Degradation of microinjected proteins: Effects of lysosomotropic agents and inhibitors of autophagy

HeLa cells, injected with radioiodinated proteins by fusion with RBC ghosts, were exposed to inhibitors of lysosomal proteolysis and autophagy. The degradation of injected [¹²⁵I]bovine serum albumin (BSA) was unaffected by chloroquine, NH₄Cl, nocodazole, colcemid, puromycin, cycloheximide, or enucleation. Although degradation of [¹²⁵I]lactate dehydrogenase (LDH) and [¹²⁵I]pyruvate kinase (PK) was inhibited one-third by chloroquine or ammonia, their degradation was unaffected by the other compounds. In contrast, enhanced degradation of ¹²⁵I-PK resulting from depriving injected HeLa cells of amino acids and serum was inhibited 70% by colcemid and abolished by chloroquine or ammonia. Similarly, degradation of [¹⁴C]sucrose-labeled BSA-polylysine conjugates that entered HeLa cells by endocytosis was inhibited as much as 80% by chloroquine and ammonia. Sensitivity of both enhanced proteolysis and degradation of exogenous proteins to ammonia or chloroquine indicates they are effective inhibitors of lysosomal proteolysis in HeLa cells. Failure of ammonia or chloroquine to inhibit degradation of injected ¹²⁵I-BSA and the modest inhibition of degradation of injected ¹²⁵I-LDH or ¹²⁵I-PK indicates that virtually all BSA molecules and most PK or LDH molecules are degraded by a nonlysosomal proteolytic system. Components of this degradative system are present in vast excess or are long lived, since inhibition of protein synthesis for 20 hr had no effect on the degradation of injected proteins.     

UBR1 promotes protein kinase quality control and sensitizes cells to Hsp90 inhibition

UBR1 and UBR2 are N-recognin ubiquitin ligases that function in the N-end rule degradation pathway. In yeast, the UBR1 homologue also functions by N-end rule independent means to promote degradation of misfolded proteins generated by treatment of cells with geldanamycin, a small molecule inhibitor of Hsp90. Based on these studies we examined the role of mammalian UBR1 and UBR2 in the degradation of protein kinase clients upon Hsp90 inhibition. Our findings show that protein kinase clients Akt and Cdk4 are still degraded in mouse Ubr1(-)/(-) cells treated with geldanamycin, but that their levels recover much more rapidly than is found in wild type cells. These findings correlate with increased induction of Hsp90 expression in the Ubr1(-)/(-) cells compared with wild type cells. We also observed a reduction of UBR1 protein levels in geldanamycin-treated mouse embryonic fibroblasts and human breast cancer cells, suggesting that UBR1 is an Hsp90 client. Further studies revealed a functional overlap between UBR1 and the quality control ubiquitin ligase, CHIP. Our findings show that UBR1 function is conserved in controlling the levels of Hsp90-dependent protein kinases upon geldanamycin treatment, and suggest that it plays a role in determining the sensitivity of cancer cells to the chemotherapeutic effects of Hsp90 inhibitors.     

Genipin-induced inhibition of uncoupling protein-2 sensitizes drug-resistant cancer cells to cytotoxic agents

Uncoupling protein-2 (UCP2) is known to suppress mitochondrial reactive oxygen species (ROS) production and is employed by drug-resistant cancer cells to mitigate oxidative stress. Using the drug-sensitive HL-60 cells and the drug-resistant MX2 subline as model systems, we show that genipin, a UCP2 inhibitor, sensitizes drug-resistant cells to cytotoxic agents. Increased MX2 cell death was observed upon co-treatment with genipin and different doses of menadione, doxorubicin, and epirubicin. DCFH-DA fluorimetry revealed that the increase in MX2 cell death was accompanied by enhanced cellular ROS levels. The drug-induced increase in ROS was linked to genipin-mediated inhibition of mitochondrial proton leak. State 4 and resting cellular respiratory rates were higher in the MX2 cells in comparison to the HL-60 cells, and the increased respiration was readily suppressed by genipin in the MX2 cells. UCP2 accounted for a remarkable 37% of the resting cellular oxygen consumption indicating that the MX2 cells are functionally reliant on this protein. Higher amounts of UCP2 protein were detected in the MX2 versus the HL-60 mitochondria. The observed effects of genipin were absent in the HL-60 cells pointing to the selectivity of this natural product for drug-resistant cells. The specificity of genipin for UCP2 was confirmed using CHO cells stably expressing UCP2 in which genipin induced an ～22% decrease in state 4 respiration. These effects were absent in empty vector CHO cells expressing no UCP2. Thus, the chemical inhibition of UCP2 with genipin sensitizes multidrug-resistant cancer cells to cytotoxic agents.     

UCP2 inhibition sensitizes breast cancer cells to therapeutic agents by increasing oxidative stress

Modulation of oxidative stress in cancer cells plays an important role in the study of the resistance to anticancer therapies. Uncoupling protein 2 (UCP2) may play a dual role in cancer, acting as a protective mechanism in normal cells, while its overexpression in cancer cells could confer resistance to chemotherapy and a higher survival through downregulation of ROS production. Thus, our aim was to check whether the inhibition of UCP2 expression and function increases oxidative stress and could render breast cancer cells more sensitive to cisplatin (CDDP) or tamoxifen (TAM). For this purpose, we studied clonogenicity, mitochondrial membrane potential (ΔΨm), cell viability, ROS production, apoptosis, and autophagy in MCF-7 and T47D (only the last four determinations) breast cancer cells treated with CDDP or TAM, in combination or without a UCP2 knockdown (siRNA or genipin). Furthermore, survival curves were performed in order to check the impact of UCP2 expression in breast cancer patients. UCP2 inhibition and cytotoxic treatments produced a decrease in cell viability and clonogenicity, in addition to an increase in ΔΨm, ROS production, apoptosis, and autophagy. It is important to note that CDDP decreased UCP2 protein levels, so that the greatest effects produced by the UCP2 inhibition in combination with a cytotoxic treatment, with regard to treatment alone, were observed in TAM+UCP2siRNA-treated cells. Moreover, this UCP2 inhibition caused autophagic cell death, since apoptosis parameters barely increased after UCP2 knockdown. Finally, survival curves revealed that higher UCP2 expression corresponded with a poorer prognosis. In conclusion, UCP2 could be a therapeutic target in breast cancer, especially in those patients treated with tamoxifen.     

Akt inhibition promotes hexokinase 2 redistribution and glucose uptake in cancer cells

Hexokinase II (HK2), the enzyme that catalyzes the first committed step of glycolysis, is overexpressed in many cancers, as is the central signaling kinase Akt. Akt activity promotes HK2 association with the mitochondria, as well as glucose uptake by cancer cells. In HeLa cervical cancer cells, Akt inhibitor IV (Ai4) increased nuclear HK2 localization, while in MDA‐MB‐231 breast cancer cells, Ai4 merely induced cytoplasmic redistribution without increased nuclear accumulation. Small interfering RNA (siRNA) directed against Akt confirmed the effect in HeLa cells. Next, we treated the cells with clotrimazole (CTZ), which detaches HK2 from the mitochondria, or leptomycin B (LMB), which promotes HK2 nuclear accumulation, and determined the effect on HK2 subcellular distribution. In both cell lines, CTZ detached HK2 from the mitochondria, without substantially increasing nuclear HK2, while LMB increased nuclear HK2, without redistributing cytoplasmic HK2. Contrary to expectations, Akt inhibition promoted glucose uptake in both cell lines, suggesting that Akt inhibition may increase glucose uptake by detaching HK2 from the mitochondria. In both cell lines, CTZ and LMB increased glucose uptake. However, the results in the HeLa cells showed greater effects: CTZ increased glucose uptake to a similar degree to Ai4, while LMB was far more effective than either. These data suggest that both detachment of HK2 from the mitochondria and increased nuclear HK2 are important for Ai4‐induced increased glucose uptake. J. Cell. Physiol. 228: 1943–1948, 2013. © 2013 Wiley Periodicals, Inc.     

Insulin processing and dissociation--effect of temperature and lysosomotropic agents

Dissociation of intact and degraded insulin from hepatocytes in monolayer culture was examined under conditions in which processing of insulin was altered by either temperature or pharmacologic agents. Conditions which increased insulin degradation or processing decreased equilibrium insulin binding whereas those conditions which inhibited processing increased equilibrium binding. The effect of lysosomotropic agents on processing was markedly temperature dependent. Not only was processing increased at higher temperatures (37 degrees C, but the effect of lysosomal inhibitors (chloroquine and methylamine) on insulin processing was abolished at this temperature. The temperature dependency of this effect may explain discrepancies between laboratories on the effect of these inhibitors in hepatocytes.     

Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy

Chloroquine (CQ) is a 4-aminoquinoline drug used for the treatment of diverse diseases. It inhibits lysosomal acidification and therefore prevents autophagy by blocking autophagosome fusion and degradation. In cancer treatment, CQ is often used in combination with chemotherapeutic drugs and radiation because it has been shown to enhance the efficacy of tumor cell killing. Since CQ and its derivatives are the only inhibitors of autophagy that are available for use in the clinic, multiple ongoing clinical trials are currently using CQ or hydroxychloroquine (HCQ) for this purpose, either alone, or in combination with other anticancer drugs. Here we show that in the mouse breast cancer cell lines, 67NR and 4T1, autophagy is induced by the DNA damaging agent cisplatin or by drugs that selectively target autophagy regulation, the PtdIns3K inhibitor LY294002, and the mTOR inhibitor rapamycin. In combination with these drugs, CQ sensitized to these treatments, though this effect was more evident with LY294002 and rapamycin treatment. Surprisingly, however, in these experiments CQ sensitization occurred independent of autophagy inhibition, since sensitization was not mimicked by Atg12, Beclin 1 knockdown or bafilomycin treatment, and occurred even in the absence of Atg12. We therefore propose that although CQ might be helpful in combination with cancer therapeutic drugs, its sensitizing effects can occur independently of autophagy inhibition. Consequently, this possibility should be considered in the ongoing clinical trials where CQ or HCQ are used in the treatment of cancer, and caution is warranted when CQ treatment is used in cytotoxic assays in autophagy research.     

Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy

Chloroquine (CQ) is a 4-aminoquinoline drug used for the treatment of diverse diseases. It inhibits lysosomal acidification and therefore prevents autophagy by blocking autophagosome fusion and degradation. In cancer treatment, CQ is often used in combination with chemotherapeutic drugs and radiation because it has been shown to enhance the efficacy of tumor cell killing. Since CQ and its derivatives are the only inhibitors of autophagy that are available for use in the clinic, multiple ongoing clinical trials are currently using CQ or hydroxychloroquine (HCQ) for this purpose, either alone, or in combination with other anticancer drugs. Here we show that in the mouse breast cancer cell lines, 67NR and 4T1, autophagy is induced by the DNA damaging agent cisplatin or by drugs that selectively target autophagy regulation, the PtdIns3K inhibitor LY294002, and the mTOR inhibitor rapamycin. In combination with these drugs, CQ sensitized to these treatments, though this effect was more evident with LY294002 and rapamycin treatment. Surprisingly, however, in these experiments CQ sensitization occurred independent of autophagy inhibition, since sensitization was not mimicked by Atg12, Beclin 1 knockdown or bafilomycin treatment, and occurred even in the absence of Atg12. We therefore propose that although CQ might be helpful in combination with cancer therapeutic drugs, its sensitizing effects can occur independently of autophagy inhibition. Consequently, this possibility should be considered in the ongoing clinical trials where CQ or HCQ are used in the treatment of cancer, and caution is warranted when CQ treatment is used in cytotoxic assays in autophagy research.     

ATM-mediated PTEN phosphorylation promotes PTEN nuclear translocation and autophagy in response to DNA-damaging agents in cancer cells

PTEN (phosphatase and tensin homolog), a tumor suppressor frequently mutated in human cancer, has various cytoplasmic and nuclear functions. PTEN translocates to the nucleus from the cytoplasm in response to oxidative stress. However, the mechanism and function of the translocation are not completely understood. In this study, topotecan (TPT), a topoisomerase I inhibitor, and cisplatin (CDDP) were employed to induce DNA damage. The results indicate that TPT or CDDP activates ATM (ATM serine/threonine kinase), which phosphorylates PTEN at serine 113 and further regulates PTEN nuclear translocation in A549 and HeLa cells. After nuclear translocation, PTEN induces autophagy, in association with the activation of the p-JUN-SESN2/AMPK pathway, in response to TPT. These results identify PTEN phosphorylation by ATM as essential for PTEN nuclear translocation and the subsequent induction of autophagy in response to DNA damage.     

AZD9291 promotes autophagy and inhibits PI3K/Akt pathway in NSCLC cancer cells

AZD9291, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), is highly selective against EGFR T790M-mutant non–small cell lung cancer (NSCLC). On investigating the growth inhibitory effects of AZD9291 on NSCLC and the underlying mechanism, we found that AZD9291 can trigger autophagy-mediated cell death in both A549 and H1975 cells by increasing the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3) and decreasing the expression of p62. In the presence of the autophagy inhibitor chloroquine, the AZD9291-induced increase in LC3 level was further augmented. AZD9291 decreased the levels of phosphoinositide-3 kinase (PI3K), protein kinase B (Akt), and phosphorylated Akt. AZD9291-induced cell death was enhanced by Akt knockdown, and the levels of both EGFR and phosphorylated EGFR were decreased by AZD9291. AZD9291 was also found to significantly suppress the tumor growth in H1975 xenograft nude mice. Thus, AZD9291 was found to induce autophagy, decrease in EGFR levels, and show a strong inhibitory effect on NSCLC both in vitro and in vivo. Furthermore, the PI3K/Akt signaling pathway was found to play a critical role in AZD9291-induced cell death.     

Akt inhibition attenuates rasfonin-induced autophagy and apoptosis through the glycolytic pathway in renal cancer cells

Rasfonin is a fungal secondary metabolite with demonstrated antitumor effects. However, the underlying mechanism of the regulatory role in autophagy initiated by rasfonin is largely unknown. Moreover, the function of Akt to positively mediate the induced autophagy remains elusive. In the present study, we observed that rasfonin induced autophagy concomitant with the upregulation of Akt phosphorylation. Both the inhibition of Akt by small molecule inhibitors and genetic modification partially reduced rasfonin-dependent autophagic flux and PARP-1 cleavage. The overexpression of myrAkts (constant active form) promoted rasfonin-induced apoptosis and autophagy in a cell type- and Akt isoform-specific manner. Using quantitative PCR and immunoblotting, we observed that rasfonin increased the expression of glycolytic gene PFKFB3, and this increased expression can be suppressed in the presence of Akt inhibitor. The inhibition of PFKFB3 suppressed rasfonin-activated autophagy with enhanced PARP-1 cleavage. In the case of glucose uptake was disrupted, which mean the glycolytic pathway was fully blocked, the rasfonin-induced autophagy and PARP-1 cleavage were downregulated. Collectively, these results demonstrated that Akt positively regulated rasfonin-enhanced autophagy and caspase-dependent apoptosis primarily through affecting the glycolytic pathway.On the basis of distinct cell morphology, three major types of cell death have been described: apoptosis, autophagic cell death, and programmed necrosis.^{1, 2, 3} Accumulating evidence suggests the existence of several molecular connections among apoptosis, necrosis, and autophagy.^{3, 4} Macroautophagy (hereafter called autophagy), an evolutionarily conserved catabolic and intracellular membrane trafficking process, is involved in the delivery of cytoplasmic contents and organelles to lysosomes for degradation.⁵ In general, the mammalian target of rapamycin (mTOR) is a negative regulator of autophagy.^{6, 7, 8} As a member of the PI3K-related kinase family, mTOR has been detected in two distinct complexes, mTORC1 and mTORC2, which regulate many aspects of cellular functions.^{9, 10} mTORC2 activates Akt (protein Kinase B), while PI3K/Akt primarily activates mTORC1.¹¹ Once activated by Akt, mTORC1 elicits a negative feedback loop to inhibit the activity of Akt. mTORC1 phosphorylates two main substrates, ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1).¹²As an upstream regulator of mTOR, Akt is usually considered to be an autophagy suppressor, and the Akt inhibitor can be used as an autophagy inducer.¹³ Three highly homologous Akt isoforms (Akt1, Akt2, and Akt3), encoded by separate genes, are expressed in mammalian cells.¹⁴ Akt is perhaps the most frequently activated oncoprotein in human cancers, and its activation contributes to the genesis of cancer through the inhibition of apoptosis and induction of proliferation.¹⁵ However, a recent study suggested that Akt isoforms showed opposite functions in tumor initiation and growth.¹⁶ Moreover, the overexpression of constitutively active Akt isoforms inhibits the proliferation of MDA-MB-231 cells.¹⁷Warburg effect, a hallmark of cancer, was first discovered by Otto Warburg.^{18, 19} In this process, cancer cells shift to glycolytic energy dependence with or without molecular oxygen. Akt activation increased the total cellular ATP content, whereas Akt deprivation reduced intracellular ATP levels.²⁰ Growing evidence indicates that Akt has a major role in the coordinated regulation of both glycolytic and oxidative metabolism.²¹ Akt augments the glycolytic flux through several mechanisms, such as increasing the expression of glucose transporters, enhancing the coupling between oxidative phosphorylation and glycolysis, promoting the accumulation of HIF1α and HK2, and activating phosphofructokinase-2 (PFK-2).¹⁸ Here, ACHN cell line was selected as the experiment material, as renal cell carcinoma (RCC) is a model for the role of Warburg effect leading to malignancy.²²In mammals, several PFK-2/FBPase-2 isoenzymes are encoded by four different genes.²³ These isoenzymes control glycolysis via the maintenance of the cellular levels of fructose-2,6-bisphosphate (F26BP), a major allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a critical rate-limiting enzyme of glycolysis. A previous study reported that the knockdown of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3), a member of the PFK-2 family, suppressed autophagy.²⁴ Given the intimate association between Akt and glycolysis, we speculated that Akt might regulate autophagy via the glycolytic pathway.Rasfonin is a natural product isolated from the fermented mycelium of Talaromyces sp. 3656-A1, named according to the biological activity of this compound against the small G-protein Ras. Recently, rasfonin was shown to induce the death of ras-mutated pancreatic tumor (Panc-1) cells.²⁵ In the present study, we demonstrated that rasfonin induces autophagy, which contributes to apoptosis. Moreover, this compound activates autophagy concomitant with the upregulation of Akt phosphorylation. API-2 and SC66, two inhibitors of Akt, attenuated both autophagy and caspase-dependent apoptosis concomitantly with an alteration in PFKFB3 expression. Although PFK-15 and 3-PO, two inhibitors of PFKFB3,²⁶ decreased the magnitude of autophagy and increased the rasfonin-induced cleavage of PARP-1, the inhibition of glucose uptake by 2-Deoxyglucose (2-DG) or glucose-free medium reduces both rasfonin-dependent autophagy and apoptosis.     

Akt and RhoA inhibition promotes anoikis of aggregated B16F10 melanoma cells

In the highly metastatic B16F10 melanoma cell line, activation of the signalling molecules that promote cell proliferation and survival on conventional adhesive culture dishes may also be responsible for the growth and resistance to anoikis of aggregates on a non-adhesive substratum. We have examined the influence of bacterial ADP-ribosyltransferases C3-like exoenzymes, which selectively modify RhoA, B and C proteins and inhibit signal pathways controlled by them. RNA interference [siRNA (small interfering RNA) Akt (also known as protein kinase B)] and a PI3K (phosphoinositide 3-kinase) inhibitor were used to analyse the changes caused by inhibiting the PI3K/Akt pathway. Inhibiting the activation of RhoA, B, C and Akt expression resulted in a decrease of the number of cells cultured in aggregates, and caspase 3 activation. RhoA activation and RhoB and RhoC expression were controlled by Akt, but not RhoA expression. Inhibiting Akt and RhoA reduced the expression of α5 integrin, and inactivated FAK (focal adhesion kinase) in B16F10 cells cultured as aggregates. Thus, inhibiting Rho subfamily proteins and Akt expression inactivates the FAK pathway and induces anoikis in anoikis-resistant cells. The activation of RhoA in melanoma cells can depend on PI3K/Akt activation, suggesting that PI3K/Akt is a suitable target for new therapeutic approaches.     

PTBP1 promotes the growth of breast cancer cells through the PTEN/Akt pathway and autophagy

Invasion and migration is the hallmark of malignant tumors as well as the major cause for breast cancer death. The polypyrimidine tract binding, PTB, protein serves as an important model for understanding how RNA binding proteins affect proliferation and invasion and how changes in the expression of these proteins can control complex programs of tumorigenesis. We have investigated some roles of polypyrimidine tract binding protein 1 (PTBP1) in human breast cancer. We found that PTBP1 was upregulated in breast cancer tissues compared with normal tissues and the same result was confirmed in breast cancer cell lines. Knockdown of PTBP1 substantially inhibited tumor cell growth, migration, and invasion. These results suggest that PTBP1 is associated with breast tumorigenesis and appears to be required for tumor cell growth and maintenance of metastasis. We further analyzed the relationship between PTBP1 and clinicopathological parameters and found that PTBP1 was correlated with her‐2 expression, lymph node metastasis, and pathological stage. This will be a novel target for her‐2(⁺) breast cancer. PTBP1 exerts these effects, in part, by regulating the phosphatase and tensin homolog‐phosphatidylinositol‐4,5‐bisphosphate 3‐kinase/protein kinase B (PTEN‐PI3K/Akt) pathway and autophagy, and consequently alters cell growth and contributes to the invasion and metastasis.     

Identification of ML-9 as a lysosomotropic agent targeting autophagy and cell death

The growing number of studies suggested that inhibition of autophagy enhances the efficacy of Akt kinase inhibitors in cancer therapy. Here, we provide evidence that ML-9, a widely used inhibitor of Akt kinase, myosin light-chain kinase (MLCK) and stromal interaction molecule 1 (STIM1), represents the ‘two-in-one'' compound that stimulates autophagosome formation (by downregulating Akt/mammalian target of rapamycin (mTOR) pathway) and inhibits their degradation (by acting like a lysosomotropic agent and increasing lysosomal pH). We show that ML-9 as a monotherapy effectively induces prostate cancer cell death associated with the accumulation of autophagic vacuoles. Further, ML-9 enhances the anticancer activity of docetaxel, suggesting its potential application as an adjuvant to existing anticancer chemotherapy. Altogether, our results revealed the complex effect of ML-9 on autophagy and indentified ML-9 as an attractive tool for targeting autophagy in cancer therapy through dual inhibition of both the Akt pathway and the autophagy.     

Guttiferone K induces autophagy and sensitizes cancer cells to nutrient stress-induced cell death

BackgroundMedicinal plants have long been an excellent source of pharmaceutical agents. Autophagy, a catabolic degradation process through lysosomes, plays an important role in tumorigenesis and cancer therapy.PurposeThrough a screen designed to identify autophagic regulators from a library of natural compounds, we found that Guttiferone K (GUTK) can activate autophagy in several cancer cell lines. The objective of this study is to investigate the mechanism by which GUTK sensitizes cancer cells to cell death in nutrient starvation condition.MethodsCell death analysis was performed by propidium iodide staining with flow cytometry or Annexin V-FITC/PI staining assay. DCFH-DA staining was used for intracellular ROS measurement. Protein levels were analyzed by western blot analysis. Cell viability was measured by MTT assay.ResultsExposure to GUTK was observed to markedly induce GFP-LC3 puncta formation and activate the accumulation of LC3-II and the degradation of p62 in HeLa cells, suggesting that GUTK is an autophagy inducer. Importantly, hydroxychloroquine, an autophagy inhibitor, was found to significantly prevent GUTK-induced cell death in nutrient starvation conditions, suggesting that the cell death observed is largely dependent on autophagy. We further provide evidence that GUTK inhibits Akt phosphorylation, thereby inhibiting the mTOR pathway in cancer cells during nutrient starvation. In addition, GUTK causes the accumulation of reactive oxygen species (ROS) and the phosphorylation of JNK in EBSS, which may mediate both autophagy and apoptosis.ConclusionThese data indicate that GUTK sensitizes cancer cells to nutrient stress-induced cell death though Akt/mTOR dependent autophagy pathway.     

Amino acid starvation sensitizes cancer cells to proteasome inhibition

We explored the crosstalk between protein degradation and synthesis in cancer cells. The tumorigenic cell line, MCF7, showed enhanced proteasome activity compared to the nontumorigenic line, MCF10A. Although there was no difference in the sensitivity of MCF7 and MCF10A cells to proteasome inhibition in complete growth medium, combining proteasome inhibition with amino acid deprivation led to reduced protein synthesis and survival of MCF7 cells, with a lesser effect on MCF10A cells. Additional cancer cell lines (including CAG and A431) could be strongly sensitized to proteasome inhibition by concomitant amino acid deprivation, whereas others were completely resistant to proteasome inhibition. We hypothesize that protein catabolism contributes to the pool of free amino acids available for protein synthesis, leading to a crucial role of the proteasome in cell survival during amino acid depletion, in some tumor cell lines.     

Compromising the constitutive p16INK4a expression sensitizes human neuroblastoma cells to Hsp90 inhibition and promotes premature senescence

The Hsp90 chaperone has become the attractive pharmacological target to inhibit tumor cell proliferation. However, tumor cells can evolve with mechanisms to overcome Hsp90 inhibition. Using human neuroblastoma, we have investigated one such limitation. Here, we demonstrate that neuroblastoma cells overcome the interference of tumor suppressor p16^INK4a in cell proliferation, which is due to its latent interaction with CDK4 and CDK6. Cells also displayed impedance to the pharmacological inhibition of cancer chaperone Hsp90 inhibition with respect to induced cytotoxicity. However, the p16^INK4a knockdown has triggered the activation of cyclin-CDK6 axis and enhanced the cell proliferation. These cells are eventually sensitized to Hsp90 inhibition by activating the DNA damage response mediated through p53-p21^WAF-1 axis and G1 cell cycle exit. While both CDK4 and CDK6 have exhibited low affinity to p16^INK4a, CDK6 has exhibited high affinity to Hsp90. Destabilizing the CDK6 interaction with Hsp90 has prolonged G2/M cell cycle arrest fostering to premature cellular senescence. The senescence driven cells exhibited compromised metastatic potential both in vitro as well as in mice xenografts. Our study unravels that cancer cells can be adapted to the constitutive expression of tumor suppressors to overcome therapeutic interventions. Our findings display potential implication of Hsp90 inhibitors to overcome such adaptations.     

LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

Lysosome-associated membrane protein type 2A (LAMP2A) is a key protein in the chaperone-mediated autophagy (CMA) pathway. LAMP2A helps in lysosomal uptake of modified and oxidatively damaged proteins directly into the lumen of lysosomes for degradation and protein turnover. Elevated expression of LAMP2A was observed in breast tumor tissues of all patients under investigation, suggesting a survival mechanism via CMA and LAMP2A. Reduced expression of the CMA substrates, GAPDH and PKM, was observed in most of the breast tumor tissues when compared with the normal adjacent tissues. Reactive oxygen species (ROS) mediated oxidative stress damages regulatory cellular components such as DNA, proteins and/or lipids. Protein carbonyl content (PCC) is widely used as a measure of total protein oxidation in cells. Ectopic expression of LAMP2A reduces PCC and thereby promotes cell survival during oxidative stress. Furthermore, inhibition of LAMP2A stimulates accumulation of GAPDH, AKT1 phosphorylation, generation of ROS, and induction of cellular apoptosis in breast cancer cells. Doxorubicin, which is a chemotherapeutic drug, often becomes ineffective against tumor cells with time due to chemotherapeutic resistance. Breast cancer cells deficient of LAMP2A demonstrate increased sensitivity to the drug. Thus, inhibiting CMA activity in breast tumor cells can be exploited as a potential therapeutic application in the treatment of breast cancer.     

LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

Lysosome-associated membrane protein type 2A (LAMP2A) is a key protein in the chaperone-mediated autophagy (CMA) pathway. LAMP2A helps in lysosomal uptake of modified and oxidatively damaged proteins directly into the lumen of lysosomes for degradation and protein turnover. Elevated expression of LAMP2A was observed in breast tumor tissues of all patients under investigation, suggesting a survival mechanism via CMA and LAMP2A. Reduced expression of the CMA substrates, GAPDH and PKM, was observed in most of the breast tumor tissues when compared with the normal adjacent tissues. Reactive oxygen species (ROS) mediated oxidative stress damages regulatory cellular components such as DNA, proteins and/or lipids. Protein carbonyl content (PCC) is widely used as a measure of total protein oxidation in cells. Ectopic expression of LAMP2A reduces PCC and thereby promotes cell survival during oxidative stress. Furthermore, inhibition of LAMP2A stimulates accumulation of GAPDH, AKT1 phosphorylation, generation of ROS, and induction of cellular apoptosis in breast cancer cells. Doxorubicin, which is a chemotherapeutic drug, often becomes ineffective against tumor cells with time due to chemotherapeutic resistance. Breast cancer cells deficient of LAMP2A demonstrate increased sensitivity to the drug. Thus, inhibiting CMA activity in breast tumor cells can be exploited as a potential therapeutic application in the treatment of breast cancer.     

The rational modulation of autophagy sensitizes colorectal cancer cells to 5-fluouracil and oxaliplatin

Colorectal cancer (CRC) is the third most common and deadliest cancer globally. Regimens using 5-fluorouracil (5FU) and Oxaliplatin (OXA) are the first-line treatment for CRC, but tumor recurrence is frequent. It is plausible to hypothesize that differential cellular responses are triggered after treatments depending on the genetic background of CRC cells and that the rational modulation of cell tolerance mechanisms like autophagy may reduce the regrowth of CRC cells. This study proposes investigating the cellular mechanisms triggered by CRC cells exposed to 5FU and OXA using a preclinical experimental design mimicking one cycle of the clinical regimen (i.e., 48 h of treatment repeated every 2 weeks). To test this, we treated CRC human cell lines HCT116 and HT29 with the 5FU and OXA, combined or not, for 48 h, followed by analysis for two additional weeks. Compared to single-drug treatments, the co-treatment reduced tumor cell regrowth, clonogenicity and stemness, phenotypes associated with tumor aggressiveness and poor prognosis in clinics. This effect was exerted by the induction of apoptosis and senescence only in the co-treatment. However, a week after treatment, cells that tolerated the treatment had high levels of autophagy features and restored the proliferative phenotype, resembling tumor recurrence. The pharmacologic suppression of early autophagy during its peak of occurrence, but not concomitant with chemotherapeutics, strongly reduced cell regrowth. Overall, our experimental model provides new insights into the cellular mechanisms that underlie the response and tolerance of CRC cells to 5FU and OXA, suggesting optimized, time-specific autophagy inhibition as a new avenue for improving the efficacy of current treatments.