Autophagy Correlates with the Therapeutic Responsiveness of Malignant Pleural Mesothelioma in 3D Models

Malignant pleural mesothelioma is a highly chemoresistant solid tumor. We have studied this apoptotic resistance using in vitro and ex vivo three-dimensional models, which acquire a high level of chemoresistance that can be reduced by PI3K/mTOR inhibitors. Here, we investigate the activity of GDC-0980, a novel dual PI3K/mTOR inhibitor, which has been proposed to be effective in mesothelioma. In this work, we aimed to identify mechanisms and markers of efficacy for GDC-0980 by utilizing 3D models of mesothelioma, both in vitro multicellular spheroids and ex vivo tumor fragment spheroids grown from patient tumor samples. We found that a subset of mesothelioma spheroids is sensitive to GDC-0980 alone and to its combination with chemotherapy. Unexpectedly, this sensitivity did not correlate with the activation of the Akt/mTOR pathway. Instead, sensitivity to GDC-0980 correlated with the presence of constitutive ATG13 puncta, a feature of autophagy, a cellular program that supports cells under stress. In tumor fragment spheroids grown from 21 tumors, we also found a subset (n = 11) that was sensitive to GDC-0980, a sensitivity that also correlated with the presence of ATG13 puncta. Interference with autophagy by siRNA of ATG7, an essential autophagic protein, increased the response to chemotherapy, but only in the sensitive multicellular spheroids. In the spheroids resistant to GDC-0980, autophagy appeared to play no role. In summary, we show that GDC-0980 is effective in mesothelioma 3D models that display ATG13 puncta, and that blockade of autophagy increases their response to chemotherapy. For the first time, we show a role for autophagy in the response to chemotherapy of 3D models of mesothelioma and propose ATG13 as a potential biomarker of the therapeutic responsiveness of mesothelioma.     

Artemin,a Member of the Glial Cell Line-derived Neurotrophic Factor Family of Ligands,Is HER2-regulated and Mediates Acquired Trastuzumab Resistance by Promoting Cancer Stem Cell-like Behavior in Mammary Carcinoma Cells

Previous studies have demonstrated that Artemin (ARTN) functions as a cancer stem cell (CSC) and metastatic factor in mammary carcinoma. Herein, we report that ARTN mediates acquired resistance to trastuzumab in HER2-positive mammary carcinoma cells. Ligands that increase HER2 activity increased ARTN expression in HER2-positive mammary carcinoma cells, whereas trastuzumab inhibited ARTN expression. Forced expression of ARTN decreased the sensitivity of HER2-positive mammary carcinoma cells to trastuzumab both in vitro and in vivo. Conversely, siRNA-mediated depletion of ARTN enhanced trastuzumab efficacy. Cells with acquired resistance to trastuzumab exhibited increased ARTN expression, the depletion of which restored trastuzumab sensitivity. Trastuzumab resistance produced an increased CSC population concomitant with enhanced mammospheric growth. ARTN mediated the enhancement of the CSC population by increased BCL-2 expression, and the CSC population in trastuzumab-resistant cells was abrogated upon inhibition of BCL-2. Hence, we conclude that ARTN is one mediator of acquired resistance to trastuzumab in HER2-positive mammary carcinoma cells.     

Potential role of HER2-overexpressing exosomes in countering trastuzumab-based therapy

Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness.     

The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance

Three-dimensional (3D) cultures are a valuable platform to study acquired multicellular apoptotic resistance of cancer. We used spheroids of cell lines and actual tumor to study resistance to the proteasome inhibitor bortezomib in mesothelioma, a highly chemoresistant tumor. Spheroids from mesothelioma cell lines acquired resistance to bortezomib by failing to upregulate Noxa, a pro-apoptotic sensitizer BH3-only protein that acts by displacing Bim, a pro-apoptotic Bax/Bak-activator protein. Surprisingly, despite their resistance, spheroids also upregulated Bim and thereby acquired sensitivity to ABT-737, an inhibitor of anti-apoptotic Bcl-2 molecules. Analysis using BH3 profiling confirmed that spheroids acquired a dependence on anti-apoptotic Bcl-2 proteins and were ‘primed for death''. We then studied spheroids grown from actual mesothelioma. ABT-737 was active in spheroids grown from those tumors (5/7, ∼70%) with elevated levels of Bim. Using immunocytochemistry of tissue microarrays of 48 mesotheliomas, we found that most (33, 69%) expressed elevated Bim. In conclusion, mesothelioma cells in 3D alter the expression of Bcl-2 molecules, thereby acquiring both apoptotic resistance and sensitivity to Bcl-2 blockade. Mesothelioma tumors ex vivo also show sensitivity to Bcl-2 blockade that may depend on Bim, which is frequently elevated in mesothelioma. Therefore, mesothelioma, a highly resistant tumor, may have an intrinsic sensitivity to Bcl-2 blockade that can be exploited therapeutically.     

Up-regulation of miR-21 mediates resistance to trastuzumab therapy for breast cancer

Trastuzumab resistance emerges to be a major issue in anti-human epidermal growth factor receptor 2 (HER2) therapy for breast cancers. Here, we demonstrated that miR-21 expression was up-regulated and its function was elevated in HER2(+) BT474, SKBR3, and MDA-MB-453 breast cancer cells that are induced to acquire trastuzumab resistance by long-term exposure to the antibody, whereas protein expression of the PTEN gene, a miR-21 target, was reduced. Blocking the action of miR-21 with antisense oligonucleotides re-sensitized the resistant cells to the therapeutic activities of trastuzumab by inducing growth arrest, proliferation inhibition, and G(1)-S cell cycle checking in the presence of the antibody. Ectopic expression of miR-21 in HER2(+) breast cancer cells confers resistance to trastuzumab. Rescuing PTEN expression with a p3XFLAG-PTEN-mut construct with deleted miR-21 targeting sequence at its 3' UTR restored the growth inhibition of trastuzumab in the resistant cells by inducing PTEN activation and AKT inhibition. In vivo, administering miR-21 antisense oligonucleotides restored trastuzumab sensitivity in the resistant breast cancer xenografts by inducing PTEN expression, whereas injection of miR-21 mimics conferred trastuzumab resistant in the sensitive breast tumors via PTEN silence. Up-regulatin of miR-21 in tumor biopsies obtained from patients receiving pre-operative trastuzumab therapy was associated with poor trastuzumab response. Therefore, miR-21 overexpression contributes to trastuzumab resistance in HER2(+) breast cancers and antagonizing miR-21 demonstrates therapeutic potential by sensitizing the malignancy to anti-HER2 treatment.     

t-Darpp Promotes Enhanced EGFR Activation and New Drug Synergies in Her2-Positive Breast Cancer Cells

Trastuzumab has led to improved survival rates of HER2⁺ breast cancer patients. However, acquired resistance remains a problem in the majority of cases. t-Darpp is over-expressed in trastuzumab-resistant cell lines and its over-expression is sufficient for conferring the resistance phenotype. Although its mechanism of action is unknown, t-Darpp has been shown to increase cellular proliferation and inhibit apoptosis. We have reported that trastuzumab-resistant BT.Her^R cells that over-express endogenous t-Darpp are sensitized to EGFR inhibition in the presence (but not the absence) of trastuzumab. The purpose of the current study was to determine if t-Darpp might modulate sensitivity to EGFR inhibitors in trastuzumab-resistant cells. Using EGFR tyrosine kinase inhibitors AG1478, gefitinib and erlotinib, we found that trastuzumab-resistant SK.Her^R cells were sensitized to EGFR inhibition, compared to SK-Br-3 controls, even in the absence of trastuzumab. t-Darpp knock-down in SK.Her^R cells reversed their sensitivity to EGFR inhibition. Increased EGFR sensitivity was also noted in SK.tDp cells that stably over-express t-Darpp. High levels of synergy between trastuzumab and the EGFR inhibitors were observed in all cell lines with high t-Darpp expression. These cells also demonstrated more robust activation of EGFR signaling and showed greater EGFR stability than parental cells. The T75A phosphorylation mutant of t-Darpp did not confer sensitivity to EGFR inhibition nor activation of EGFR signaling. The over-expression of t-Darpp might facilitate enhanced EGFR signaling as part of the trastuzumab resistance phenotype. This study suggests that the presence of t-Darpp in HER2⁺ cancers might predict the enhanced response to dual HER2/EGFR targeting.     

Targeting the autophagy promoted antitumor effect of T-DM1 on HER2-positive gastric cancer

Trastuzumab emtansine (T-DM1), an antibody-drug conjugate consisted of the HER2-targeted monoclonal antibody trastuzumab and the tubulin inhibitor emtansine, has shown potent therapeutic value in HER2-positive breast cancer (BC). However, a clinical trial indicated that T-DM1 exerts a limited effect on HER2-positive gastric cancer (GC), but the underlying mechanism is inconclusive. Our research attempted to reveal the probable mechanism and role of autophagy in T-DM1-treated HER2-positive GC. In this study, our results showed that T-DM1 induced apoptosis and exhibited potent therapeutic efficacy in HER2-positive GC cells. In addition, autophagosomes were observed by transmission electron microscopy. Autophagy was markedly activated and exhibited the three characterized gradations of autophagic flux, consisting of the formation of autophagosomes, the fusion of autophagosomes with lysosomes, and the deterioration of autophagosomes in autolysosomes. More importantly, autophagic inhibition by the suppressors 3-methyladenine (3-MA) and LY294002 significantly potentiated cytotoxicity and apoptosis in HER2-positive GC cells in vitro, while the combined use of LY294002 and T-DM1 elicited potent anti-GC efficacy in vivo. In mechanistic experiments, immunoblot analysis indicated the downregulated levels of Akt, mTOR, and P70S6K and confocal microscopy analysis clearly showed that autophagic inhibition promoted the fusion of T-DM1 molecules with lysosomes in GC cells. In conclusion, our research demonstrated that T-DM1 induced apoptosis as well as cytoprotective autophagy, and autophagic inhibition could potentiate the antitumor effect of T-DM1 on HER2-positive GC. Furthermore, autophagic inhibition might increase the fusion of T-DM1 with lysosomes, which might accelerate the release of the cytotoxic molecule emtansine from the T-DM1 conjugate. These findings highlight a promising therapeutic strategy that combines T-DM1 with an autophagy inhibitor to treat HER-positive GC more efficiently.Subject terms: Targeted therapies, Tumour biomarkers     

Metformin inhibits melanoma development through autophagy and apoptosis mechanisms

Metformin is the most widely used antidiabetic drug because of its proven efficacy and limited secondary effects. Interestingly, recent studies have reported that metformin can block the growth of different tumor types. Here, we show that metformin exerts antiproliferative effects on melanoma cells, whereas normal human melanocytes are resistant to these metformin-induced effects. To better understand the basis of this antiproliferative effect of metformin in melanoma, we characterized the sequence of events underlying metformin action. We showed that 24 h metformin treatment induced a cell cycle arrest in G0/G1 phases, while after 72 h, melanoma cells underwent autophagy as demonstrated by electron microscopy, immunochemistry, and by quantification of the autolysosome-associated LC3 and Beclin1 proteins. In addition, 96 h post metformin treatment we observed robust apoptosis of melanoma cells. Interestingly, inhibition of autophagy by knocking down LC3 or ATG5 decreased the extent of apoptosis, and suppressed the antiproliferative effect of metformin on melanoma cells, suggesting that apoptosis is a consequence of autophagy. The relevance of these observations were confirmed in vivo, as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers. Taken together, our data suggest that metformin has an important impact on melanoma growth, and may therefore be beneficial in patients with melanoma.     

Triazole analog 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol induces reactive oxygen species and autophagy-dependent apoptosis in both in vitro and in vivo breast cancer models

Autophagy is considered as an important cell death mechanism that closely interacts with other common cell death programs like apoptosis. Critical role of autophagy in cell death makes it a promising, yet challenging therapeutic target for cancer. We identified a series of 1,2,3-triazole analogs having significant breast cancer inhibition property. Therefore, we attempted to study whether autophagy and apoptosis were involved in the process of cancer cell inhibition. The lead molecule, 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol (T-12) induced significant cell cycle arrest, mitochondrial membrane depolarization, apoptosis and autophagy in MCF-7 and MDA-MB-231 cells. T-12 increased reactive oxygen species and its inhibition by N-acetyl-l-cysteine protected breast cancer cells from autophagy and apoptosis. Autophagy inhibitor, 3-methyladenine abolished T-12 induced apoptosis, mitochondrial membrane depolarization and reactive oxygen species generation. This suggested that T-12 induced autophagy facilitated cell death rather than cell survival. Pan-caspase inhibition did not abrogate T-12 induced autophagy, suggesting that autophagy precedes apoptosis. In addition, T-12 inhibited cell survival pathway signaling proteins, Akt, mTOR and Erk1/2. T-12 also induced significant regression of tumor with oral dose of as low as 10 mg/kg bodyweight in rat mammary tumor model without any apparent toxicity. In presence of reactive oxygen species inhibitor (N-acetyl-l-cysteine) and autophagy inhibitor (chloroquine), T-12 induced tumor regression was significantly decreased. In conclusion, T-12 is a potent inducer of autophagy-dependent apoptosis in breast cancer cells both in vitro and in vivo and can serve as an important lead in development of new anti-tumor therapy.     

Modulation of MicroRNA-194 and cell migration by HER2-targeting trastuzumab in breast cancer

Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of the HER2 oncoprotein, can effectively target HER2-positive breast cancer through several mechanisms. Although the effects of trastuzumab on cancer cell proliferation, angiogenesis and apoptosis have been investigated in depth, the effect of trastuzumab on microRNA (miRNA) has not been extensively studied. We have performed miRNA microarray profiling before and after trastuzumab treatment in SKBr3 and BT474 human breast cancer cells that overexpress HER2. We found that trastuzumab treatment of SKBr3 cells significantly decreased five miRNAs and increased three others, whereas treatment of BT474 cells significantly decreased two miRNAs and increased nine. The only change in miRNA expression observed in both cell lines following trastuzumab treatment was upregulation of miRNA-194 (miR-194) that was further validated in vitro and in vivo. Forced expression of miR-194 in breast cancer cells that overexpress HER2 produced no effect on apoptosis, modest inhibition of proliferation, significant inhibition of cell migration/invasion in vitro and significant inhibition of xenograft growth in vivo. Conversely, knockdown of miR-194 promoted cell migration. Increased miR-194 expression markedly reduced levels of the cytoskeletal protein talin2 and specifically inhibited luciferase reporter activity of a talin2 wild-type 3'-untranslated region, but not that of a mutant reporter, indicating that talin2 is a direct downstream target of miR-194. Trastuzumab treatment inhibited breast cancer cell migration and reduced talin2 expression in vitro and in vivo. Knockdown of talin2 inhibited cell migration/invasion. Knockdown of trastuzumab-induced miR-194 expression with a miR-194 inhibitor compromised trastuzumab-inhibited cell migration in HER2-overexpressing breast cancer cells. Consequently, trastuzumab treatment upregulates miR-194 expression and may exert its cell migration-inhibitory effect through miR-194-mediated downregulation of cytoskeleton protein talin2 in HER2-overexpressing human breast cancer cells.     

Antiproliferation activity of Devil's club (Oplopanax horridus) and anticancer agents on human pancreatic cancer multicellular spheroids

Devil's club (DC, Oplopanax horridus) is an important medicinal herb of the Pacific Northwest which has significant antiproliferation activity against a variety of human tumor cell lines in vitro. This study compared the antiproliferation activity of DC extract alone, and in combination with chemotherapeutic agents gemcitabine (GEM), cisplatin (CDDP), and paclitaxel (PTX) on human pancreatic cancer PANC-1 3D spheroids and 2D monolayer cells. 3D tumor spheroids were prepared with a rotary cell culture system. PANC-1 3D spheroids were significantly more resistant to killing by DC extract, GEM and PTX compared to 2D cells, with IC₅₀ levels closer to that observed in vivo. DC extract significantly enhanced the antiproliferation activity of CDDP and GEM at some concentrations. The bioactive compound identified as a polyacetylene showed strong antiproliferation activity against PANC-1 2D cells and 3D spheroids with IC₅₀ at 0.73 ± 0.04 and 3.15 ± 0.16 μM, respectively. 3D spheroids and 2D cells differentially expressed a number of apoptosis related genes. Cell cycle analysis showed that the proportion of cells in S phase was increased and in G2/M phase reduced in 3D spheroids compared to 2D cells. DC extract can potentially be used to enhance the activity of chemotherapeutic agents against pancreatic cancer cells. Use of 3D spheroid model for screening of natural products can potentially increase the efficiency in discovering in vivo bioactive compounds.     

Dual functions of autophagy in the response of breast tumor cells to radiation: cytoprotective autophagy with radiation alone and cytotoxic autophagy in radiosensitization by vitamin D 3

In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D 3, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D 3 enhanced radiosensitivity and promoted autophagy in MCF-7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D 3 failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D 3 was not attenuated by a genetic block to autophagy due largely to the promotion of apoptosis via the collateral suppression of protective autophagy. However, MCF-7 cells were protected from the combination of 1,25D 3 with radiation using a concentration of chloroquine that produced minimal sensitization to radiation alone. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D 3). As both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental system in response to radiation, this type of model could be utilized to distinguish biochemical, molecular and/or functional differences in these dual functions of autophagy.     

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.     

Shed HER2 extracellular domain in HER2‐mediated tumor growth and in trastuzumab susceptibility

The question of the serum HER2 extracellular domain (HER2/ECD) measurement for prediction of response to the anti‐HER2 antibody Trastuzumab is still an open and current matter of clinical debate. To elucidate the involvement of shed HER2/ECD in HER2‐driven tumor progression and in guiding therapy of individual patients, we examined biological effects exerted by elevated HER2/ECD in cancer growth and in response to Trastuzumab. To this purpose SKOV3 tumor cells were stably transfected to release a recombinant HER2/ECD molecule (rECD). Transfectants releasing high levels of 110‐kDa rECD, identical in size to native HER2/ECD (nECD), grew significantly slower than did controls, which constitutively released only basal levels of nECD. While transmembrane HER2 and HER1 were expressed at equal levels by both controls and transfected cells, activation of these molecules and of downstream ERK2 and Akt was significantly reduced only in rECD transfectants. Surface plasmon resonance analysis revealed heterodimerization of the rECD with HER1, ‐2, and ‐3. In cell growth bioassays in vitro, shed HER2 significantly blocked HER2‐driven tumor cell proliferation. In mice, high levels of circulating rECD significantly impaired HER2‐driven SKOV3 tumor growth but not that of HER2‐negative tumor cells. In vitro and in mice, Trastuzumab significantly inhibited tumor growth due to the rECD‐facilitated accumulation of the antibody on tumor cells. Globally our findings sustain the biological relevance of elevated HER2/ECD levels in the outcome of HER2‐disease and in the susceptibility to Trastuzumab‐based therapy. J. Cell. Physiol. 225: 256–265, 2010. © 2010 Wiley‐Liss, Inc.     

Coordinate Autophagy and mTOR Pathway Inhibition Enhances Cell Death in Melanoma

The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway promotes melanoma tumor growth and survival while suppressing autophagy, a catabolic process through which cells collect and recycle cellular components to sustain energy homeostasis in starvation. Conversely, inhibitors of the PI3K/AKT/mTOR pathway, in particular the mTOR inhibitor temsirolimus (CCI-779), induce autophagy, which can promote tumor survival and thus, these agents potentially limit their own efficacy. We hypothesized that inhibition of autophagy in combination with mTOR inhibition would block this tumor survival mechanism and hence improve the cytotoxicity of mTOR inhibitors in melanoma. Here we found that melanoma cell lines of multiple genotypes exhibit high basal levels of autophagy. Knockdown of expression of the essential autophagy gene product ATG7 resulted in cell death, indicating that survival of melanoma cells is autophagy-dependent. We also found that the lysosomotropic agent and autophagy inhibitor hydroxychloroquine (HCQ) synergizes with CCI-779 and led to melanoma cell death via apoptosis. Combination treatment with CCI-779 and HCQ suppressed melanoma growth and induced cell death both in 3-dimensional (3D) spheroid cultures and in tumor xenografts. These data suggest that coordinate inhibition of the mTOR and autophagy pathways promotes apoptosis and could be a new therapeutic paradigm for the treatment of melanoma.     

Autophagy initiation correlates with the autophagic flux in 3D models of mesothelioma and with patient outcome

Understanding the role of autophagy in cancer has been limited by the inability to measure this dynamic process in formalin-fixed tissue. We considered that 3-dimensional models including ex vivo tumor, such as we have developed for studying mesothelioma, would provide valuable insights. Using these models, in which we could use lysosomal inhibitors to measure the autophagic flux, we sought a marker of autophagy that would be valid in formalin-fixed tumor and be used to assess the role of autophagy in patient outcome. Autophagy was studied in mesothelioma cell lines, as 2-dimensional (2D) monolayers and 3-dimensional (3D) multicellular spheroids (MCS), and in tumor from 25 chemonaive patients, both as ex vivo 3D tumor fragment spheroids (TFS) and as formalin-fixed tissue. Autophagy was evaluated as autophagic flux by detection of the accumulation of LC3 after lysosomal inhibition and as autophagy initiation by detection of ATG13 puncta. We found that autophagic flux in 3D, but not in 2D, correlated with ATG13 positivity. In each TFS, ATG13 positivity was similar to that of the original tumor. When tested in tissue microarrays of 109 chemonaive patients, higher ATG13 positivity correlated with better prognosis and provided information independent of known prognostic factors. Our results show that ATG13 is a static marker of the autophagic flux in 3D models of mesothelioma and may also reflect autophagy levels in formalin-fixed tumor. If confirmed, this marker would represent a novel prognostic factor for mesothelioma, supporting the notion that autophagy plays an important role in this cancer.     

Mycophenolic acid potentiates HER2-overexpressing SKBR3 breast cancer cell line to induce apoptosis: involvement of AKT/FOXO1 and JAK2/STAT3 pathways

Trastuzumab has been successfully used as a first-line therapy specific for HER2-overexressing breast cancer patients. However, despite the effectiveness of trastuzumab, the occurrence of inherent and acquired resistance remains as the main challenge of the therapy. Thus, this has motivated efforts toward finding new therapeutic strategies including combining trastuzumab with other drugs to enhance its therapeutic efficacy. In that line, we investigated the capability of mycophenolic acid (MPA), an inhibitor of de novo guanine nucleotide synthesis with potential anti-cancer activity, on improving the response to trastuzumab among SKBR3 cells as well as trastuzumab resistant SKBR3-TR cells. Our data indicated that irrespective to trastuzumab sensitivity of cells, MPA effectively inhibited cell growth through inducing adipocyte-like cell differentiation as well as blocking cell cycle progression at G₁ phase along with augmentation of p27^kip expression level. Furthermore, combined treatment with trastuzumab and MPA was more potent in cell growth inhibition, cell cycle arrest and apoptosis induction, as evident by flow cytometric analyses and caspase-3 production, in both trastuzumab sensitive and resistant SKBR3 cells. Besides, western blot analysis showed that elevated apoptosis induction in both cell groups was associated with attenuation in phosphorylation of some key elements of HER2 signaling pathway including AKT, ERK, STAT3 and consequently augmentation in FOXO1 expression level in response to combination of trastuzumab and MPA. These data suggest that manipulation of intracellular GTP level by MPA and consequent molecular perturbation in some of the cell survival and pro-apoptotic relevant signaling pathways might provide an alternative clinical strategy for chemosensitization of resistant breast cancer cells to anti- HER2 therapy.