PTEN-deficient Cells Research Articles

Abstract PD13-08: An ENT2-dependent, cell-penetrating, and DNA-damaging lupus autoantibody crosses the blood-brain barrier to target breast cancer brain metastases

Abstract Introduction: Antibody-based immunotherapy has potential to change paradigms in the management of breast cancer brain metastases, if the blood-brain barrier (BBB) can be overcome. The autoimmune disease systemic lupus erythematosus offers an unexpected new approach to this problem. 3E10 is a lupus anti-DNA autoantibody that localizes to DNA at tumors, penetrates cells via the ENT2 nucleoside transporter, inhibits DNA repair, and is synthetically lethal to BRCA1/2 or PTEN-deficient cancer cells with impaired homologous recombination (HR). Breast cancer brain metastases exhibit increased HR defects and PTEN loss compared to primary breast tumors, and ENT2 expressed in brain endothelial cells (BECs) regulates nucleoside flux at the BBB. Further, 3E10 has previously delivered cargo protein to ischemic brain. We hypothesized that ENT2 may facilitate transport of 3E10 across the BBB, and that 3E10 could be used to treat breast cancer brain metastases. Deoxymab-1 (also known as PAT-DX1 or DX1) is a re-engineered and optimized fragment of 3E10 in pre-clinical development for use against HR-deficient tumors. In the present study we examined the ability of DX1 to cross the BBB and suppress breast cancer brain metastases. BBB study results: ENT2-dependent transport of DX1 across the BBB was evaluated in vitro and in vivo. DX1 penetrated hCMEC/D3 BECs and crossed from apical to basolateral chambers in an hCMEC/D3 transwell model of the BBB. Integrity of the model was confirmed by measuring transendothelial electrical resistance and demonstrating the barrier prevented movement of control protein into the basolateral chamber. Expression of ENT2 in hCMEC/D3 cells was confirmed by immunostaining, and the ENT2 inhibitor dipyridamole (DP) inhibited both penetration by DX1 into the cells and its transport across the BBB transwell model. Immunodeficient mice with orthotopic GBM tumors were treated with IV and IP control buffer (n=2), IV DX1 (20 mg/kg) and IP control buffer (n=4), or IV DX1 (20 mg/kg) and IP DP (ENT2 inhibitor, 70 mg/kg) (n=4). DX1 was labeled with Alexa Fluor 750 (AF750) to allow detection by IVIS. Twenty-four hours after treatment, mice treated with DX1 in the absence of DP exhibited strong AF750 signal in the brain correlating to tumor. Co-treatment with DP reduced uptake of DX1 into the brain tumors by ~78% (P<0.001). These findings are consistent with DX1 crossing the BBB and localizing into brain tumors in an ENT2-dependent manner. Efficacy study results: The 231-BR brain-seeking subclone of the MDA-MB-231 triple negative breast cancer cell line exhibits PTEN loss relative to parental cells. DX1 penetrated and killed 231-BR cells in vitro. For in vivo testing, brain metastases were generated in immunodeficient mice by intracardiac injection of 231-BR cells engineered for expression of luciferase. Brain metastases were confirmed by IVIS one week later, and then mice were treated with IV control buffer (PBS, n=7) or DX1 (20 mg/kg, n=7). In separate studies DX1 was delivered as a single cycle or as four consecutive cycles, with one cycle defined as control or DX1 3X/week. DX1 significantly suppressed tumor growth, evidenced by weekly IVIS. At week 5 brain radiance efficiencies (x105) in the single cycle study in control and DX1-treated mice were 264.8±72.0 and 71.9±31.3 (P<0.04), and 320±66 and 20.2±8.5 (P≤0.01) in the four-cycle study. One cycle of DX1 yielded a non-significant increase in median survival from 30 to 35 days (P=0.42). Four cycles of DX1 had greater impact, with median survival increased by 14 days (from 31 to 45) (P<0.002). DX1 was not associated with nonspecific toxicity. Conclusion: Our findings support an ENT2-mediated mechanism of BBB penetration by DX1 and establish proof of concept for use of a DNA-targeting autoantibody against breast cancer brain metastases. Citation Format: James E Hansen, Shenqi Zhang, Anupama Shirali, Christopher May, Benedette Cuffari, Valentina Dubljevic, James A Campbell, Jiangbing Zhou. An ENT2-dependent, cell-penetrating, and DNA-damaging lupus autoantibody crosses the blood-brain barrier to target breast cancer brain metastases [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD13-08.

Feedback activation of STAT3 limits the response to PI3K/AKT/mTOR inhibitors in PTEN-deficient cancer cells

The PI3K/AKT/mTOR signaling pathway is constitutively active in PTEN-deficient cancer cells, and its targeted inhibition has significant anti-tumor effects. However, the efficacy of targeted therapies is often limited due to drug resistance. The relevant signaling pathways in PTEN-deficient cancer cells treated with the PI3K/mTOR inhibitor BEZ235 were screened using a phosphokinase array, and further validated following treatment with multiple PI3K/AKT/mTOR inhibitors or AKT knockdown. The correlation between PTEN expression levels and STAT3 kinase phosphorylation in the tissue microarrays of gastric cancer patients was analyzed by immunohistochemistry. Cell proliferation and clonogenic assays were performed on the suitably treated PTEN-deficient cancer cells. Cytokine arrays, small molecule inhibition and knockdown assays were performed to identify related factors. PTEN-deficient tumor xenografts were established in nude mice that were treated with PI3K/AKT/mTOR and/or STAT3 inhibitors. PTEN deficiency was positively correlated with low STAT3 activity. PI3K/mTOR inhibitors increased the expression and secretion of macrophage migration inhibitory factor (MIF) and activated the JAK1/STAT3 signaling pathway. Both cancer cells and in vivo tumor xenografts showed that the combined inhibition of PI3K/AKT/mTOR and STAT3 activity enhanced the inhibitory effect of BEZ235 on the proliferation of PTEN-deficient cancer cells. Our findings provide a scientific basis for a novel treatment strategy in cancer patients with PTEN deficiency.

Spatial Epitope Barcoding Reveals Subclonal Tumor Patch Behaviors

Intratumoral variability is a seminal feature of human tumors contributing to tumor progression and response to treatment. Current technologies are unsuitable to accurately track phenotypes and subclonal evolution within tumors, especially in response to genetic manipulations. Here, we developed epitope combinatorial tags (EpicTags), which we coupled to multiplex ion beam imaging (EpicMIBI) for in situ tracking of barcodes within tissue microenvironments. Using this platform, we dissected the spatial components of cell lineages and phenotypes in a xenograft model of small-cell lung cancer. We observed emergent properties from mixed clones leading to the preferential expansion of subclonal patches for both neuroendocrine and non-neuroendocrine cancer cell states in this model. In tumors harboring a fraction of PTEN-deficient cancer cells, we uncovered a non-autonomous increase of subclonal patch size in PTEN wild-type cancer cells. EpicMIBI can facilitate in situ interrogation of cell-intrinsic and cell-extrinsic processes involved in intratumoral heterogeneity.

ATM Kinase Inhibition Preferentially Sensitises PTEN-Deficient Prostate Tumour Cells to Ionising Radiation.

Simple SummaryProstate cancer is the most frequently diagnosed cancer in men. Despite the importance of radical radiotherapy for the management of this disease, recurrence remains a challenge. PTEN is a tumour suppressor that is frequently inactivated in advanced prostate cancer and has been associated with relapse following radiotherapy. The present study shows that the role of PTEN in response to ionizing radiation is complex. Furthermore, it demonstrates that in the absence of PTEN, an increased response to combined treatment using radiotherapy and the ATM inhibitor KU-60019 can be observed. Our findings provide a strong rationale for evaluating loss of PTEN in prostate cancer as a therapeutic target for ATM inhibitor in combination with radiotherapy in the clinical setting.Radical radiotherapy, often in combination with hormone ablation, is a safe and effective treatment option for localised or locally-advanced prostate cancer. However, up to 30% of patients with locally advanced PCa will go on to develop biochemical failure, within 5 years, following initial radiotherapy. Improving radiotherapy response is clinically important since patients exhibiting biochemical failure develop castrate-resistant metastatic disease for which there is no curative therapy and median survival is 8–18 months. The aim of this research was to determine if loss of PTEN (highly prevalent in advanced prostate cancer) is a novel therapeutic target in the treatment of advanced prostate cancer. Previous work has demonstrated PTEN-deficient cells are sensitised to inhibitors of ATM, a key regulator in the response to DSBs. Here, we have shown the role of PTEN in cellular response to IR was both complex and context-dependent. Secondly, we have confirmed ATM inhibition in PTEN-depleted cell models, enhances ionising radiation-induced cell killing with minimal toxicity to normal prostate RWPE-1 cells. Furthermore, combined treatment significantly inhibited PTEN-deficient tumour growth compared to PTEN-expressing counterparts, with minimal toxicity observed. We have further shown PTEN loss is accompanied by increased endogenous levels of ROS and DNA damage. Taken together, these findings provide pre-clinical data for future clinical evaluation of ATM inhibitors as a neoadjuvant/adjuvant in combination with radiation therapy in prostate cancer patients harbouring PTEN mutations.

Anti-Tumor Effects of Sodium Meta-Arsenite in Glioblastoma Cells with Higher Akt Activities.

Glioblastoma is a type of aggressive brain tumor that grows very fast and evades surrounding normal brain, lead to treatment failure. Glioblastomas are associated with Akt activation due to somatic alterations in PI3 kinase/Akt pathway and/or PTEN tumor suppressor. Sodium meta-arsenite, KML001 is an orally bioavailable, water-soluble, and trivalent arsenical and it shows antitumoral effects in several solid tumor cells via inhibiting oncogenic signaling, including Akt and MAPK. Here, we evaluated the effect of sodium meta-arsenite, KML001, on the growth of human glioblastoma cell lines with different PTEN expression status and Akt activation, including PTEN-deficient cells (U87-MG and U251) and PTEN-positive cells (LN229). The growth-inhibitory effect of KML001 was stronger in U87-MG and U251 cells, which exhibited higher Akt activity than LN229 cells. KML001 deactivated Akt and decreased its protein levels via proteasomal degradation in U87-MG cells. KML001 upregulated mutant PTEN levels via inhibition of its proteasomal degradation. KML001 inhibited cell growth more effectively in active Akt-overexpressing LN229 cells than in mock-expressing LN229 cells. Consistent with these results, KML001 sensitized PTEN-deficient cells more strongly to growth inhibition than it did PTEN-positive cells in prostate and breast cancer cell lines. Finally, we illustrated in vivo anti-tumor effects of KML001 using an intracranial xenograft mouse model. These results suggest that KML001 could be an effective chemotherapeutic drug for the treatment of glioblastoma cancer patients with higher Akt activity and PTEN loss.

RAD51-Mediated DNA Homologous Recombination Is Independent of PTEN Mutational Status.

Simple SummaryPTEN is an important tumor suppressor that is frequently mutated in malignancy. PTEN mutational loss has been associated with reduced RAD51 expression and homologous recombination deficiency (HRD), however; recent studies have failed to recapitulate these findings. Here, we show that RAD51 expression, foci formation and homologous recombination repair activity are unaltered in normal and tumorigenic PTEN-deficient cells and patient samples. Furthermore, we show that PTEN-deficient tumor cell lines do not synergize with the clinical PARP inhibitor olaparib, underscoring a need to discontinue its use in treating patients with PTEN-deficient tumors that do not otherwise exhibit HRD.PTEN mutation occurs in a variety of aggressive cancers and is associated with poor patient outcomes. Recent studies have linked mutational loss of PTEN to reduced RAD51 expression and function, a key factor involved in the homologous recombination (HR) pathway. However, these studies remain controversial, as they fail to establish a definitive causal link to RAD51 expression that is PTEN-dependent, while other studies have not been able to recapitulate the relationship between the PTEN expression and the RAD51/HR function. Resolution of this apparent conundrum is essential due to the clinically-significant implication that PTEN-deficient tumors may be sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) commonly used in the clinical management of BRCA-mutated and other HR-deficient (HRD) tumors. Methods: Primary Pten-deficient (and corresponding wild-type) mouse embryonic fibroblasts (MEFs) and astrocytes and PTEN-null human tumor cell lines and primary cells were assessed for RAD51 expression (via the Western blot analysis) and DNA damage repair analyses (via alkali comet and γH2AX foci assays). RAD51 foci analysis was used to measure HR-dependent DNA repair. Xrcc2-deficient MEFs served as an HR-deficient control, while the stable knockdown of RAD51 (shRAD51) served to control for the relative RAD51/HR-mediated repair and the phospho-53BP1 foci analysis served to confirm and measure non-homologous end joining (NHEJ) activity in PTEN-deficient and shRAD51-expressing (HRD) lines. Cell proliferation studies were used to measure any potential added sensitivity of PTEN-null cells to the clinically-relevant PARPi, olaparib. RAD51 levels and DNA damage response signaling were assessed in PTEN-mutant brain tumor initiating cells (BTICs) derived from primary and recurrent glioblastoma multiforme (GBM) patients, while expression of RAD51 and its paralogs were examined as a function of the PTEN status in the RNA expression datasets isolated from primary GBM tumor specimens and BTICs. Results: Pten knockout primary murine cells display unaltered RAD51 expression, endogenous and DNA strand break-induced RAD51 foci and robust DNA repair activity. Defective HR was only observed in the cells lacking Xrcc2. Likewise, human glioblastoma multiforme (GBM) cell lines with known PTEN deficiency (U87, PTEN-mutated; U251 and U373, PTEN-null) show apparent expression of RAD51 and display efficient DNA repair activity. Only GBM cells stably expressing shRNAs against RAD51 (shRAD51) display dysfunctional DNA repair activity and reduced proliferative capacity, which is exacerbated by PARPi treatment. Furthermore, GBM patient-derived BTICs displayed robust RAD51 expression and intact DNA damage response signaling in spite of PTEN-inactivating mutations. RNA expression analysis of primary GBM tissue specimens and BTICs demonstrate stable levels of RAD51 and its paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and DMC1), regardless of the PTEN mutational status. Conclusions: Our findings demonstrate definitively that PTEN loss does not alter the RAD51 expression, its paralogs, or the HR activity. Furthermore, deficiency in PTEN alone is not sufficient to impart enhanced sensitivity to PARPi associated with HRD. This study is the first to unequivocally demonstrate that PTEN deficiency is not linked to the RAD51 expression or the HR activity amongst primary neural and non-neural Pten-null cells, PTEN-deficient tumor cell lines, and primary PTEN-mutant GBM patient-derived tissue specimens and BTICs.

Targeting the Synthetic Vulnerability of PTEN-Deficient Glioblastoma Cells with MCL1 Inhibitors.

PTEN deletion or mutation occurs in 30% to 60% of patients with glioblastoma (GBM) and is associated with poor prognosis. Efficacious therapy for this subgroup of patients is currently lacking. To identify potential target(s) to selectively suppress PTEN-deficient GBM growth, we performed a three-step synthetic lethal screen on LN18 PTEN wild-type (WT) and knockout (KO) isogeneic GBM cell lines using a library containing 606 target-selective inhibitors. A MCL1 inhibitor UMI-77 identified in the screen exhibited excellent suppression on the proliferation, colony formation, 3D spheroid, and neurosphere formation of PTEN-deficient GBM cells. Mechanistically, loss of PTEN in GBM cells led to upregulation of MCL1 in posttranslational level via inhibition of GSK3β, and consequently confer cells resistance to apoptosis. Pharmacologic inhibition or knockdown of MCL1 blocked this PI3K-GSK3β-MCL1 axis and caused reduction of several antiapoptotic proteins, finally induced massive caspase-3 cleavage and apoptosis. In both subcutaneous and orthotopic GBM models, knockdown of MCL1 significantly impaired the in vivo growth of PTEN-deficient xenografts. Moreover, the combination of UMI-77 and temozolomide synergistically killed PTEN-deficient GBM cells. Collectively, our work identified MCL1 as a promising target for PTEN-deficient GBM. For future clinical investigations, priority should be given to the development of a selective MCL1 inhibitor with efficient brain delivery and minimal in vivo toxicity.

Exploiting Chromosomal Instability of PTEN-Deficient Triple-Negative Breast Cancer Cell Lines for the Sensitization against PARP1 Inhibition in a Replication-Dependent Manner.

Simple SummaryThe poor prognosis of patients with TNBC have fostered a major effort to identify more patients who would benefit from targeted therapies. Here we recognize PTEN as a potential CIN-causing gene in TNBC and consider PTEN-deficient TNBC for the treatment with PARP1 inhibitors due to the protective role of PTEN during DNA replication.Chromosomal instability (CIN) is an emerging hallmark of cancer and its role in therapeutic responses has been increasingly attracting the attention of the research community. To target the vulnerability of tumors with high CIN, it is important to identify the genes and mechanisms involved in the maintenance of CIN. In our work, we recognize the tumor suppressor gene Phosphatase and Tensin homolog (PTEN) as a potential gene causing CIN in triple-negative breast cancer (TNBC) and show that TNBC with low expression levels of PTEN can be sensitized for the treatment with poly-(ADP-ribose)-polymerase 1 (PARP1) inhibitors, independent of Breast Cancer (BRCA) mutations or a BRCA-like phenotype. In silico analysis of mRNA expression data from 200 TNBC patients revealed low expression of PTEN in tumors with a high CIN70 score. Western blot analysis of TNBC cell lines confirm lower protein expression of PTEN compared to non TNBC cell lines. Further, PTEN-deficient cell lines showed cellular sensitivity towards PARP1 inhibition treatment. DNA fiber assays and examination of chromatin bound protein fractions indicate a protective role of PTEN at stalled replication forks. In this study, we recognize PTEN as a potential CIN-causing gene in TNBC and identify its important role in the replication processes.

KLF5-mediated COX2 upregulation contributes to tumorigenesis driven by PTEN deficiency

Tumor suppressor gene PTEN is frequently mutated in a wide variety of cancers. However, the downstream targets or signal transduction pathways of PTEN remain not fully understood. By analyzing Pten-null mouse embryonic fibroblasts (MEFs) cell lines and their isogenic counterparts, we showed that loss of PTEN led to increased cyclooxygenase2 (COX2) expression in an AKT-independent manner. Moreover, we demonstrated that PTEN deficiency promotes the transcription of COX2 via upregulation of the transcription factor Krüppel-like factor 5 (KLF5). Knocked down the expression of COX2 suppressed proliferation, migration and tumoral growth of Pten-null cells. Further experiments revealed that COX2 enhanced Pten-null MEFs growth and migration through upregulation of NADPH oxidase 4 (NOX4). In addition, MK-2206, a specific inhibitor of AKT, in combination with celecoxib, a COX2 inhibitor, strongly inhibited Pten-deficient cell growth. We concluded that KLF5/COX2/NOX4 signaling pathway is critical for cell growth and migration caused by the loss of PTEN, and the combination of MK-2206 and celecoxib may be an effective new approach to treating PTEN deficiency related tumors.

Chromatin Regulator CHD1 Remodels the Immunosuppressive Tumor Microenvironment in PTEN-Deficient Prostate Cancer.

Genetic inactivation of PTEN is common in prostate cancer and correlates with poorer prognosis. We previously identified CHD1 as an essential gene in PTEN-deficient cancer cells. Here, we sought definitive in vivo genetic evidence for, and mechanistic understanding of, the essential role of CHD1 in PTEN-deficient prostate cancer. In Pten and Pten/Smad4 genetically engineered mouse models, prostate-specific deletion of Chd1 resulted in markedly delayed tumor progression and prolonged survival. Chd1 deletion was associated with profound tumor microenvironment (TME) remodeling characterized by reduced myeloid-derived suppressor cells (MDSC) and increased CD8+ T cells. Further analysis identified IL6 as a key transcriptional target of CHD1, which plays a major role in recruitment of immunosuppressive MDSCs. Given the prominent role of MDSCs in suppressing responsiveness to immune checkpoint inhibitors (ICI), our genetic and tumor biological findings support combined testing of anti-IL6 and ICI therapies, specifically in PTEN-deficient prostate cancer. SIGNIFICANCE: We demonstrate a critical role of CHD1 in MDSC recruitment and discover CHD1/IL6 as a major regulator of the immunosuppressive TME of PTEN-deficient prostate cancer. Pharmacologic inhibition of IL6 in combination with immune checkpoint blockade elicits robust antitumor responses in prostate cancer.This article is highlighted in the In This Issue feature, p. 1241.

Abstract LB-005: Senescence reprogramming in primary tumors initiates prostate cancer metastases

Abstract Recent studies have raised fundamental questions about the possibility that senescence induced either by oncogene activation or chemotherapy, while initially restricting tumorigenesis, may promote cancer metastases at a late stage. This coexisting dichotomy remains unclear due to the lack of evidence on the mechanism by which senescent tumor cells initiate metastases, a fatal and therapeutically challenging hallmark of cancer. Here, we put forward the hypothesis that the genetic make-up of the tumors accounts for the distinct outcomes of senescence in cancer. Using different genetically engineered mouse models we demonstrate that inactivation of Timp1 is capable to switch senescence driven by the loss of PTEN and chemotherapy from a tumor-suppressive into a tumor-promoting response that drives metastases. Of note, depletion of senescent tumor cells by the senolytic compound ABT263 or p53 genetic inactivation significantly impeded metastases formation in Ptenpc-/-; Timp1-/- mice, indicating the involvement of senescence in the metastatic process. Moreover, enhancement of senescence by Docetaxel therapy decreased the proliferation of prostate tumors in Ptenpc-/- mice, whereas it accelerated metastases formation in Ptenpc-/-; Timp1-/- mice. Mechanistically, TIMP1-loss in PTEN-deficient cells reprogrammed the senescent-secretome significantly changing the levels of GDF15, FGF and IGFBP thereby promoting cellular invasion, migration and the up-regulation of a metastases-associated gene signature. In prostate cancer patients, we found that TIMP1 is commonly lost in combination with PTEN in advanced and metastatic prostate tumors and that TIMP1 inactivation is associated with TIMP1 promoter methylation. Altogether, these findings highlight the crucial role of TIMP1 as the gatekeeper that limits the pro-metastatic function of senescent tumor cells, implying that senescence inducing therapies should be administered with caution in patients affected by cancers with TIMP1 inactivation. Citation Format: Ilaria Guccini, Ajinkya Revandkar, Mariantonietta D'Ambrosio, Manuel Colucci, Emiliano Pasquini, Simone Mosole, Martina Troiani, Andrea Alimonti. Senescence reprogramming in primary tumors initiates prostate cancer metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-005.

Combined p53- and PTEN-deficiency activates expression of mesenchyme homeobox 1 (MEOX1) required for growth of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive cancer subtype for which effective therapies are unavailable. TNBC has a high frequency of tumor protein p53 (Tp53/p53)- and phosphatase and tensin homolog (PTEN) deficiencies, and combined p53- and PTEN-deficiency is associated with poor prognosis and poor response to anticancer therapies. In this study, we discovered that combined p53- and PTEN-deficiency in TNBC activates expression of the transcription factor mesenchyme homeobox 1 (MEOX1). We found that MEOX1 is expressed only in TNBC cells with frequent deficiencies in p53 and PTEN, and that its expression is undetectable in luminal A, luminal B, and HER2+ subtypes, as well as in normal breast cells with wild-type (WT) p53 and PTEN. Notably, siRNA knockdown of both p53 and PTEN activated MEOX1 expression in breast cancer cells, whereas individual knockdowns of either p53 or PTEN had only minimal effects on MEOX1 expression. MEOX1 knockdown abolished cell proliferation of p53- and PTEN-deficient TNBC in vitro and inhibited tumor growth in vivo, but had no effect on the proliferation of luminal and HER2+ cancer cells and normal breast cells. RNA-Seq and immunoblotting analyses showed that MEOX1 knockdown decreased expression of tyrosine kinase 2 (TYK2), signal transducer and activator of transcription 5B (STAT5B), and STAT6 in p53- and PTEN-deficient TNBC cells. These results reveal the effects of combined p53- and PTEN-deficiency on MEOX1 expression and TNBC cell proliferation, suggesting that MEOX1 may serve as a potential therapeutic target for managing p53- and PTEN-deficient TNBC.

PTEN suppresses epithelial\u2013mesenchymal transition and cancer stem cell activity by downregulating Abi1

The epithelial–mesenchymal transition (EMT) is an embryonic program frequently reactivated during cancer progression and is implicated in cancer invasion and metastasis. Cancer cells can also acquire stem cell properties to self-renew and give rise to new tumors through the EMT. Inactivation of the tumor suppressor PTEN has been shown to induce the EMT, but the underlying molecular mechanisms are less understood. In this study, we reconstituted PTEN-deficient breast cancer cells with wild-type and mutant PTEN, demonstrating that restoration of PTEN expression converted cancer cells with mesenchymal traits to an epithelial phenotype and inhibited cancer stem cell (CSC) activity. The protein rather than the lipid phosphatase activity of PTEN accounts for the reversal of the EMT. PTEN dephosphorylates and downregulates Abi1 in breast cancer cells. Gain- and loss-of-function analysis indicates that upregulation of Abi1 mediates PTEN loss-induced EMT and CSC activity. These results suggest that PTEN may suppress breast cancer invasion and metastasis via dephosphorylating and downregulating Abi1.

Leflunomide triggers synthetic lethality in PTEN-deficient prostate cancer.

BACKGROUNDThe loss of PTEN function presents in up to 50% of late-stage prostate cancers, and is therefore a potential target for therapeutics. PTEN-deficient cells depend on de novo pyrimidine synthesis, a feature which can present a vulnerability.METHODSWe utilized in vitro growth assays and in vivo xenograft models to test the effect of de novo pyrimidine synthesis inhibition on prostate cell lines.RESULTSHere, we demonstrate that PTEN-deficient prostate cancer cell lines are susceptible to inhibition of de novo pyrimidine synthesis by leflunomide. Tumor growth inhibition was observed in vitro and in vivo following leflunomide treatment, and is likely due to an overwhelming accumulation of DNA damage.CONCLUSIONSOur work highlights that synthetic lethality arises upon the combination of PTEN loss and leflunomide treatment in prostate cancer, and may present a therapeutic opportunity for this patient population.

Clinical and functional characterization of CXCR1/CXCR2 biology in the relapse and radiotherapy resistance of primary PTEN-deficient prostate carcinoma

Functional impairment of the tumour suppressor PTEN is common in primary prostate cancer and has been linked to relapse post-radiotherapy (post-RT). Pre-clinical modelling supports elevated CXC chemokine signalling as a critical mediator of PTEN-depleted disease progression and therapeutic resistance. We assessed the correlation of PTEN deficiency with CXC chemokine signalling and its association with clinical outcomes. Gene expression analysis characterized a PTENLOW/CXCR1HIGH/CXCR2HIGH cluster of tumours that associates with earlier time to biochemical recurrence [hazard ratio (HR) 5.87 and 2.65, respectively] and development of systemic metastasis (HR 3.51). In vitro, CXCL signalling was further amplified following exposure of PTEN-deficient prostate cancer cell lines to ionizing radiation (IR). Inhibition of CXCR1/2 signalling in PTEN-depleted cell-based models increased IR sensitivity. In vivo, administration of a CXCR1/2-targeted pepducin (x1/2pal-i3), or CXCR2-specific antagonist (AZD5069), in combination with IR to PTEN-deficient xenografts attenuated tumour growth and progression compared to control or IR alone. Post-mortem analysis confirmed that x1/2pal-i3 administration attenuated IR-induced CXCL signalling and anti-apoptotic protein expression. Interventions targeting CXC chemokine signalling may provide an effective strategy to combine with RT in locally advanced prostate cancer patients with known presence of PTEN-deficient foci.

A DNA Repair Inhibitor Isolated from an Ecuadorian Fungal Endophyte Exhibits Synthetic Lethality in PTEN-Deficient Glioblastoma.

Disruption of the tumor suppressor PTEN, either at the protein or genomic level, plays an important role in human cancer development. The high frequency of PTEN deficiency reported across several cancer subtypes positions therapeutic approaches that exploit PTEN loss-of-function with the ability to significantly impact the treatment strategies of a large patient population. Here, we report that an endophytic fungus isolated from a medicinal plant produces an inhibitor of DNA double-strand-break repair. Furthermore, the novel alkaloid product, which we have named irrepairzepine (1), demonstrated synthetic lethal targeting in PTEN-deficient glioblastoma cells. Our results uncover a new therapeutic lead for PTEN-deficient cancers and an important molecular tool toward enhancing the efficacy of current cancer treatments.

N6-methyladenosine mRNA methylation of PIK3CB regulates AKT signalling to promote PTEN-deficient pancreatic cancer progression

ObjectivePancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Thus far, most drugs have failed to significantly improve patient survival. N6-methyladenosine (m6A) plays an important role in...

Discovery of an Atropisomeric PI3Kβ Selective Inhibitor through Optimization of the Hinge Binding Motif.

A series of PI3Kβ selective inhibitors derived from a novel 4-(1H-benzo[d]imidazol-1-yl)quinoline chemotype has been rationally designed. Crucial to achieving the desired selectivity over the other class I PI3K isoforms, including the challenging δ-isoform, was the identification of a subset of substituted pyridine hinge binders. This work led to the discovery of (P)-14, a highly selective and orally bioavailable PI3Kβ inhibitor displaying an excellent pharmacokinetic profile in addition to great cellular potency in various PTEN-deficient tumor cell lines. Results from a dog toxicology study revealing structure-related, off-target ocular toxicity are also briefly discussed.

Targeting PI3Kβ alone and in combination with chemotherapy or immunotherapy in tumors with PTEN loss.

Background: PTEN-deficient tumors are dependent on PI3Kβ activity, making PI3Kβ a compelling target. We evaluated the efficacy of PI3Kβ inhibitor AZD8186 on tumors with PTEN loss. Results: In vitro cell viability assay and immunoblotting demonstrated that PTEN loss was significantly correlated with AZD8186 sensitivity in triple negative breast cancer (TNBC) cell lines. Colony formation assay confirmed sensitivity of PTEN-deficient cell lines to AZD8186. AZD8186 inhibited PI3K signaling in PTEN loss TNBC cells. AZD8186 in combination with paclitaxel, eribulin had synergistic effects on growth inhibition in PTEN loss cells. AZD8186 promoted apoptosis in PTEN loss cells which was synergized by paclitaxel. In vivo, AZD8186 had limited activity as a single agent, but enhanced antitumor activity when combined with paclitaxel in MDA-MB-436 and MDA-MB-468 cell-line xenografts. AZD8186 significantly enhanced antitumor efficacy of anti-PD1 antibodies in the PTEN-deficient BP murine melanoma xenograft model, but not in the PTEN-wild-type CT26 xenograft model. Methods: In vitro, cell proliferation and colony formation assays were performed to determine cell sensitivity to AZD8186. Immunoblotting was performed to assess PTEN expression and PI3K signaling activity. FACS was performed to evaluate apoptosis. In vivo, antitumor efficacy of AZD8186 and its combinations were evaluated. Conclusions: AZD8186 has single agent efficacy in PTEN-deficient TNBC cell lines in vitro, but has limited single agent efficacy in vivo. However, AZD8186 has enhanced efficacy when combined with paclitaxel and anti-PD1 in vivo. Further study is needed to determine optimal combination therapies for PTEN-deficient solid tumors.

PTEN-deficient cells prefer glutamine for metabolic synthesis

Abstract PTEN loss-of-function mutations frequently occur in gliomas and lead to poor overall survival. PTEN deficiency induces metabolic reprogramming, which may provide therapeutic targets. PTEN is known to impact the Warburg effect and glutaminolysis. To uncover essential glutamine-related metabolic changes specific in PTEN-deficient cells and thus provide potential therapeutic targets, we performed capillary electrophoresis–mass spectrometry-based metabolomics analysis and metabolic flux analysis under different glutamine culture conditions and PTEN alteration status. Glu, Asn, Gly, Ala, and 1-methylnicotinamide were decreased in PTEN-deficient cells under normal culture conditions. Meanwhile, under Gln-deprived culture conditions, Glu, citrate, and UTP synthesis were reduced and acetyl carnitine was increased in PTEN-deficient cells. The reliance on Gln was increased for metabolic intermediates synthesis but decreased for energy production in PTEN-deficient cells. However, the reliance on Gln for UTP synthesis cannot be targeted due to anaplerotic synthesis of UTP from other sources. How to target these metabolic addictions needs further research.