- Email: [email protected]
mTOR axis
abstracts
2005P
Aptamer-mediated exosomes detection for early breast cancer identification
C. Quintavalle1, C.L. Esposito2, F. Ingenito3, A. Affinito1, G. Roscigno3, I. Scognamiglio3, S. Nuzzo4, S. Catuogno2, R. Thomas5, G. Condorelli3 1 Percuros BV, Leiden, Netherlands, 2IEOS CNR, Naples, Italy, 3Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy, 4 IRCCS SDN SpA, Naples, Italy, 5Breast Unit, Clinica Mediterranea Spa, Naples, Italy Background: Increasing evidence indicates that the release of exosomes by tumor cells play a key role in tumor progression, drug resistance, immune surveillance escape, angiogenesis, tumor invasion, and metastasis. For this reason, cancer-derived exosomes are emerging as very interesting targets for early diagnosis and therapy in cancer, including breast cancer. Indeed, progresses in developing nucleic acids-based therapeutic compounds, including antisense DNAs, aptamers, short interfering/microRNAs, and short activator RNAs, have attracted great interest as emerging platforms for precise cancer treatment. Methods: We have recently developed a novel differential SELEX (Systematic Evolution of Ligands by Exponential enrichment) strategy by using exosomes purified from epithelial BC primary cells in the positive selection step and exosome-derived from primary normal epithelial breast cell lines in the negative selection step. Results: Three aptamers have been shown to be enriched in different families, and their ability to selectively bind BC cells-derived exosomes has been proven. Interestingly, two of them can selectively bind triple negative-derived exosomes, compared to more differentiated BC cells. To optimize the best aptamers, shortened version of aptamers (about 35mer) have been generated and their binding ability has been tested. Moreover, the aptamers showed no binding affinity for lung cancer and glioblastomaderived exosomes. We also tested by binding assay the ability of one of the identified aptamers, named ex-50sh, to selective recognize exosomes isolated from serum of breast cancer patients. Proteomic analysis of the putative target is in progress. Immunofluorescent experiments showed that the short aptamers can also block the uptake of MDA-231-derived exosomes on MDA-231 cell lines. Moreover, ex-50sh is able to block the EMT transformation induced by MDA-231 exosomes in low malignant MCF7. Conclusions: Altogether, the selected aptamers will provide new insights in the molecular characterization of breast cancer exosomes and, most importantly, innovative tools for early breast cancer diagnosis. Legal entity responsible for the study: Gerolama Condorelli. Funding: University of Naples Federico II. Disclosure: All authors have declared no conflicts of interest.
2006P
MicroRNA-181c promotes tamoxifen resistance in breast cancer cells via upregulation Akt/mTOR axis
A.M. Scherbakov1, Y. Shchegolev2, D. Sorokin2, A. Shunaev2, O.E. Andreeva2, M. Krasil’nikov2 1 Laboratory of Oncoproteomics, N.N. Blokhin National Medical Research Center of Oncology, Moscow, Russian Federation, 2Department of Experimental Tumor Biology, N.N. Blokhin National Medical Research Center of Oncology, Moscow, Russian Federation
based on our previous data, which demonstrated the effect of exosome-mediated transferring of hormonal resistance in in vitro cultured MCF-7 breast cancer cells. Methods: Estrogen-dependent breast cancer cells MCF-7 and the tamoxifen-resistant subline MCF-7 /T were used as an experimental model. The analysis of exosomal microRNAs was performed by HiSeq2500 and at least 5 million reads per samples were obtained. MicroRNA was extracted from by PureLink RNA Micro Kit; library preparaR Small RNA Library Prep Set for IlluminaV R. tion was carried out with NEBNextV Transfection of the RNA oligonucleotides was performed using Metafectene PRO (Biontex) to result in the final RNA concentration of 50 nM. Results: A comparative analysis of exosomal miRNAs of MCF-7 and resistant MCF-7/ T cells was carried out. In total, 2588 miRNAs have been identified in the exosomes. Among them, mir-181 family, which is one of the negative regulators of estrogendependent growth, was identified as the group of miRs, hyperexpressed in the resistant exosomes. Following this, we analysed the role of mir-181c, one of the main members of miR-181 family, in the regulation of cell growth and hormonal response. Mir-181c transfection was found to induce the estrogen-independent growth and partial tamoxifen resistance of MCF-7 cells. The study of the signaling proteins showed that mir-181c transfection, in contrast to scrambled RNA transfection, caused the increase of the amount of Raptor, phosphorylated forms of mTOR and Akt that correlated with increased AP-1 transcriptional activity. Conclusions: We have demonstrated the involvement of miR-181c in the development of hormonal resistance of breast cancer cells that allows us to consider mir-181 as the perspective target of the treatment of hormone- independent cancers. The research was supported by the Russian Science Foundation (19-15-00245, miRNA analysis) and RFBR (#18-29-09017, tamoxifen resistance). Legal entity responsible for the study: The authors. Funding: Russian Science Foundation, project 19-15-00245. Disclosure: All authors have declared no conflicts of interest.
2007P
Spatio-temporal separation of tumor infiltrating CD81 T-cells and HER2/neu1 tumor cells in tumor-immune milieu of infiltrating ductal carcinoma of the breast
S. Sreedharan1, M. Biswas1, S. Thiyagarajan2, N.P. Basak2, A. Basu2, P.S. Ghosh3 Histopathology, Mitra RxDx Inv., Bangalore, India, 2Cancer Biology, Mitra RxDx INC., Bangalore, India, 3Surgical Oncology, IPGME&R & SSKM Hospital, Kolkata, India
1
Background: Designated tumor infiltrating lymphocytes (TIL) within tumor microenvironment (TME) comprised of both regulatory cells and effector cells, and their interaction with tumor cells account for the immune escape and immunosurveillance mechanisms respectively. The number and location of CD8þ tumor infiltrating lymphocytes (TILs) are the measures of immune response and carries prognostic significance and predictive potential in the era of immuno-oncology. The present study was an attempt to find the relation between the treatment response and the spatio-temporal separation of tumor infiltrating CD8þ T-cells and HER2/neuþ tumor cells in breast cancer TME. Methods: In this study, a total of 7 breast cancer patients were enrolled and surgically removed fresh tumor tissues were evaluated for the baseline HER2/neu and CD8 expression by immunohistochemistry (IHC) using dual stain (duplex IHC). The tumor tissues were also tested for the response to HER2 blocker in a novel humanized ex vivo platform that preserves tumour microenvironment using tumour explants, maintained in defined tumour grade-matched matrix support and autologous patient serum. Results: Tumors responded to HER2 blockade had a majority of the CD8þ T cells (6080%) concentrated around the HER2/neuþ tumor area (within 50-100mm). While, the non-responding tumors had significantly lesser number of the CD8þT cells (20-40%) proximal to HER2/neuþ zone compared to the responders. The present study indeed delineated a preferential spatial relationship of tumor infiltrating CD8þ cells in HER2/ neuþ tumor region especially for the recognition of tumor antigen, cross presentation and tumor cell killing. Conclusions: Assessment of distribution pattern of effector cells surrounding tumor area had a correlation on the efficacy of HER2 inhibition. It warrants validation in the larger cohort of patient tumors to underpin its clinical relevance for possible immunotherapies like combination of check point inhibitors with HER2/neu blocker and others. Identification of other co-stimulatory and pathway molecules on the tumors not responded to HER2 blocker is being studied to understand the mechanism of resistance. Legal entity responsible for the study: The authors. Funding: Mitra RxDx Inc. Disclosure: All authors have declared no conflicts of interest.
Background: The main purpose of the work was to study the key exosomal factors involved in the development of hormonal resistance of breast cancer cells. The work is
v804 | Tumour Biology and Pathology
Volume 30 | Supplement 5 | October 2019
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz269.024/5577479 by guest on 24 October 2019
Methods: We isolated organoids from breast cancer patients and optimized the organoids growing conditions. By immunofluorescence assay we confirmed the receptor status, and the preservation of epithelial (E-cadherin) or the fibroblasts components included in the tissue microenvironment (TME) (alfa-SMA and FAP). Here we used organoids as mimic of tumor burden for drug penetration studies. In particular, we studied doxorubicin (DOX) penetration and biodistribution, by several means: immunofluorescence studies, caspase assay, and cell viability. Results: We demonstrated by IF studies, that miR-340, an oncosuppressor miR, was able to modulate DOX penetration. At the same time, miR-340 induced an increase in DOX sensitivity assessed by caspase-3 assay, PARP cleavage, as well as p38 phosphorylation. Breast cancer organoids have also been used to better understand the role of the TME in breast cancer response to therapy. We assessed drug sensitivity in the presence or absence of conditioned media obtained from cancer activated fibroblasts (CAFs). We found that organoids treated with conditioned media exhibit lower level of caspase 3 activation compared to the control upon palbociclib treatment, suggesting the importance role of TME in drug resistance. Conclusions: Thus, organoids can be considered as a new tool for studying breast cancer and developing personalized medicine approaches and to test possible use of RNA therapeutics. Legal entity responsible for the study: Gerolama Condorelli. Funding: AIRC. Disclosure: All authors have declared no conflicts of interest.
Annals of Oncology
2005P
Aptamer-mediated exosomes detection for early breast cancer identification
C. Quintavalle1, C.L. Esposito2, F. Ingenito3, A. Affinito1, G. Roscigno3, I. Scognamiglio3, S. Nuzzo4, S. Catuogno2, R. Thomas5, G. Condorelli3 1 Percuros BV, Leiden, Netherlands, 2IEOS CNR, Naples, Italy, 3Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy, 4 IRCCS SDN SpA, Naples, Italy, 5Breast Unit, Clinica Mediterranea Spa, Naples, Italy Background: Increasing evidence indicates that the release of exosomes by tumor cells play a key role in tumor progression, drug resistance, immune surveillance escape, angiogenesis, tumor invasion, and metastasis. For this reason, cancer-derived exosomes are emerging as very interesting targets for early diagnosis and therapy in cancer, including breast cancer. Indeed, progresses in developing nucleic acids-based therapeutic compounds, including antisense DNAs, aptamers, short interfering/microRNAs, and short activator RNAs, have attracted great interest as emerging platforms for precise cancer treatment. Methods: We have recently developed a novel differential SELEX (Systematic Evolution of Ligands by Exponential enrichment) strategy by using exosomes purified from epithelial BC primary cells in the positive selection step and exosome-derived from primary normal epithelial breast cell lines in the negative selection step. Results: Three aptamers have been shown to be enriched in different families, and their ability to selectively bind BC cells-derived exosomes has been proven. Interestingly, two of them can selectively bind triple negative-derived exosomes, compared to more differentiated BC cells. To optimize the best aptamers, shortened version of aptamers (about 35mer) have been generated and their binding ability has been tested. Moreover, the aptamers showed no binding affinity for lung cancer and glioblastomaderived exosomes. We also tested by binding assay the ability of one of the identified aptamers, named ex-50sh, to selective recognize exosomes isolated from serum of breast cancer patients. Proteomic analysis of the putative target is in progress. Immunofluorescent experiments showed that the short aptamers can also block the uptake of MDA-231-derived exosomes on MDA-231 cell lines. Moreover, ex-50sh is able to block the EMT transformation induced by MDA-231 exosomes in low malignant MCF7. Conclusions: Altogether, the selected aptamers will provide new insights in the molecular characterization of breast cancer exosomes and, most importantly, innovative tools for early breast cancer diagnosis. Legal entity responsible for the study: Gerolama Condorelli. Funding: University of Naples Federico II. Disclosure: All authors have declared no conflicts of interest.
2006P
MicroRNA-181c promotes tamoxifen resistance in breast cancer cells via upregulation Akt/mTOR axis
A.M. Scherbakov1, Y. Shchegolev2, D. Sorokin2, A. Shunaev2, O.E. Andreeva2, M. Krasil’nikov2 1 Laboratory of Oncoproteomics, N.N. Blokhin National Medical Research Center of Oncology, Moscow, Russian Federation, 2Department of Experimental Tumor Biology, N.N. Blokhin National Medical Research Center of Oncology, Moscow, Russian Federation
based on our previous data, which demonstrated the effect of exosome-mediated transferring of hormonal resistance in in vitro cultured MCF-7 breast cancer cells. Methods: Estrogen-dependent breast cancer cells MCF-7 and the tamoxifen-resistant subline MCF-7 /T were used as an experimental model. The analysis of exosomal microRNAs was performed by HiSeq2500 and at least 5 million reads per samples were obtained. MicroRNA was extracted from by PureLink RNA Micro Kit; library preparaR Small RNA Library Prep Set for IlluminaV R. tion was carried out with NEBNextV Transfection of the RNA oligonucleotides was performed using Metafectene PRO (Biontex) to result in the final RNA concentration of 50 nM. Results: A comparative analysis of exosomal miRNAs of MCF-7 and resistant MCF-7/ T cells was carried out. In total, 2588 miRNAs have been identified in the exosomes. Among them, mir-181 family, which is one of the negative regulators of estrogendependent growth, was identified as the group of miRs, hyperexpressed in the resistant exosomes. Following this, we analysed the role of mir-181c, one of the main members of miR-181 family, in the regulation of cell growth and hormonal response. Mir-181c transfection was found to induce the estrogen-independent growth and partial tamoxifen resistance of MCF-7 cells. The study of the signaling proteins showed that mir-181c transfection, in contrast to scrambled RNA transfection, caused the increase of the amount of Raptor, phosphorylated forms of mTOR and Akt that correlated with increased AP-1 transcriptional activity. Conclusions: We have demonstrated the involvement of miR-181c in the development of hormonal resistance of breast cancer cells that allows us to consider mir-181 as the perspective target of the treatment of hormone- independent cancers. The research was supported by the Russian Science Foundation (19-15-00245, miRNA analysis) and RFBR (#18-29-09017, tamoxifen resistance). Legal entity responsible for the study: The authors. Funding: Russian Science Foundation, project 19-15-00245. Disclosure: All authors have declared no conflicts of interest.
2007P
Spatio-temporal separation of tumor infiltrating CD81 T-cells and HER2/neu1 tumor cells in tumor-immune milieu of infiltrating ductal carcinoma of the breast
S. Sreedharan1, M. Biswas1, S. Thiyagarajan2, N.P. Basak2, A. Basu2, P.S. Ghosh3 Histopathology, Mitra RxDx Inv., Bangalore, India, 2Cancer Biology, Mitra RxDx INC., Bangalore, India, 3Surgical Oncology, IPGME&R & SSKM Hospital, Kolkata, India
1
Background: Designated tumor infiltrating lymphocytes (TIL) within tumor microenvironment (TME) comprised of both regulatory cells and effector cells, and their interaction with tumor cells account for the immune escape and immunosurveillance mechanisms respectively. The number and location of CD8þ tumor infiltrating lymphocytes (TILs) are the measures of immune response and carries prognostic significance and predictive potential in the era of immuno-oncology. The present study was an attempt to find the relation between the treatment response and the spatio-temporal separation of tumor infiltrating CD8þ T-cells and HER2/neuþ tumor cells in breast cancer TME. Methods: In this study, a total of 7 breast cancer patients were enrolled and surgically removed fresh tumor tissues were evaluated for the baseline HER2/neu and CD8 expression by immunohistochemistry (IHC) using dual stain (duplex IHC). The tumor tissues were also tested for the response to HER2 blocker in a novel humanized ex vivo platform that preserves tumour microenvironment using tumour explants, maintained in defined tumour grade-matched matrix support and autologous patient serum. Results: Tumors responded to HER2 blockade had a majority of the CD8þ T cells (6080%) concentrated around the HER2/neuþ tumor area (within 50-100mm). While, the non-responding tumors had significantly lesser number of the CD8þT cells (20-40%) proximal to HER2/neuþ zone compared to the responders. The present study indeed delineated a preferential spatial relationship of tumor infiltrating CD8þ cells in HER2/ neuþ tumor region especially for the recognition of tumor antigen, cross presentation and tumor cell killing. Conclusions: Assessment of distribution pattern of effector cells surrounding tumor area had a correlation on the efficacy of HER2 inhibition. It warrants validation in the larger cohort of patient tumors to underpin its clinical relevance for possible immunotherapies like combination of check point inhibitors with HER2/neu blocker and others. Identification of other co-stimulatory and pathway molecules on the tumors not responded to HER2 blocker is being studied to understand the mechanism of resistance. Legal entity responsible for the study: The authors. Funding: Mitra RxDx Inc. Disclosure: All authors have declared no conflicts of interest.
Background: The main purpose of the work was to study the key exosomal factors involved in the development of hormonal resistance of breast cancer cells. The work is
v804 | Tumour Biology and Pathology
Volume 30 | Supplement 5 | October 2019
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz269.024/5577479 by guest on 24 October 2019
Methods: We isolated organoids from breast cancer patients and optimized the organoids growing conditions. By immunofluorescence assay we confirmed the receptor status, and the preservation of epithelial (E-cadherin) or the fibroblasts components included in the tissue microenvironment (TME) (alfa-SMA and FAP). Here we used organoids as mimic of tumor burden for drug penetration studies. In particular, we studied doxorubicin (DOX) penetration and biodistribution, by several means: immunofluorescence studies, caspase assay, and cell viability. Results: We demonstrated by IF studies, that miR-340, an oncosuppressor miR, was able to modulate DOX penetration. At the same time, miR-340 induced an increase in DOX sensitivity assessed by caspase-3 assay, PARP cleavage, as well as p38 phosphorylation. Breast cancer organoids have also been used to better understand the role of the TME in breast cancer response to therapy. We assessed drug sensitivity in the presence or absence of conditioned media obtained from cancer activated fibroblasts (CAFs). We found that organoids treated with conditioned media exhibit lower level of caspase 3 activation compared to the control upon palbociclib treatment, suggesting the importance role of TME in drug resistance. Conclusions: Thus, organoids can be considered as a new tool for studying breast cancer and developing personalized medicine approaches and to test possible use of RNA therapeutics. Legal entity responsible for the study: Gerolama Condorelli. Funding: AIRC. Disclosure: All authors have declared no conflicts of interest.
Annals of Oncology