Pten null cell line

Hence, intracellular localization plays an important role s in the regulation of PTEN function s [ 16 ]. These various phosphatase-dependent and -independent functions of PTEN contribute to tumor suppression, and highlight the complexity of strategies to therapeutically target PTEN-deficient cancers.

Loss of PTEN function is a major determinant that affects tumor development across tissues. PTEN function and expression are modulated by germline and somatic PTEN mutations, genomic deletion, epigenetic and transcriptional silencing, post-transcriptional regulation, post-translational regulation, and protein-protein interactions [ 3 ].

Some patients with Cowden Syndrome harbor germline mutations in the PTEN promoter, or in possibly splice donor and acceptor sites [ 23 ]. PHTS patients have an increased lifetime risk of developing cancer [ 25 — 27 ]. Although the distribution of these mutations is mostly sporadic, several mutational hotspots have been identified at amino acids Arg, Arg and Arg Fig. However, PTEN mutations are not limited to a specific cancer subtype. Somatic mutations in PTEN occur throughout the coding region.

Domain mapping was obtained from UniProt [ ]. PTEN loss-of-function can also result from epigenetic and transcriptional silencing.

This hypermethylation has been observed in breast, colorectal, endometrial, gastric, hematological, liver, lung, skin melanoma , and prostate cancers, and glioma [ 46 — 55 ].

The p53 tumor suppressive transcription factor promotes PTEN expression. Accordingly, Sp1 overexpression upregulated PI3K pathway activation assessed by AKT phosphorylation , and promoted migration and invasion of human salivary adenoid cystic cancer cells [ 61 ]. Usually such miRNAs are specific to a particular cancer subtype; however, miR represses PTEN expression in many cancer subtypes and metabolic diseases [ 45 ].

Post-translational modifications including phosphorylation, acetylation, oxidation, and ubiquitylation have been shown to cause loss of PTEN function. The phosphatase activity of PTEN can be inhibited by phosphorylation of several serine and threonine resides in its C-terminal tail [ 64 , 65 ], which may be driven by the kinase CK2 [ 66 ]. PTEN can be also inhibited by oxidation and acetylation. PTEN contains a residue characteristic of protein tyrosine phosphatases termed a catalytic cysteine nucleophile which is prone to oxidation at Cys [ 67 ].

Reactive oxygen species can contribute to the formation of a disulfide bond between Cys71 and Cys in PTEN to inhibit its catalytic activity [ 68 , 69 ]. PTEN monoubiquitination at Lys13 and Lys promotes its nuclear localization and suppresses its phosphatase activity [ 17 ]. Several proteins have been shown to interact with PTEN to repress its tumor suppressive functions [ 3 ]. These proteins make attractive therapeutic targets to reverse loss of PTEN phosphatase activity in cancer cells.

In cells expressing wild-type p53, PTEN inhibits cell proliferation and increases apoptosis. In contrast, PTEN promotes proliferation and suppresses apoptosis in cells expressing mutant p PTEN knockdown in mutant pexpressing cells decreases proliferation and tumor growth in mice [ 83 ].

Thus, p53 status should be considered when PTEN may be involved in a pathway of therapeutic interest. As described above, PTEN expression may be lost by many non-genomic mechanisms. It is therefore necessary to determine PTEN status in tumors by both protein quantification and DNA sequencing, as neither method alone will provide comprehensive information.

Ideally, tumor PTEN phosphatase activity would also be quantified, although current technologies may not permit routine clinical implementation of such an assay. Methods to determine PTEN status by IHC were historically difficult and continue to be refined, which may contribute to conflicting results between studies. These issues are likely due to antibody and scoring variability. The most commonly used control for PTEN positivity is adjacent normal cells i.

Reverse-phase protein array RPPA analysis, which is analogous to quantitative high-throughput dot-blotting, of lysates from breast tumors showed that PTEN levels are strongly, inversely correlated with levels of activated AKT [ 86 ].

Efforts are underway to employ RPPA in routine clinical use; this method may be useful to accurately determine PTEN protein content, and is amenable to multiplex analysis of a panel of proteins and post-translational modifications [ 87 ].

Further confounding is the fact that tumors exhibited genetic heterogeneity. Biopsies of seven regions from a primary tumor and one from a liver metastasis within a patient with renal cell carcinoma were analyzed by DNA exome sequencing. Given that PTEN is often silenced at the transcriptional level, it is possible that the remaining intact PTEN allele in hemizygous prostate tumors was transcriptionally silenced.

It should be considered that different regions within a tumor may exhibit convergent phenotypic evolution i. Hence, determination of PTEN status may not be as important as determination of the resultant phenotype e. As such, therapeutics targeting several nodes of this pathway are under development.

Statistically significant PTEN -related sensitizing or resistance effects are indicated by green circles and red circles, respectively. In addition to promoting tumorigenesis, loss of PTEN drives resistance to anti-cancer therapeutics. These data suggest that targeted agents acting at the level of PI3K or downstream may be most effective for treatment of PTEN-deficient cancers, while agents targeting signaling nodes upstream of PI3K will be less useful.

GSIs are used to treat patients with melanoma because of their ability to prevent cleavage and activation of the Notch receptor, which is usually de-regulated in melanoma.

RO was only effective at inducing senescence and apoptosis in human melanoma cell lines with wild-type PTEN. Post-translational modifications in PTEN also confer resistance to targeted therapies. Several drugs are currently in clinical trials for the treatment of patients with PTEN-deficient cancers Table 2.

Drug sensitivity information gleaned from preclinical studies has been partially validated in the clinic. First, we must consider the putative mechanisms of Trastuzumab anti-tumor action: Trastuzumab binds to the extracellular domain of HER2 on the cell surface, disrupts HER2-HER3 heterodimers which activate PI3K [ 99 ], inhibits cleavage of the HER2 extracellular domain which removes the Trastuzumab-binding epitope from HER2 [ ], and induces antibody-dependent cellular cytotoxicity [ , ].

Several groups found that PTEN loss was generally associated with poor response to Trastuzumab therapy, whether this agent was administered in the neoadjuvant, adjuvant, or metastatic settings [ — ]. Contrary to these smaller studies, results from a recent phase III trial NCCTG N with 1, patients treated with adjuvant chemotherapy plus Trastuzumab sequential or concurrent revealed that PTEN status did not have an impact on disease-free-survival [ ].

In two phase I trials, patients with metastatic breast cancer that had progressed on Trastuzumab were treated with Trastuzumab plus Everolimus, with or without Vinorelbine chemotherapy.

These studies did not report PTEN status of tumors, but encouraging anti-tumor activity was observed [ , ]. Notably, all of the aforementioned trials have been conducted with patients with advanced breast cancer.

In a meta-analysis of five small clinical studies, PTEN loss in tumors was associated with decreased objective response, shorter progression-free survival, and shorter overall survival in patients with primary or metastatic CRC treated with Cetuximab-based therapy [ — ].

While PTEN deficiency has been associated with increased sensitivity to PI3K pathway inhibitors in preclinical studies in select cancer subtypes [ , — ], early clinical data from patients receiving single-agent therapies give mixed results. In a phase II study with patients with metastatic castration-resistant prostate cancer treated with Everolimus, PTEN loss determined by FISH was associated with improved response and longer progression-free survival [ ].

Such individuals exhibit macrocephaly, delayed mental development, skin lesions, vascular abnormalities, and cancer predisposition. The lifetime risks for cancer among PHTS individuals versus the general population are: female breast A gain-of-function somatic mutation in AKT1 was recently discovered in patients with Proteus Syndrome [ ].

Individuals with Tuberous Sclerosis carry germline mutations in TSC1 or TSC2 , exhibit benign tumors, and have an elevated risk of developing subependymal giant cell astrocytoma [ ].

Such signaling is known to promote cell growth and proliferation, leading to tissue overgrowth and the formation of benign tumors. However, DNA replication promotes the acquisition of additional genetic lesions, PTEN deficiency can increase genomic instability, and tissue overgrowth promotes inflammation.

These processes likely contribute to malignant transformation and cancer development. Trials testing TORC1 inhibitors Rapamycin, Everolimus in patients with Tuberous Sclerosis have shown promising results and tumor shrinkage in many cases [ — ]. This presents a challenge with the use of TORC1 inhibitors because the effects of long-term treatment are unknown, and these drugs elicit significant adverse events e.

Nuclear PTEN is important for the regulation of genome stability, homologous recombination, and apoptosis [ 15 , 16 ]. PTEN loss or disruption of nuclear import leads to severe chromosomal alterations [ 16 ]. Several recent studies have proposed that the role of PTEN in regulation of homologous recombination can be harnessed therapeutically.

PTEN deficiency incites a defect in homologous recombination in tumor cells. This defect sensitizes tumor cells to inhibitors of polyadenosine diphosphate ribose polymerase PARP , an enzyme important for repair of DNA double-strand breaks [ ]. In line with these findings, a mouse model with T cell-specific deletion of Pten develops T cell receptor Tcr - Myc translocations caused by a defect in Tcr recombination [ ].

PARP inhibitors are in widespread clinical testing for a variety of cancer subtypes with deficiencies in genome integrity, mainly in patients with breast or ovarian cancers harboring mutations in BRCA1 or BRCA2. Synthetic lethality occurs when aberrations in two genes cause cell death, but individually these aberrations do not.

Putz et al. Hopkins et al. This membrane-permeable lipid phosphatase enters other cells and antagonizes PI3K signaling [ ]. Other therapeutic strategies have been proposed to target tumors with PTEN loss. The recent development of targeted anti-cancer therapeutics has been focused primarily on oncogenes and tumor promoters, most commonly in the form of kinase inhibitors. Designing anti-cancer therapeutics directed at loss of tumor suppressors has traditionally been more difficult. However, this concentration of ZVAD-FMK completely blocked caspase 3 activation in response to anti-Fas antibody, indicating that this concentration of peptide was effective in blocking the effector caspase cascade in these cells Figure 3d.

That increased caspase 3 activity need not necessarily lead to substantial increases in apoptosis in T cells has been demonstrated previously Alam et al. In contrast to these previous studies, the percentage of cells present within the different stages of the cell cycle was largely unaffected by PTEN expression in Jurkat T cells Figure 4a.

These results are in accordance with the recently published data by Xu et al. On this basis, Xu et al. Such an effect would manifest as a slower rate of cell division, with no change in the static DNA profile of the cells. In the absence of dox induction, both cell lines showed the characteristic decline in CFDA-SE fluorescence that is indicative of cell division Figure 5. However, the degree of the shift was retarded in dox-treated PIJ cells compared to control cells. The delay in the leftward shift associated with proliferation was seen as early as day 2 not shown and was pronounced by day 5.

PTEN expression does not alter the cell cycle profile. The fluorescence intensity of the cells was evaluated daily for 5 days by flow cytometry. To confirm that the primary antiproliferative effect of PTEN expression in Jurkat T cells is due to delayed cell cycle progression, cell cycle passage was measured in a BrdU pulse-chase experiment. PTEN expressing cells progressed through the cell cycle without apparent arrest at any stage.

However, the percentage of cells entering and exiting G1 indicates that PTEN exerts a global inhibitory effect on the rate of cell cycle progression Figure 6a. The rate at which BrdU positive cells progress into G1 G1 entry provides an indication of the rate of transit through late S, G2 and M.

Likewise, the rate at which BrdU negative cells become depleted from the G1 pool G1 exit indicates the transit rate through G1. PTEN expression slows progression through the cell cycle. To determine G1 exit, the percentage of BrdU-negative cells in G1 is plotted against time middle panel. The percentage of cells in G2 left panel , G1 middle panel or S right panel is plotted against time. Cells were released from G2 block and analysed by propidium iodide PI staining at various time points to assess the percentage of cells in each phase of the cell cycle.

This correlated with a reduced rate of entry into G1 Figure 6b , middle panel. Thus, by these multiple measures, PTEN expression results in delayed progression through all stages of the cell cycle. The fact that the static DNA profile remains unchanged by PTEN expression indicates that the delay is proportional in all cell cycle stages. In order to elucidate the molecular mechanisms that may contribute to the effect of PTEN upon the rate of cell cycle progression, we carried out Northern blot analyses for a variety of cell cycle regulators.

Cyclin D1 is not expressed in T cells and could not be detected in our Jurkat T cells Ajchenbaum et al. PTEN affects expression of cell cycle regulatory genes.

The level of 18S ribosomal RNA was used as a gel loading control. Equal protein loading was confirmed by reblotting for ZAP Similar results were obtained using three different anti-p27 KIP1 blotting antibodies. ZAP was blotted on the same membrane to confirm equal loading of protein on the gels Figure 7b , lower panel. Quantitation of the signal in a similar experiment revealed a 4. The reason for the decrease in p27 KIP1 expression seen in the Con18 control cells is unclear.

In order to test if the novel effects of PTEN expression upon Jurkat T cells are mediated by an unusual biochemical pathway, or by the same pathway that mediates the effects of PTEN upon cell cycle arrest and apoptosis in other cell lines, constitutively activated Akt was expressed in PIJ and Con18 cells.

Expression of active Akt in the dox-treated Con18 cells had only a marginal effect upon the rate of proliferation; however, Akt dramatically accelerated proliferation in dox-induced PIJ cells, returning the rate of proliferation to that of a PTEN-null Jurkat T-cell line Figure 8.

These results indicate that PTEN slows cell cycle progression through the same biochemical pathways linked to cell cycle arrest or apoptosis in other systems. Akt reverses effects of PTEN on cell proliferation. Cell number was determined as for Figure 2. PTEN is inactivated in a high percentage of human tumours, and there has been considerable interest in understanding what role the loss of PTEN function plays in tumorigenesis.

Previous investigations have strongly supported a role for PTEN in mediating cell cycle, apoptosis, cell migration and cell growth for comprehensive reviews on the role of PTEN in solid tumours see Leslie and Downes , Waite and Eng , Wishart and Dixon For example, PTEN expression in glioma cell lines, prostate cancer cell lines and endometrial carcinomas leads to G1 arrest alone Furnari et al.

However, with the exception of a recent study by Xu et al. In contrast to what has been reported for solid tumours, expression of PTEN in these T-cell leukaemias, at levels sufficient to restore basal D3-phosphoinositide levels, neither caused cell cycle arrest nor stimulated significant increases in the rate of cell death, either by apoptosis or necrosis.

Instead, PTEN expression slowed progression through all phases of the cell cycle, thereby reducing the rate of cell division, while maintaining a static DNA profile with the same percentage of cells in each phase of the cycle. These results suggest that the importance of PTEN loss in the transformation of T lymphocytes may be largely independent of its antiapoptotic effects, and may instead be primarily related to its acceleration of the proliferation rate.

On the other hand, mature haematopoietic cells are not contact dependent and are not subject to anoikis. We speculate that, in cells that are not primed for apoptosis upon withdrawal of activated Akt anoikis-insensitive cells , the ability of PTEN to uniformly delay progression throughout the cell cycle may represent the dominant mode of action of PTEN.

Such a model, wherein PTEN expression has more of a modulatory function, would seem to make sense given that PTEN is known to be expressed in normal cells that are neither apoptotic nor arrested in G0 or G1, and is more in keeping with the effects that modulation of dPTEN expression has in Drosophila Stocker and Hafen, PTEN and myotubularin: novel phosphoinositide phosphatases.

Joining Europe PMC. Tools Tools overview. ORCID article claiming. Journal list. Grant finder. External links service. Annotations submission service. Developers Developer resources. API case studies. SOAP web service. Annotations API. OAI service. Bulk downloads. Developers Forum. Search syntax reference. Contact us Helpdesk. Tech blog. Developer forum. Let us know how we are doing. Red dash line marks the boundary between cancer acini and stroma areas. D RNA-seq analysis shows T cell inflamed phenotype after drug withdrawal.

The relative expression levels of indicated genes in each sample were determined and the statistical analysis was performed based on the average of expression levels of each cohort. RNA-seq analysis of the bulk tumor tissues revealed that BAY-I treatment could increase the T cell-inflamed gene expression profile 12 and the expressions of MHC class I and II molecules, which were well maintained in 4-week drug withdrawal group Fig. Importantly, the dendritic cell DC -associated genes were significantly upregulated in the drug withdrawal group Fig.

Together, these results demonstrated that BAY-I treatment can prime the tumor and generate a persistent T cell inflammatory environment even in the absence of continued drug administration. Effective priming of tumor antigen-specific T cells requires secondary lymphoid organs such as lymph nodes 6. In Pten -null prostate tumor tissues, we found tertiary lymphoid structures TLS with clear B and T cell zones, resembling germinal center morphology 45 Fig. Castrated Pten -null mice were treated with 4 cycles of BAY-I then drug was withdrawal for 4—10 weeks.

The same staining were performed with 3 mice and similar results were observed. Castrated Pten -null mice were treated with 4 cycles of vehicle, BAY-I then withdrawal for 4 weeks, or treated with anti-PD-1 antibody as indicated in Fig. Correlation between TLS score and Cd8a expression was calculated. Statistic was performed by Pearson correlation coefficient. The TLS may also account for persistent T cell-inflamed phenotype after drug withdrawal.

On the other hand, late exhaustion marker TIM-3 and CTLA-4 were not expressed on the cell surface even through their gene expressions were upregulated Figs. A schematic illustration of treatment strategy. The same staining were performed with 6 mice in all cohort and similar results were observed. Data were presented as mean with dot plots; N. To test this, we first treated castrated Pten -null prostate cancer model with BAY-I for 4 weeks, then dosed with control or PD-1 antibody for 4 weeks Fig.

The mouse body weights did not change significantly Supplementary Fig. Sequential BAY-I and anti-PD-1 combination treatment significantly decreased cancer cell areas than isotype treated cohort, while this combination effect was absent, in the Pten -null; Cd8- KO prostate Fig.

As successful immunotherapy often associated with long-lasting durable therapeutic effects 50 , we investigated the potential long-term effect of sequential combination of BAY-I and anti-PD-1 therapy. Together, these results indicated that a sequential combination of BAY-I and anti-PD-1 therapy could lead to a long-lasting, durable immune cell-mediated anti-tumor effect even after complete drug withdrawal. We demonstrate in this study that a carefully designed isoform-specificity and dosing schedule for PI3K inhibitor, and sequential administration of targeted and anti-PDmediated ICT can effectively overcome resistance to ICT in a preclinical prostate cancer setting and achieve a long-last durable immune cell-mediated therapeutic effect even after drug withdrawal.

These are cancer cell intrinsic property and are reversible upon BAY withdrawal 30 Fig. Our study showed that not only the isoform profile but also the dosing schedule of BAY is critically important for promoting anti-cancer immunity.

The differential effects of daily vs. Intermittent treatment could also minimize aberrant immune activation in non-cancerous organs, avoiding adverse side-effects Supplementary Fig.

Intermittent dosing of PI3K inhibitors have been reported by other works and shown to reach successful therapeutic effect while improve drug tolerance 35 , 53 , Optimization the dosing schedule of these inhibitors may also improve their therapeutic effects as monotherapies or in combination with ICT.

A recent report demonstrates a cloning replacement of tumor-specific T cells following ICT in human basal or squamous cell carcinoma, and suggest that pre-existing tumor-specific T cells may have limited role in ICT The different conclusions on the origins of tumor-specific T cells in our study and those by Yost et al.

However, other immune cells may also contribute to the overall therapeutic outcome. Together, the superior effects of intermittent BAY treatment support the idea that drugs that can co-target both cancer cell-intrinsic and microenvironment pathways may have considerably more clinical benefit than single-target drugs.

Treatments that can improve T cell infiltration may augment ICT efficacy 2 , 5 , Intriguingly, once the tumor has become T cell-inflamed, it stays in T cell-inflamed status even after drug withdrawal Figs. This T cell-inflamed state paves the way for successful anti-PD1 treatment.

Importantly, the T cell-inflamed state and memory T cell signature remain even after 4 weeks of anti-PD1 antibody withdrawal, demonstrating long-last and durable anti-tumor immunity Fig. Although the detailed mechanisms associated with this prolonged response require further investigation, our study provides a successful pre-clinical case for sequential, instead of simultaneous, anti-PI3K and ICT combination treatment to avoid potential combined toxicity when both drugs are used together.

Future works are need to test the effects of BAY-I and sequential BAY-I and anti-PD-1 treatment strategies on other models with different cancer-initiating and immune evasion mechanisms, and in humanized mouse cancer models before moving to clinical settings.

In summary, our results demonstrate that a carefully designed anti-PI3K treatment, both in its specificity and dosing schedule, to inhibit cancer cell growth while promote anti-tumor immunity, is critically important for successful combination of anti-PI3K targeted therapy and ICT. For surgical castration, mouse was firstly anaesthetized by avertin. Then, a midline ventral skin incision was made into the scrotum and testicles were removed from both sides by sealing off the blood vessels using suture line Jinhuan Medical, CR The skin incision was then sutured using suture line.

The animals were closely monitored during the procedure for signs of pain or bleeding and placed in a clean cage for post-surgery recovery. The signal was detected by using ECL western blotting substrate kit Thermo fisher, and The following secondary antibodies were used: HRP-conjugated anti-mouse antibody Jackson ImmunoResearch Laboratories, ; Image Lab 5. Prostates were dissected and photographed, weighted and minced in sterile tissue culture dishes, and subjected to collagenase A 1.

Data was analyzed by using flowjo software. Cells were seeded at a density of 2. Four days later, cells were harvested, live cell numbers were counted by cell counter, and T cell proliferation rate proliferated cell was determined by CFSE staining was determined by FACS. Cell growth rate was calculated by the percentage of live, proliferated cell number at each drug concentration vs. BAY was dissolved in 0. The slide was washed in PBS pH 7. The antibodies used and dilutions were listed in Supplementary Table 2.

All antibodies used are listed in Supplementary Table 2. Primers were listed in Supplementary Table 2. All RNA-seq data were aligned to the mm10 genome using Tophat version v2. Differentially expressed genes were identified by Cuffdiff version v2. FPKM was used for following analysis and comparison. GSEA analysis was performed as software suggested The pathway activity score was calculated with GSVA The expression levels of TLS score genes 46 were scaled to the range of 0 to 1, and TLS score was defined as the mean scaled value of related genes for each individual sample,.

Further information on research design is available in the Nature Research Reporting Summary linked to this article. Source data are provided with this paper. All RNAseq raw data used in Figs. Sharma, P. Immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential.

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