Inhibition of pten and activation of akt by menadione

However, rapalogs have shown objective responses in only a subset of patients and unfortunately responses are frequently short lived. Therefore, there is a pressing need to identify predictors and pharmacodynamic markers of rapamycin response, and mechanisms of therapy resistance.

Activation of Akt has been proposed to be a predictor of rapamycin response 2 , 3. Rapamycin and its analogs have been shown to induce Akt activation 4 — 7. Rapamycin-induced Akt activation has been primarily attributed to the loss of this negative-feedback loop.

This feedback loop activation of Akt was not only seen in vitro , but was also observed in a Phase I clinical trial of rapamycin analog everolimus 8. There is concern that Akt activation may limit the antitumor efficacy of rapamycin and analogs.

The purpose of this study was to determine whether PI3K pathway mutations or Akt activation at baseline is a predictor of rapamycin sensitivity, and whether rapamycin-induced Akt activation is associated with resistance to rapamycin and analogs in vitro and in the clinic.

Cell lines used are described in the Supplementary Methods. Cells were plated in triplicate at densities of to 5, cells per well depending on growth characteristics of the cell lines. After adhering overnight, rapamycin response was determined by treating with six concentrations based on a fold dilution series range 0— nM. Cell growth was measured 5 days later using sulforhodamine B SRB assay as previously described 9.

The half maximal inhibitory concentration IC50 of rapamycin was determined based on dose-response curve Cells were treated with different concentrations of rapamycin 0 [vehicle], 1, 10 and nM , and harvested at various time points 2-, , and hour to capture dose and time effects. Two biological replicates per condition were used. Protein levels were expressed as the mean expression values in Log2. Immunohistochemistry IHC was performed on 25 archival samples, and pre- and on-treatment core biopsies.

The details of IHC technique has already been published Immunostaining was scored dichotomously staining intensity 0 vs. MCF7 xenografts were formed by inoculating 1. Mice were euthanized 24 hours after the first or fourth weekly injection day In rapamycin treatment studies, after tumors were formed, mice 12 mice for treatment and 11 mice for control groups were treated and euthanized as above. Consistent with recommendations from Veterinary Medicine at MD Anderson Cancer Center regarding ethical research of animals, treatment was ceased and animals were euthanized when average tumor burden in untreated control mice reached approximately mm 3 day In all three experiments, tumor growth was followed by caliper measurements and tumor volumes were calculated as previously described The primary objective of the trial was to assess the clinical activity of everolimus plus depot octreotide by progression free survival in treated and untreated patients with metastatic, unresectable low grade neuroendocrine carcinoma.

Sixty patients 30 carcinoid, 30 islet cell were enrolled on the trial. In the second half of the study, patients were approached to undergo pre and on-therapy tumor biopsies as an optional procedure. Two patients did not have tumor in one of the two core biopsies, and were eliminated from matched-pair analysis.

Sixteen patients who had paired evaluable biopsies received 10 mg everolimus po per day, one patient with matched biopsies received 5 mg po per day.

The cell line RPPA slide data consisted of samples and proteins slides with replicates , and were collected from 43 cell lines, with 4 treatments DMSO, Rapa1, Rapa10 and Rapa per cell line, 3 time points 2-, or hour come with per treatment, and 2 biological replicates set A and B.

To determine the differences in expression between RS and RR cell lines, we fitted a linear mixed model to each baseline protein expression level in the control vehicle DMSO treated cells. In this model, rapamycin sensitivity group and time were entered as fixed effects, and replicate was considered as a random effect Supplementary Methods.

To determine differences in pharmacodynamic response to rapamycin treatment in RS versus RR cells, we also used a linear mixed model incorporating an interaction term. Explicit mathematical formulas for the models are presented in the Appendix.

Means are reported for baseline measures and pharmacodynamic changes. We used the FDR to address the multiple comparison issue in our study.

The FDR, defined as the expected proportion of false positives among all significant test, is a statistical method frequently used to correct for multiple comparisons. Control and treatment groups were compared using unpaired t- or Mann Whitney U tests, where appropriate. For the neuroendocrine trial, paired t-test and two-sample t-test analysis were performed as appropriate to compare the protein expression of pre- vs. Pearson correlations were calculated between protein expression and progression-free survival of all patients.

ANOVA test were conducted to find the protein signature that manifests different expressions among response groups. Analyses were performed using R 2. To identify determinants of rapamycin sensitivity and mechanisms of resistance, we established a panel of 43 human cancer cell lines with differing genetic backgrounds, including different aberrations in the PI3K signaling pathway, including PIK3CA and PTEN mutations Supplementary Table 1. This panel was specifically enriched for cell lines reported to be rapamycin-resistant, based on published literature.

All forty-three human cancer cell lines were treated with increasing doses of rapamycin for hours and SRB assay was used to determine rapamycin half maximal inhibitory concentration. An IC50 of nM, a clinically achievable concentration 17 , 18 , was selected as a threshold for rapamycin sensitivity. Rapamycin sensitivity in cancer cell lines. Rapamycin responses were determined by treating 43 cell lines in triplicate with six concentrations based on a fold dilution series range 0— nM.

Cell growth was measured 5 days later using SRB assay. WT, wild-type; MT, mutant. To determine which proteins were differentially expressed between RS and RR cell lines, we measured the functional proteomic profile in cells cultured in the presence of vehicle only, and collected after 2, 24 and 72 hours of culture Supplementary Figure 1.

Rapamycin sensitivity and baseline Akt activation. All cell lines were cultured in the presence of vehicle only and collected after 2, 24 and 72 hours of culture. Using functional proteomics analysis, log transformed p-Akt S and p-Akt T baseline expression and their change in three time points for RS and RR cell lines are shown. A protein signature of PI3K pathway activity and rapamycin sensitivity.

Heat map showing pre-ordered 43 cancer cell lines and 17 PI3K hierarchically clustered pathway proteins. Bar at the left of the heat map indicate whether a cell line was RS green or RR red. Red, high expression; green, low expression according to z scores.

The mid-line of each box is at the median for the data represented by that box. The data presented here contrasts with that previously reported by Khan et al. Possible explanations for these discrepancies are that in the study of Khan et al. Unlike Khan et al. Together, our data satisfies the criteria laid out by Manning and Cantley 1 for a protein to constitute a PKB substrate. However, in the studies cited above, it appears that unless stimulated appropriately little InsP 3 R phosphorylation is detected.

The remarkable degree of sequence conservation through evolution of the PKB consensus site in InsP 3 Rs is suggestive of a fundamental role of phosphorylation of this region in InsP 3 R function. The COOH terminus of InsP 3 Rs is critically important in the regulation of channel gating and is a hotspot for interactions with other proteins, including huntingtin-associated protein, Bcl-X L , protein phosphatase 1a, and cytochrome c 12 , 14 , 26 — Using an in situ proximity ligation technique, we detected this interaction between endogenous PKB and InsP 3 Rs in cells either maintained in serum-containing medium or stimulated with insulin.

Activated PKB was, however, detected throughout the cell. Although PIP 3 has previously been localized to the ER 30 , we have shown a clear visualization of endogenous activated PKB interacting with one of its substrates in a location distal from the plasma membrane. The relevance of these findings to disease was shown in the U87 glioblastoma cancer cell line that exhibits increased PKB activity From these data we conclude that phosphorylation of InsP 3 Rs by hyperactive PKB was significantly responsible for the lack of sensitivity of the U87 cells to apoptotic stimuli.

In summary, we provide evidence for a mechanism by which InsP 3 R-mediated apoptosis is inhibited and by which PKB elicits its prosurvival effects Fig. This model also predicts that PKB constitutes a link between the cellular environment and growth factor status and can dynamically control IICR to determine cell fate.

Indeed, IICR is an upstream signal in the activation of other proapoptotic pathways such as those mediated by Bad and calpains 9. The recent descriptions of role for IICR in controlling autophagy 32 and cellular metabolism 12 , which are also targets for the PKB pathway, support this idea. In addition, our data contribute to the developing model that regulation of IICR is a nexus at which multiple signaling pathways converge to determine the physiological output of a given cellular stimulus.

Primary antibodies, unless otherwise stated, were from Cell Signaling Technology. All other chemicals, unless stated otherwise, were purchased from Sigma. Mikoshiba, University of Tokyo, Tokyo as template. Constructs were verified by sequencing. Tet suppressor HeLa cells were from Invitrogen and maintained as prescribed. Scrambled siRNA was used as control. Cells were infected at a multiplicity of infection of plaque-forming units 24 h before analysis or treatment.

A GFP-expressing adenovirus was used as control. Adenoviral vectors were prepared according to the supplier's instructions Microbix. Fura-2 imaging was performed as described Experiments were performed on three coverslips of 20 or more cells per day on 3 separate days.

UAPO oil immersion objective Figs. Image analysis was performed with ImageJ. Immunoblots were performed essentially as described Proteins were detected with polyclonal or monoclonal PKB antibodies both diluted , InsP 3 R1 was detected by using a polyclonal antibody generated in house against the COOH terminus of the protein as described Labeling experiments were performed similar to that described This assay was performed as described Proximity probes were constructed by conjugating oligonucleotides to a monoclonal PKB antibody and a polyclonal InsP 3 R1 antibody.

A probe for the Max oncogenic transcription factor has been described 19 and was used as a control. Cells were counterstained with an anticalnexin CLNX polyclonal antibody Sigma and visualized by using an AlexaFluor conjugated secondary antibody.

To this end, mean pixel intensity of region of interest of the image not covered by a cell was subtracted from the mean pixel intensity of a region of interest that was drawn around each cell. The data are presented as mean pixel intensity. The brightness of sample images was increased for presentation. Pearson's colocalization analysis was performed with Volocity software using the region of interest threshold option 37 version 4.

Lysates were prepared as for immunoblotting. When experiments involved transfection or adenoviral infection, growth media were replaced with fresh media or media containing apoptosis-inducing agent after 24 h.

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Provided by the Springer Nature SharedIt content-sharing initiative. By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Advanced search. In vitro, PTEN inhibition promoted their migration, but not the proliferation capacity. In addition, PTEN inhibitor treatment also improved the recovery of corneal nerve fiber density and sensitivity that was impaired in diabetic mice.