RESUMO
BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive neoplasm and often acquires chemoresistance by increasing stemness in tumour tissue, thereby generating cancer stem cells (CSCs). CSCs escape treatment by deploying metabolic pathways to trigger dormancy or proliferation, also gaining the ability to exit and re-enter the cell cycle to hide their cellular identity. METHODS: We employed various cellular and biochemical assays to identify the role of the glycolytic enzyme PFKFB3, by knocking it down and pharmacologically inhibiting it with PFK158, to determine its anticancer effects in vitro and in vivo by targeting the CSC population in MPM. RESULTS: Here, we have identified PFKFB3 as a strategic player to target the CSC population in MPM and demonstrated that both pharmacologic (PFK158) and genetic inhibition of PFKFB3 destroy the FAK-Stat3-SOX2 nexus resulting in a decline in conspicuous stem cell markers viz. ALDH, CD133, CD44, SOX2. Inhibition of PFKFB3 accumulates p21 and p27 in the nucleus by decreasing SKP2. Lastly, PFK158 diminishes tumour-initiating cells (TICs) mediated MPM xenograft in vivo. CONCLUSIONS: This study confers a comprehensive and mechanistic function of PFKFB3 in CSC maintenance that may foster exceptional opportunities for targeted small molecule blockade of the TICs in MPM.
Assuntos
Mesotelioma Maligno , Quinolinas , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Células-Tronco Neoplásicas/patologia , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Piridinas/farmacologia , Quinolinas/farmacologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/farmacologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
PFKFB3 (6-phosphofructo-2-kinase) is the rate-limiting enzyme of glycolysis and is overexpressed in several human cancers that are associated with poor prognosis. High PFKFB3 expression in cancer stem cells promotes glycolysis and survival in the tumor microenvironment. Inhibition of PFKFB3 by the glycolytic inhibitor PFK158 and by shRNA stable knockdown in small cell lung carcinoma (SCLC) cell lines inhibited glycolysis, proliferation, spheroid formation, and the expression of cancer stem cell markers CD133, Aldh1, CD44, Sox2, and ABCG2. These factors are also associated with chemotherapy resistance. We found that PFK158 treatment and PFKFB3 knockdown enhanced the ABCG2-interacting drugs doxorubicin, etoposide, and 5-fluorouracil in reducing cell viability under conditions of enriched cancer stem cells (CSC). Additionally, PFKFB3 inhibition attenuated the invasion/migration of SCLC cells by downregulating YAP/TAZ signaling while increasing pLATS1 via activation of pMST1 and NF2 and by reducing the mesenchymal protein expression. PFKFB3 knockdown and PFK158 treatment in a H1048 SCLC cancer stem cell-enriched mouse xenograft model showed significant reduction in tumor growth and weight with reduced expression of cancer stem cell markers, ABCG2, and YAP/TAZ. Our findings identify that PFKFB3 is a novel target to regulate cancer stem cells and its associated therapeutic resistance markers YAP/TAZ and ABCG2 in SCLC models.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Animais , Linhagem Celular Tumoral , Proliferação de Células , Glicólise , Via de Sinalização Hippo , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Fosfofrutoquinase-2/metabolismo , Piridinas , Quinolinas , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Microambiente TumoralRESUMO
Dissemination of ovarian cancer cells can lead to inoperable metastatic lesions in the bowel and omentum that cause patient death. Here we show that LRRC15, a type-I 15-leucine-rich repeat-containing membrane protein, highly overexpressed in ovarian cancer bowel metastases compared with matched primary tumors and acts as a potent promoter of omental metastasis. Complementary models of ovarian cancer demonstrated that LRRC15 expression leads to inhibition of anoikis-induced cell death and promotes adhesion and invasion through matrices that mimic omentum. Mechanistically, LRRC15 interacted with ß1-integrin to stimulate activation of focal adhesion kinase (FAK) signaling. As a therapeutic proof of concept, targeting LRRC15 with the specific antibody-drug conjugate ABBV-085 in both early and late metastatic ovarian cancer cell line xenograft models prevented metastatic dissemination, and these results were corroborated in metastatic patient-derived ovarian cancer xenograft models. Furthermore, treatment of 3D-spheroid cultures of LRRC15-positive patient-derived ascites with ABBV-085 reduced cell viability. Overall, these data uncover a role for LRRC15 in promoting ovarian cancer metastasis and suggest a novel and promising therapy to target ovarian cancer metastases.Significance: This study identifies that LRRC15 activates ß1-integrin/FAK signaling to promote ovarian cancer metastasis and shows that the LRRC15-targeted antibody-drug conjugate ABBV-085 suppresses ovarian cancer metastasis in preclinical models.
Assuntos
Imunoconjugados , Neoplasias Ovarianas , Carcinoma Epitelial do Ovário , Adesão Celular , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/farmacologia , Integrinas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologiaRESUMO
Mesothelioma is a rare cancer with disproportionately higher death rates for shipping and mining populations. These patients have few treatment options, which can be partially attributed to limited chemotherapy responses for tumors. We initially hypothesized that quinacrine could be combined with cisplatin or pemetrexed to synergistically eliminate mesothelioma cells. The combination with cisplatin resulted in synergistic cell death and the combination with pemetrexed was not synergistic, although novel artificially-generated pemetrexed-resistant cells were more sensitive to quinacrine. Unexpectedly, we discovered cells with NF2 mutations were very sensitive to quinacrine. This change of quinacrine sensitivity was confirmed by NF2 ectopic expression and knockdown in NF2 mutant and wildtype cell lines, respectively. There are few common mutations in mesothelioma and inactivating NF2 mutations are present in up to 60% of these tumors. We found quinacrine alters the expression of over 3000 genes in NF2-mutated cells that were significantly different than quinacrine-induced changes in NF2 wildtype cells. Changes to NF2/hippo pathway biomarkers were validated at the mRNA and protein levels. Additionally, quinacrine induces a G1 phase cell cycle arrest in NF2-mutated cells versus the S phase arrest in NF2-wildtype cells. This study suggests quinacrine may have repurposing potential for a large subset of mesothelioma patients.
RESUMO
A considerable subset of gynecologic cancer patients experience disease recurrence or acquired resistance, which contributes to high mortality rates in ovarian cancer (OC). Our prior studies showed that quinacrine (QC), an antimalarial drug, enhanced chemotherapy sensitivity in treatment-refractory OC cells, including artificially generated chemoresistant and high-grade serous OC cells. In this study, we investigated QC-induced transcriptomic changes to uncover its cytotoxic mechanisms of action. Isogenic pairs of OC cells generated to be chemoresistant and their chemosensitive counterparts were treated with QC followed by RNA-seq analysis. Validation of selected expression results and database comparison analyses indicated the ribosomal biogenesis (RBG) pathway is inhibited by QC. RBG is commonly upregulated in cancer cells and is emerging as a drug target. We found that QC attenuates the in vitro and in vivo expression of nucleostemin (NS/GNL3), a nucleolar RBG and DNA repair protein, and the RPA194 catalytic subunit of Pol I that results in RBG inhibition and nucleolar stress. QC promotes the redistribution of fibrillarin in the form of extranuclear foci and nucleolar caps, an indicator of nucleolar stress conditions. In addition, we found that QC-induced downregulation of NS disrupted homologous recombination repair both by reducing NS protein levels and PARylation resulting in reduced RAD51 recruitment to DNA damage. Our data suggest that QC inhibits RBG and this inhibition promotes DNA damage by directly downregulating the NS-RAD51 interaction. Additionally, QC showed strong synergy with PARP inhibitors in OC cells. Overall, we found that QC downregulates the RBG pathway, induces nucleolar stress, supports the increase of DNA damage, and sensitizes cells to PARP inhibition, which supports new therapeutic stratagems for treatment-refractory OC. Our work offers support for targeting RBG in OC and determines NS to be a novel target for QC.
RESUMO
The advanced or recurrent endometrial cancer (EC) has a poor prognosis because of chemoresistance. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a glycolytic enzyme, is overexpressed in a variety of human cancers and plays important roles in promoting tumor cell growth. Here, we showed that high expression of PFKFB3 in EC cell lines is associated with chemoresistance. Pharmacological inhibition of PFKFB3 with PFK158 and or genetic downregulation of PFKFB3 dramatically suppressed cell proliferation and enhanced the sensitivity of EC cells to carboplatin (CBPt) and cisplatin (Cis). Moreover, PFKFB3 inhibition resulted in reduced glucose uptake, ATP production, and lactate release. Notably, we found that PFK158 with CBPt or Cis exerted strong synergistic antitumor activity in chemoresistant EC cell lines, HEC-1B and ARK-2 cells. We also found that the combination of PFK158 and CBPt/Cis induced apoptosis- and autophagy-mediated cell death through inhibition of the Akt/mTOR signaling pathway. Mechanistically, we found that PFK158 downregulated the CBPt/Cis-induced upregulation of RAD51 expression and enhanced CBPt/Cis-induced DNA damage as demonstrated by an increase in γ-H2AX levels in HEC-1B and ARK-2 cells, potentially revealing a means to enhance PFK158-induced chemosensitivity. More importantly, PFK158 treatment, either as monotherapy or in combination with CBPt, led to a marked reduction in tumor growth in two chemoresistant EC mouse xenograft models. These data suggest that PFKFB3 inhibition alone or in combination with standard chemotherapy may be used as a novel therapeutic strategy for improved therapeutic efficacy and outcomes of advanced and recurrent EC patients.
Assuntos
Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias do Endométrio/tratamento farmacológico , Fosfofrutoquinase-2/genética , Apoptose/efeitos dos fármacos , Carboplatina/farmacologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Neoplasias do Endométrio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fosfofrutoquinase-2/antagonistas & inibidores , Piridinas/farmacologia , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The sulfated glycolipid PG545 shows promising antitumor activity in various cancers. This study was conducted to explore the effects and the mechanism of PG545 action in endometrial cancer (EC). PG545 exhibited strong synergy as assessed by the Chou-Talalay-Method in vitro when combined with cisplatin, or paclitaxel in both type I (Hec1B) and type II (ARK2) EC cell lines. While PG545 showed antitumor activity as monotherapy, a combination of PG545 with paclitaxel and cisplatin was highly effective in reducing the tumor burden and significantly prolonged survival of both Hec1B and ARK2 xenograft bearing mice. Mechanistically, PG545 elicits ER stress as an early response with resultant induction of autophagy. Our data demonstrated an increase in pERK, Bip/Grp78, IRE1α, Calnexin and CHOP/GADD153 within 6-24 hrs of PG545 treatment in EC cells. In parallel, PG545 also blocked FGF2 and HB-EGF mediated signaling in EC cells. Moreover, melatonin-mediated ER stress inhibition reduced PG545-mediated autophagy and PG545 in combination with cisplatin further heightened this stress response. Collectively these data indicate that PG545 exhibits strong synergistic effects with chemotherapeutics in vitro and showed promising antitumor activity in vivo. Our preclinical data indicates that in future studies PG545 can be a useful adjunct to chemotherapy in endometrial cancer.
Assuntos
Antineoplásicos/administração & dosagem , Autofagia/efeitos dos fármacos , Neoplasias do Endométrio/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glicolipídeos/administração & dosagem , Saponinas/administração & dosagem , Animais , Autofagia/fisiologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Neoplasias do Endométrio/patologia , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/fisiologia , Feminino , Humanos , Camundongos , Camundongos Nus , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The metabolic signatures of cancer cells are often associated with elevated glycolysis. Pharmacological (PFK158 treatment) and genetic inhibition of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a critical control point in the glycolytic pathway, decreases glucose uptake, ATP production, and lactate dehydrogenase activity and arrests malignant pleural mesothelioma (MPM) cells in the G0/G1 phase to induce cell death. To overcome this nutrient stress, inhibition of PFKFB3 activity led to an escalation in endoplasmic reticulum (ER) activity and aggravated ER stress mostly by upregulating BiP and GADD153 expression and activation of the endocytic Rac1-Rab5-Rab7 pathway resulting in a unique form of cell death called "methuosis" in both the sarcomatoid (H28) and epithelioid (EMMeso) cells. Transmission electron microscopy (TEM) analysis showed the formation of nascent macropinocytotic vesicles, which rapidly coalesced to form large vacuoles with compromised lysosomal function. Both immunofluorescence microscopy and co-immunoprecipitation analyses revealed that upon PFKFB3 inhibition, two crucial biomolecules of each pathway, Rac1 and Calnexin interact with each other. Finally, PFK158 alone and in combination with carboplatin-inhibited tumorigenesis of EMMeso xenografts in vivo. Since most cancer cells exhibit an increased glycolytic rate, these results provide evidence for PFK158, in combination with standard chemotherapy, may have a potential in the treatment of MPM.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Neoplasias Pulmonares/genética , Mesotelioma/genética , Fosfofrutoquinase-2/antagonistas & inibidores , Piridinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Glicólise , Xenoenxertos , Humanos , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Mesotelioma Maligno , Camundongos , Camundongos Nus , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Pinocitose , Neoplasias Pleurais/genética , Neoplasias Pleurais/metabolismoRESUMO
Metabolic alterations are increasingly recognized as important novel anti-cancer targets. Among several regulators of metabolic alterations, fructose 2,6 bisphosphate (F2,6BP) is a critical glycolytic regulator. Inhibition of the active form of PFKFB3ser461 using a novel inhibitor, PFK158 resulted in reduced glucose uptake, ATP production, lactate release as well as induction of apoptosis in gynecologic cancer cells. Moreover, we found that PFK158 synergizes with carboplatin (CBPt) and paclitaxel (PTX) in the chemoresistant cell lines, C13 and HeyA8MDR but not in their chemosensitive counterparts, OV2008 and HeyA8, respectively. We determined that PFK158-induced autophagic flux leads to lipophagy resulting in the downregulation of cPLA2, a lipid droplet (LD) associated protein. Immunofluorescence and co-immunoprecipitation revealed colocalization of p62/SQSTM1 with cPLA2 in HeyA8MDR cells uncovering a novel pathway for the breakdown of LDs promoted by PFK158. Interestingly, treating the cells with the autophagic inhibitor bafilomycin A reversed the PFK158-mediated synergy and lipophagy in chemoresistant cells. Finally, in a highly metastatic PTX-resistant in vivo ovarian mouse model, a combination of PFK158 with CBPt significantly reduced tumor weight and ascites and reduced LDs in tumor tissue as seen by immunofluorescence and transmission electron microscopy compared to untreated mice. Since the majority of cancer patients will eventually recur and develop chemoresistance, our results suggest that PFK158 in combination with standard chemotherapy may have a direct clinical role in the treatment of recurrent cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Autofagia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Fosfofrutoquinase-2/antagonistas & inibidores , Piridinas/farmacologia , Quinolinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Carboplatina/administração & dosagem , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/uso terapêutico , Feminino , Glicólise/efeitos dos fármacos , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Camundongos Nus , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , Fosfofrutoquinase-2/metabolismo , Piridinas/uso terapêutico , Quinolinas/uso terapêuticoRESUMO
Endoplasmic reticulum (ER) stress results from protein unfolding/misfolding during cellular maturation, which requires a coordinated action of several chaperones and enzymes and Ca2+ signalling. ER-stress possibly has a positive effect on survival of pancreatic cancer cell. Therefore, detailed insights into this complex signaling network are urgently needed. Here, we systematically analyzed the impact of ER stress-mediated unfolded protein response (UPR) and Ca2+-signaling cross-talk for the survival of pancreatic adenocarcinoma (PDAC) cells. We observed enhanced ER activity and initiation of UPR signaling induced by a carbazole alkaloid (mahanine). This event triggers a time-dependent increase of intracellular Ca2+ leakage from ER and subsequently Ca2+ signaling induced by enhanced reactive oxygen species (ROS) produced by this pro-oxidant agent. In addition, we observed an altered glycosylation, in particular with regard to reduced linkage-specific sialic acids possibly due to decreased sialyltransferase activity. Changes in sialylation entailed enhanced expression of the ganglioside GD3 in the treated cells. GD3, an inducer of apoptosis, inhibited pancreatic xenograft tumor. Taken together, our study describes a molecular scenario how PDAC cells are driven into apoptosis by mahanine by UPR-driven ER stress-associated and ROS-mediated calcium signaling and possibly defective sialylation.