MAPK signaling. These results provide a plausible mechanism to explain the apparent opposing effects 15-LOX-1 and 15-LOX-2 play in prostate cancer.     

Loss of HSulf-1 up-regulates heparin-binding growth factor signaling in cancer

Emerging data suggest that signaling by heparin-binding growth factors is influenced by the sulfation state of N-acetylglucosamine residues of heparan sulfate proteoglycans (HSPGs). Here we report that the recently identified protein HSulf-1, a heparin-degrading endosulfatase, encodes a cell surface-associated enzyme that diminishes sulfation of cell surface HSPGs. The message encoding this enzyme is readily detectable in a variety of normal tissues, including normal ovarian surface epithelial cells, but is undetectable in 5 of 7 ovarian carcinoma cell lines and markedly diminished or undetectable in approximately 75% of ovarian cancers. Similar down-regulation is also observed in breast, pancreatic, renal cells, and hepatocellular carcinoma lines. Re-expression of HSulf-1 in ovarian cancer cell lines resulted in diminished HSPG sulfation, diminished phosphorylation of receptor tyrosine kinases that require sulfated HSPGs as co-receptors for their cognate ligands, and diminished downstream signaling through the extracellular signal-regulated kinase pathway after treatment with fibroblast growth factor-2 or heparin-binding epidermal growth factor. Consistent with these changes, HSulf-1 re-expression resulted in reduced proliferation as well as sensitivity to induction of apoptosis by the broad spectrum kinase inhibitor staurosporine and the chemotherapeutic agent cisplatin. Collectively, these observations provide evidence that HSulf-1 modulates signaling by heparin-binding growth factors, and HSulf-1 down-regulation represents a novel mechanism by which cancer cells can enhance growth factor signaling.     

Hypoxia-induced SM22α in A549 cells activates the IGF1R/PI3K/Akt pathway, conferring cellular resistance against chemo- and radiation therapy

Chemo- or radiation-resistance in tumors caused by hypoxia often undermines efficacy of cancer therapy. Thus, therapies that overcome cellular resistance during hypoxia are necessary. SM22α is an actin-binding protein found in smooth muscle, fibroblasts, and some epithelium. We demonstrate that SM22α is induced in A549 non-small cell lung carcinoma cells by hypoxia and its overexpression increased chemo- and radiation-resistance. Hypoxia-mediated induction of SM22α expression is hypoxia-inducible factor-independent. Moreover, SM22α overexpression enhances tumor cell growth and activates the IGF1R/PI3K/Akt pathway via direct interaction with IGF1Rβ. Our results suggest SM22α as a novel regulator of hypoxic survival pathway of A549 NSCLC cells.     

Inhibition of mitogen-activated protein kinase kinase selectively inhibits cell proliferation in human breast cancer cells displaying enhanced insulin-like growth factor I-mediated mitogen-activated protein kinase activation.

Mitogen-activated protein (MAP) kinase mediates cell proliferation, cell differentiation, and cell survival by regulating signaling pathways activated by receptor protein tyrosine kinases (RPTKs), including the insulin-like growth factor 1 receptor (IGF-IR). We analyzed the upstream signaling components of the MAP kinase pathway, including RPTKs, in human breast cancer cell lines and found that some of those components were overexpressed. Importantly, signaling molecules such as IGF-IR, insulin receptor, and insulin receptor substrate 1, leading to the MAP kinase pathway, were found to be concomitantly overexpressed within certain tumor lines, i.e., MCF-7 and T-47D. When compared with the nonmalignant and other breast tumor lines examined, MCF-7 and T-47D cells displayed a more rapid, robust, and sustained MAP kinase activation in response to insulin-like growth factor I (IGF-I) stimulation. By contrast, IGF-I treatment led to a sustained down-regulation of MAP kinase in those lines overexpressing ErbB2-related RPTKs. Interestingly, blocking the MAP kinase pathway with PD098059 had the greatest antiproliferative effect on MCF-7 and T-47D among the normal and tumor lines tested. Furthermore, addition of an IGF-IR blocking antibody to growth medium attenuated the ability of PD098059 to suppress the growth of MCF-7 and T-47D cells. Thus, our study suggests that concomitant overexpression of multiple signaling components of the IGF-IR pathway leads to the amplification of IGF-I-mediated MAP kinase signaling and resultant sensitization to PD098059. The enhanced sensitivity to PD098059 implies an increased requirement for the MAP kinase pathway in those breast cancer cells, making this pathway a potential target in the treatment of selected breast malignancies.     

Synergistic Signaling of Tumor Cell Invasiveness by Hepatocyte Growth Factor and Hypoxia

Hepatocyte growth factor (HGF) signaling promotes tumor invasiveness in renal cell carcinoma (RCC) and other cancers. In clear cell RCC, VHL loss generates pseudohypoxia that exacerbates HGF-driven invasion through β-catenin deregulation. Hypoxia also enhances HGF-driven invasiveness by papillary RCC cells, but in the absence of VHL, loss signaling integration involves three parallel routes: 1) hypoxia-induced reactive oxygen species production and decreased DUSP2 expression, leading to enhanced mitogen-activated protein kinase (MAPK) cascade activation; 2) reactive oxygen species-induced diacylglycerol production by phospholipase Cγ, leading to protein kinase C activation and increased protein phosphatase-2A activity, thereby suppressing HGF-induced Akt activation; and 3) a profound shift from HGF-enhanced, proliferation-oriented metabolism to autophagy-dependent invasion and suppression of proliferation. This tripartite signaling integration was not unique to RCC or HGF; in RCC cells, invasive synergy induced by the combination of hypoxia and epidermal growth factor occurred through the same mechanism, and in estrogen receptor-positive breast cancer cells, this mechanism was suppressed in the absence of estrogen. These results define the molecular basis of growth factor and hypoxia invasive synergy in VHL-competent papillary RCC cells, illustrate the plasticity of invasive and proliferative tumor cell states, and provide signaling profiles by which they may be predicted.     

Protein kinase B, P34cdc2 kinase, and p21 ras GTP-binding in kidneys of aging rats

Renal nephropathy present in male Wistar rats more than 13 months of age was reported as an indication that the rats were in renal failure. In this study, the renal tissue damage at 14 months of age in male Munich Wistar rats was similar to that reported for Wistar rats, indicating that Munich Wistar rats could be another model for study of kidney function in the aging rat. The usual renal response to injury involves increased cell division and/or reparative processes that involve tyrosine kinase activity (TyrK) and/or guanosine triphosphate-binding (G) protein signal trans-duction pathways. This study reveals the presence of renal tissue damage coinciding with significantly reduced activity of Ras, Akt, and p34cdc2 kinase, the signaling proteins that regulate cell division and/or growth, in renal cortical tissues of aging rats compared to young rats (P < 0.005, P < 0.005, and P< 0.001, respectively). These results suggest that proteins involved in signal transduction pathways associated with cell replication are downregulated in the aging kidney cortex at a time when renal cellular damage is also present.