The PI3K/AKT Pathway and Renal Cell Carcinoma

The phosphatidylinositol 3 kinase(PI3K)/AKT pathway is genetically targeted in more pathway components and in more tumor types than any other growth factor signaling pathway,and thus is frequently activated as a cancer driver.More importantly,the PI3K/AKT pathway is composed of multiple bifurcating and converging kinase cascades,providing many potential targets for cancer therapy.Renal cell carcinoma(RCC) is a high-risk and high-mortality cancer that is notoriously resistant to traditional chemotherapies or radiotherapies.The PI3K/AKT pathway is modestly mutated but highly activated in RCC,representing a promising drug target.Indeed,PI3 K pathway inhibitors of the rapalog family are approved for use in RCC.Recent large-scale integrated analyses of a large number of patients have provided a molecular basis for RCC,reiterating the critical role of the PI3K/AKT pathway in this cancer.In this review,we summarize the genetic alterations of the PI3K/AKT pathway in RCC as indicated in the latest large-scale genome sequencing data,as well as treatments for RCC that target the aberrant activated PI3K/AKT pathway.     

Molecular analysis of sunitinib resistant renal cell carcinoma cells after sequential treatment with RAD001 (everolimus) or sorafenib

Sequential application of target drugs is standard procedure after renal cell carcinoma (RCC) patients develop resistance. To optimize the sequence, antitumour effects of the mTOR inhibitor RAD001 or the tyrosine kinase inhibitor (TKI) sorafenib on RCC cells with acquired resistance to the TKI sunitinib was evaluated. RCC cells were exposed to 1 μM sunitinib for 24 hrs (as control) and for 8 weeks (to induce resistance) and then switched to RAD001 (5 nM) or sorafenib (5 μM) for a further 8 weeks. Tumour cell growth, cell cycle progression, cell cycle regulating proteins and intracellular signalling were then investigated. Short‐term application of sunitinib (24 hrs) induced cell growth blockade with accumulation in the G2/M phase. RCC cells became resistant to sunitinib after 8 weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated distinct cross‐resistance. In contrast, full response was achieved when the cancer cells were treated with RAD001. p19 and p27 strongly increased, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR‐inhibitor for second‐line therapy could be the strategy of choice after first‐line sunitinib failure.     

Molecular pathology and targeted therapy of clear cell renal cancer

The golden standard of care for advanced renal cell cancer (RCC) was until now the cytokine therapy with relatively low response rates. Advances of molecular genetics in RCC revealed several molecular targets such as VHL and angiogenic genotype or EGFR in the clear cell variant. Among the novel targeted agents, multiple tyrosine kinase inhibitors were proved to be clinically effective against advanced clear cell renal cancer, changing the standard of care. It is a further question how molecular diagnostics can improve these results by the detection of these targets or gene defects in individual tumors.     

Acquired resistance to tyrosine kinase inhibitors during cancer therapy

Selective tyrosine kinase inhibitors have emerged as important therapeutic agents in the treatment of a variety of human malignancies. Although several of these inhibitors have marked clinical activity, it is widely recognized that the overall value of these agents is substantially limited by the acquisition of drug resistance, which eventually arises in most, if not all treated patients. Mechanisms of drug resistance are beginning to be elucidated through the molecular analysis of clinical specimens as well as through cell culture modeling. By identifying resistance mechanisms, it should be possible to develop 'second-generation' inhibitors as well as rational drug combinations that can overcome or even prevent acquired resistance to kinase inhibitors, thereby enhancing clinical benefit.     

Autophagy orchestrates adaptive responses to targeted therapy in endometrial cancer

Targeted therapies in endometrial cancer (EC) using kinase inhibitors rarely result in complete tumor remission and are frequently challenged by the appearance of refractory cell clones, eventually resulting in disease relapse. Dissecting adaptive mechanisms is of vital importance to circumvent clinical drug resistance and improve the efficacy of targeted agents in EC. Sorafenib is an FDA-approved multitarget tyrosine and serine/threonine kinase inhibitor currently used to treat hepatocellular carcinoma, advanced renal carcinoma and radioactive iodine-resistant thyroid carcinoma. Unfortunately, sorafenib showed very modest effects in a multi-institutional phase II trial in advanced uterine carcinoma patients. Here, by leveraging RNA-sequencing data from the Cancer Cell Line Encyclopedia and cell survival studies from compound-based high-throughput screenings we have identified the lysosomal pathway as a potential compartment involved in the resistance to sorafenib. By performing additional functional biology studies we have demonstrated that this resistance could be related to macroautophagy/autophagy. Specifically, our results indicate that sorafenib triggers a mechanistic MAPK/JNK-dependent early protective autophagic response in EC cells, providing an adaptive response to therapeutic stress. By generating in vivo subcutaneous EC cell line tumors, lung metastatic assays and primary EC orthoxenografts experiments, we demonstrate that targeting autophagy enhances sorafenib cytotoxicity and suppresses tumor growth and pulmonary metastasis progression. In conclusion, sorafenib induces the activation of a protective autophagic response in EC cells. These results provide insights into the unopposed resistance of advanced EC to sorafenib and highlight a new strategy for therapeutic intervention in recurrent EC.     

PI3K(p110α) Inhibitors as Anti-Cancer Agents: Minding the Heart

The central role of phosphatidylinositol 3-kinase (PI3K, p110α) signaling in allowing cancer cells to bypass normal growth-limiting controls has led to the development of PI3K(p110α) inhibitors. A challenge in targeting PI3K(p110α) relates to the diverse actions of the PI3K pathway in numerous cell types. Recent findings in mice deficient in PI3K(p110α) activity in the heart, demonstrate the critical role of this pathway in protecting the heart against pathological insults. Mice deficient in PI3K(p110α) displayed accelerated heart failure in response to dilated or hypertrophic cardiomyopathy. These results help explain the association of cardiomyopathy in cancer patients given tyrosine kinase inhibitors and raise concerns for the use of PI3K(p110α) inhibitors in cancer patients with cardiovascular risk factors. Interestingly, an inhibitor of the mammalian target of rapamycin (a downstream effector of PI3K), did not have adverse effects on the heart. A more complete understanding of the complex arms and interactions of the PI3K pathway will hopefully lead to the development of anti-cancer agents without cardiac complications.     

Hypothyroidism during treatment with tyrosine kinase inhibitors

Tyrosine kinase inhibitors are relatively new targeted therapy drugs used for the treatment of metastatic clear cell kidney carcinoma, gastrointestinal stromal tumours, thyroid carcinoma and pancreatic neuroendocrine tumours during the progression of the disease. Hypothyroidism or thyroid dysfunction is often a side effect of this treatment. Therefore, monitoring of thyroid hormone levels before the beginning and during the treatment of tyrosine kinase inhibitors is a necessity. Hypothyroidism correlates with objective response to the treatment. Sunitinib. This is the most described tyrosine kinase inhibitor which causes hypothyroidism. The mechanism of hypothyroidism is still unclear. Sorafenib. Symptoms of hypothyroidism occur in 18% of patients treated with sorafenib due to metastatic renal cell carcinoma. Imatinib. Hypothyroidism is one of the most frequent side effects of the treatment. Emergent tracheotomy was necessary due to larynx swelling during marked hypothyroidism. Motesanib. Hypothyroidism or increased TSH level is diagnosed in 22% to 69% of patients with metastatic differentiated or medullary thyroid carcinomas. The management of patients with thyroid dysfunction and related symptoms such as fatigue is undoubtedly a challenge to an oncologist.     

TOPK/PBK is phosphorylated by ERK2 at serine 32, promotes tumorigenesis and is involved in sorafenib resistance in RCC

TOPK/PBK (T-LAK Cell-Originated Protein Kinase) is a serine/threonine kinase that is highly expressed in a variety of human tumors and is associated with poor prognosis in many types of human malignancies. Its activation mechanism is not yet fully understood. A bidirectional signal transduced between TOPK and ERK2 (extracellular signal-regulated kinase 2) has been reported, with ERK2 able to phosphorylate TOPK at the Thr9 residue. However, mutated TOPK at Thr9 cannot repress cellular transformation. In the present study, Ser32 was revealed to be a novel phosphorylated site on TOPK that could be activated by ERK2. Phospho-TOPK (S32) was found to be involved in the resistance of renal cell carcinoma (RCC) to sorafenib. Herein, combined a TOPK inhibitor with sorafenib could promoted the apoptosis of sorafenib-resistant RCC. High expression of HGF/c-met contributes to activation of p-TOPK (S32) during the development of sorafenib resistance in RCC. The current research presents a possible mechanism of sorafenib resistance in RCC and identifies a potential diagnostic marker for predicting sorafenib resistance in RCC, providing a valuable supplement for the clinically targeted treatment of advanced RCC.Subject terms: Predictive markers, Renal cancer     

Inhibition of the PI3K/Akt/GSK3 Pathway Downstream of BCR/ABL,Jak2-V617F,or FLT3-ITD Downregulates DNA Damage-Induced Chk1 Activation as Well as G2/M Arrest and Prominently Enhances Induction of Apoptosis

Constitutively-activated tyrosine kinase mutants, such as BCR/ABL, FLT3-ITD, and Jak2-V617F, play important roles in pathogenesis of hematopoietic malignancies and in acquisition of therapy resistance. We previously found that hematopoietic cytokines enhance activation of the checkpoint kinase Chk1 in DNA-damaged hematopoietic cells by inactivating GSK3 through the PI3K/Akt signaling pathway to inhibit apoptosis. Here we examine the possibility that the kinase mutants may also protect DNA-damaged cells by enhancing Chk1 activation. In cells expressing BCR/ABL, FLT3-ITD, or Jak2-V617F, etoposide induced a sustained activation of Chk1, thus leading to the G2/M arrest of cells. Inhibition of these kinases by their inhibitors, imatinib, sorafenib, or JakI-1, significantly abbreviated Chk1 activation, and drastically enhanced apoptosis induced by etoposide. The PI3K inhibitor GD-0941 or the Akt inhibitor MK-2206 showed similar effects with imatinib on etoposide-treated BCR/ABL-expressing cells, including those expressing the imatinib-resistant T315I mutant, while expression of the constitutively activated Akt1-myr mutant conferred resistance to the combined treatment of etoposide and imatinib. GSK3 inhibitors, including LiCl and SB216763, restored the sustained Chk1 activation and mitigated apoptosis in cells treated with etoposide and the inhibitors for aberrant kinases, PI3K, or Akt. These observations raise a possilibity that the aberrant kinases BCR/ABL, FLT3-ITD, and Jak2-V617F may prevent apoptosis induced by DNA-damaging chemotherapeutics, at least partly through enhancement of the Chk1-mediated G2/M checkpoint activation, by inactivating GSK3 through the PI3K/Akt signaling pathway. These results shed light on the molecular mechanisms for chemoresistance of hematological malignancies and provide a rationale for the combined treatment with chemotherapy and the tyrosine kinase or PI3K/Akt pathway inhibitors against these diseases.     

Reduced Immunosuppressive Properties of Axitinib in Comparison with Other Tyrosine Kinase Inhibitors

The multikinase inhibitors sunitinib, sorafenib, and axitinib have an impact not only on tumor growth and angiogenesis, but also on the activity and function of immune effector cells. In this study, a comparative analysis of the growth inhibitory properties and apoptosis induction potentials of tyrosine kinase inhibitors on T cells was performed. Tyrosine kinase inhibitor treatment resulted in a dramatic decrease in T cell proliferation along with distinct impacts on the cell cycle progression. This was at least partially associated with an enhanced induction of apoptosis although triggered by distinct apoptotic mechanisms. In contrast to sunitinib and sorafenib, axitinib did not affect the mitochondrial membrane potential (Δψ_m) but resulted in an induction or stabilization of the induced myeloid leukemia cell differentiation protein (Mcl-1), leading to an irreversible arrest in the G₂/M cell cycle phase and delayed apoptosis. Furthermore, the sorafenib-mediated suppression of immune effector cells, in particular the reduction of the CD8⁺ T cell subset along with the down-regulation of key immune cell markers such as chemokine CC motif receptor 7 (CCR7), CD26, CD69, CD25, and CXCR3, was not observed in axitinib-treated immune effector cells. Therefore, axitinib rather than sorafenib seems to be suitable for implementation in complex treatment regimens of cancer patients including immunotherapy.     

Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation

Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury.     

Tyrosine protein kinase inhibition and cancer

The various aspects of the research on tyrosine protein kinase inhibition and its connections with cancer are presented. The emphasis was made on the theoretical low toxic side effects of specific tyrosine protein kinase inhibitors. Particularly, the strategy of finding peptidic substrate-derived inhibitors or modulators is discussed, with an almost complete compendium of the tyrosine protein kinase peptidic substrates published so far. A series of data has been gathered that may serve as a basis for the discovery of selective and specific tyrosine protein kinase inhibitors by screening on molecular and cellular models. The potential of SH2 domain-interfering agents are also presented as a promising route to new anticancer compounds.     

Functional characterization of peanut serine/threonine/tyrosine protein kinase: molecular docking and inhibition kinetics with tyrosine kinase inhibitors

Serine/threonine/tyrosine (STY) protein kinase from peanut is developmentally regulated and is induced by abiotic stresses. In addition, STY protein kinase activity is regulated by tyrosine phosphorylation. Kinetic mechanism of plant dual specificity protein kinases is not studied so far. Recombinant STY protein kinase occurs as a monomer in solution as shown by gel filtration chromatography. The relative phosphorylation rate of kinase against increasing enzyme concentrations follows a first-order kinetics indicating an intramolecular phosphorylation mechanism. Moreover, the active recombinant STY protein kinase could not transphosphorylate a kinase-deficient mutant of STY protein kinase. Molecular docking studies revealed that the tyrosine kinase inhibitors bind the protein kinase at the same region as ATP. STY protein kinase activity was inhibited by the tyrosine kinase inhibitors, and the inhibitor potency series against the recombinant STY protein kinase was tyrphostin > genistein > staurosporine. The inhibition constant (K(i)), and the IC(50) value of STY protein kinase for tyrosine kinase inhibitors with ATP and histone are discussed. All the inhibitors competed with ATP. Genistein was an uncompetitive inhibitor with histone, whereas staurosporine and tyrphostin were linear mixed type noncompetitive inhibitors with histone. Molecular docking and kinetic analysis revealed that Y148F mutant of the "ATP-binding loop" and Y297F mutant of the "activation loop" showed a dramatic increase in K(i) values for genistein and tyrphostin with respect to wild-type STY protein kinase. Data presented here provide the direct evidence on the mechanism of inhibition of plant protein kinases by tyrosine kinase inhibitors. This study also suggests that tyrosine kinase inhibitors may be useful in unraveling the plant tyrosine phosphorylation signaling cascades.     

Long non-coding RNA GAS5 sensitizes renal cell carcinoma to sorafenib via miR-21/SOX5 pathway

Although the use of sorafenib appears to increase the survival rate of renal cell carcinoma (RCC) patients, there is also a proportion of patients who exhibit a poor primary response to sorafenib treatment. Therefore, it is critical to elucidate the mechanisms underlying sorafenib resistance and find representative biomarkers for sorafenib treatment in RCC patients. Herein, we identified that a long noncoding RNA GAS5 was downregulated in sorafenib nonresponsive RCCs. GAS5 overexpression conferred sorafenib sensitive to nonresponsive RCC cells, whereas knockdown of GAS5 promoted responsive RCC cells resistant to sorafenib treatment in vitro and in vivo. Mechanistically, GAS5 functioned as competing endogenous RNA to repress miR-21, which controlled its down-stream target SOX5. We proposed that GAS5 was responsible for sorafenib resistance in RCC cells and GAS5 exerted its function through the miR-21/ SOX5 axis. Our findings suggested that GAS5 downregulation may be a new marker of poor response to sorafenib and GAS5 could be a potential therapeutic target for sorafenib treatment in RCC.     

BCRP/ABCG2 Inhibition Sensitizes Hepatocellular Carcinoma Cells to Sorafenib

The multikinase inhibitor, sorafenib (Nexavar®, BAY43-9006), which inhibits both the Raf/MEK/ERK pathway and several receptor tyrosine kinases (RTKs), has shown significantly therapeutic benefits in advanced hepatocellular carcinoma (HCC). However, not all HCC patients respond to sorafenib well and new therapeutic strategies to optimize the efficacy of sorafenib are urgently required. Overexpression of breast cancer resistance protein (BCRP/ABCG2) mediates the drug-efflux of several tyrosine kinase inhibitors (TKIs) to attenuate their efficacy. This study aimed to investigate the role of BCRP/ABCG2 in the sensitivity of HCC to sorafenib. Our data showed that BCRP/ABCG2 mediated the efflux of sorafenib. Co-treatment with a BCRP/ABCG2 inhibitor greatly augmented the cytotoxicity of sorafenib in HCC cells. Similar results were also achieved by the competitive inhibitor of BCRP/ABCG2, gefitinib, in combination with sorafenib. These results suggest not only that BCRP/ABCG2 is a potential predictor for the sorafenib sensitivity in HCC, but also that blockage of BCRP/ABCG2 may be a potential strategy to increase the response of HCC cells to sorafenib.     

Vascular endothelial growth factor inhibition: Conflicting roles in tumor growth

Angiogenesis, the physiological process of sprouting of new blood vessels from pre-existing ones, is a key biological feature of almost all cancers. Among the multitude of factors driving tumor angiogenesis, vascular endothelial growth factor (VEGF) is the most potent, exerting myriad effects on vascular pruning and sprouting, permeability, network formation, proliferation, and cell death. Despite the initial unimpressive clinical performance of anti-VEGF antibody (bevacizumab) as cancer monotherapy, clear improvements in clinical outcomes following combination bevacizumab and chemotherapy regimens and multi-targeted VEGF receptor tyrosine kinase inhibitors (sorafenib and sunitinib) in select tumor types have established VEGF-targeted agents as an effective means of controlling cancer growth. Prolongation of overall survival and cure with these agents, however, remains elusive. Moreover, recent data has revealed key differences in the therapeutic and biological tumor response to antibody versus receptor kinase VEGF inhibitors and suggested, at least pre-clinically, that VEGF blockade in certain circumstances may actually promote more aggressive tumor growth. Given the diverse mechanisms and potentially opposing roles of VEGF neutralization in cancer biology, identification of novel biomarkers predictive of in vivo angiogenic responses may hold the key to optimizing therapeutic outcomes of anti-VEGF therapy in future cancer patients.     

Connexin43 is associated with the progression of clear cell renal carcinoma and is regulated by tangeretin to sygergize with tyrosine kinase inhibitors

BackgroundThe roles of Connexin43 (Cx43) in clear cell renal cell carcinoma (ccRCC) microenviroment remains to be poorly defined.MethodsThe expression profile, prognosis and immune analysis of Cx43 in various cancers, particularly in ccRCC were performed using TCGA database, and various biological function assays were applied to explore the physiological role of Cx43 and tangeretin in ccRCC. Western blot were applied to examine the protein expression and Kunming mice were used to evaluate preliminary safety or anti-tumor activity of tangeretin and sunitinib.ResultsCompared with the normal group, higher expression levels of Cx43 in ccRCC, and distinct associations between Cx43 expression and ccRCC prognosis or immune infiltration, were found. Notably, the expression of Cx43 was found to be highly correlated with that of receptor tyrosine kinases (RTKs), particularly with VEGFR1, VEGFR2 and VEGFR3. The expression of Cx43 and EGFR was also found to be higher in ccRCC than that in the para-cancerous specimens. Knocking down Cx43 expression decreased RCC cell viability, cell migration, p-EGFR, MMP-9 and survivin expression. Using 14 Chinese medicine monomers, tangeretin was screened and found to inhibit tumor cell viability and Cx43 expression. Tangeretin also enhanced the sensitivity of RCC cells to tyrosine kinase inhibitors (TKIs) sunitinib and sorafenib. However, the same concentration of tangeretin exerted a less prominent effect on normal renal cell viability.ConclusionsCx43 is strongly associated with RTK expression and ccRCC progression, while tangeretin can inhibit RCC cell malignancy by inhibiting Cx43 expression and enhance the sensitivity of RCC cells to TKIs.