{'fraction_non_alphanumeric': 0.04061543918769121, 'fraction_numerical': 0.02598323948033521, 'mean_word_length': 4.820387096774193, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Tumours with PI3K activation are resistant to dietary restriction

Dietary restriction (DR) delays the incidence and decreases the growth of various types of tumours, but the mechanisms underlying the sensitivity of tumours to food restriction remain unknown. We find that certain human cancer cell lines, when grown as tumour xenografts in mice, are highly sensitive to the anti-growth effects of DR, while others are resistant. Cancer cells that form DR-resistant tumours carry mutations that cause constitutive activation of the PI3K pathway and in culture proliferate in the absence of insulin or IGF1. Substitution of an activated mutant allele of PI3K with wild-type PI3K in otherwise isogenic cancer cells, or the restoration of PTEN expression in a PTEN-null cancer cell line, is sufficient to convert a DR-resistant tumour into one that is DR-sensitive. DR does not affect a PTEN-null mouse model of prostate cancer, but significantly decreases tumour burden in a mouse model of lung cancer lacking constitutive PI3K signaling. Thus, the PI3K pathway is a major determinant of the sensitivity of tumours to DR and activating mutations in the pathway may influence the response of cancers to DR-mimetic therapies.

The life-prolonging anti-tumorigenic effects of dietary restriction (DR) were first described in the beginning of the twentieth century [1][2][3] . To date, reports indicate that the incidence, as well as the growth rate, of various tumour types is reduced in laboratory rodents undergoing DR, the latter being achieved by a 10 to 50% decrease in caloric intake [4][5][6][7][8][9][10][11] . Interestingly, the effects of DR are not uniform, with tumours from different tissues responding to restriction to different degrees, and a fraction of tumours originating in the same tissues being resistant to DR 4,7,[10][11][12][13][14] .
A consistent response of animals to dietary restriction is a reduction in the levels of circulating factors influencing cellular and organismal growth, notably insulin and insulin-like growth factor 1 (IGF1) 8,11,12,15,16 . Despite the common systemic changes that accompany DR and its general anti-tumorigenic effects, the question of whether all tumours, under similar experimental conditions, respond equally to DR remains unanswered. Here we investigate the responsiveness of different types of human and mouse tumours to DR. We identify the activation status of the PI3K pathway as a molecular signature that largely predicts the sensitivity of a tumour to DR and show that by modulating PI3K activation we can convert a DR-resistant tumour into one that is DR-sensitive.
Differential sensitivities of tumour xenografts to DR To investigate the responsiveness of different types of tumours to dietary restriction, we injected six established human cancer cell lines derived from the brain (U87-MG), colon (SW620), prostate (PC3) and breast (MDA-MB-231, MDA-MB-435, and MCF10DCIS.com 17 , hereafter described as MCF10DCIS) subcutaneously into NOD/ SCID (non-obese diabetic, severe combined immunodeficient) mice. MDA-MB-231 and MCF10DCIS cells were also injected orthotopically into the mammary fat pads of the mice. Xenograft-bearing mice were then fed ad libitum a standard rodent diet for 3-4 days (20 days in the case of U87-MG and orthotopic MDA-MB-231), after which they were subdivided into ad libitum fed or DR (dietary-restricted) groups for 2 to 3 weeks. All DR mice received daily meals amounting to 60% of the caloric intake of their ad libitum counterparts (that is, 40% DR), and underwent similar decreases in body weight (20-30%) at the end of the restriction period ( Supplementary Fig. 1a, b). In contrast, the tumours formed showed differential sensitivities to DR and could be classified into two main categories: DR-sensitive tumours, which showed significant decreases in tumour volume (MDA-MB-435, MDA-MB-231 and SW620), and DR-resistant tumours, which grew to similar sizes in ad libitum and DR mice (PC3, U87-MG and MCF10DCIS) ( Fig. 1a and Supplementary Fig.  1g). As with their subcutaneous counterparts, DR caused a significant reduction in the volume of orthotopically generated MDA-MB-231 tumour xenografts, whereas MCF10DCIS tumours were completely resistant to DR (Fig. 1b). Consistent with earlier reports 15,16 , in response to DR all mice experienced notable decreases in plasma levels of insulin and IGF1 ( Supplementary Fig. 1c-f). These findings suggest that factors other than systemic changes in the host underlie the differential sensitivities of the tumour xenografts to DR.

Constitutive PI3K signalling in DR-resistant tumours
Because the xenografted tumours responded differentially to DR despite similar decreases in insulin and IGF1 levels, we determined whether the six cancer cell lines studied have differential requirements for these factors for their growth in tissue culture. Indeed, in cell lines that form DR-sensitive tumours (MDA-MB-231, MDA-MB-435 and SW620) insulin or IGF1 caused a dose-dependent increase in cell numbers (Fig. 2a). In contrast, cell lines that generate DR-resistant tumours (MCF10DCIS, U87-MG and PC3) grew in culture in an insulin-and IGF1-independent fashion (Fig. 2b).
These results indicated that the cancer cell lines forming DRresistant tumours have a deregulation in an insulin/IGF1-activated signalling pathway, with the PI3K/Akt pathway 18 being an attractive candidate. Acting through receptor tyrosine kinases, insulin and IGF1 recruit PI3K to the cell membrane 19 , where its activity, which is antagonized by the PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor, leads to the recruitment and activation of Akt, a serine/threonine kinase. In turn, Akt phosphorylates and regulates several targets that enhance cellular growth and inhibit apoptosis. Consistent with a deregulation in PI3K signalling in cell lines that form DR-resistant tumours, serum withdrawal for 1 or 24 h did not affect Akt S473 phosphorylation, a marker of Akt activation, in MCF10DCIS, PC3 and U87-MG cells, but did in MDA-MB-231, MDA-MB-435 and SW620 cells (Fig. 2c). Analysis of PTEN expression showed that PTEN loss could account for the serum-insensitive Akt activity 20 in PC3 and U87-MG, but not in MCF10DCIS cells (Fig. 2d). Activating mutations in the PIK3CA gene, which encodes the catalytic subunit of PI3K, also lead to constitutive Akt activity in cancer cells [21][22][23][24] . By sequencing in all the cell lines exons 9 and 20 of PI3KCA-which can be the sites of the 'hotspot' E545K and H1047R mutations 21,25 , respectively-we found that the MCF10DCIS cells harbour a previously unknown H1047R mutation. None of the cell lines that form DR-sensitive tumours have lost PTEN function or carry PI3K activating mutations 26 (Fig. 2e). Furthermore, no other common oncogene-activating (for example, HRAS, KRAS or BRAF) or tumour suppressor-inactivating (for example, TP53) mutations correlated with tumour cell sensitivity to insulin/IGF1 in vitro or tumour sensitivity to DR in vivo 26 (Fig. 2e). Although PI3K can be directly activated by Ras 27 and this is necessary for the initiation of Ras-driven tumours 28 , the MDA-MB-231 and SW620 cell lines carrying activated KRAS alleles form DR-sensitive tumours (Fig. 2e). These results indicate that tumour resistance to DR correlates with constitutive activation (for example, through PIK3CA mutations or PTEN loss), rather than with hyperactivation of the PI3K pathway.

DR increases apoptosis in DR-sensitive tumours
Although phospho-S473 Akt could not be detected in DR-sensitive tumours by immunohistochemistry, it was readily detectable in DRresistant tumours and its levels did not decrease after DR (data not shown). Thus, to assay for changes in PI3K signalling in DR-sensitive tumours after restriction, we examined the FOXO1 transcription factor-a major downstream target of Akt that is selectively phosphorylated as a result of PI3K activation 21 . Phosphorylation of FOXO1 by Akt leads to its cytoplasmic sequestration and degradation so that a decrease in Akt signalling causes relocalization of FOXO1 to the nucleus, where it induces transcription of pro-apoptotic and anti-proliferative genes 29 . In the DR-resistant tumours (PC3 and MCF10DCIS), FOXO1 was predominantly localized to the cytoplasm of tumour cells in both ad libitum and DR mice ( Supplementary Fig. 2a, b). In contrast, DR caused a significant relocalization of FOXO1 from the cytoplasm to the nucleus in the cells of DR-sensitive tumours (MDA-MB-435, SW620 and MDA-MB-231) ( Supplementary Fig. 2a, b), indicating that in these tumours DR inhibited PI3K signalling.
Suppression by DR of the growth of DR-sensitive tumours could be the result of enhanced cellular death, decreased cellular proliferation, or both. To distinguish between these possibilities, we measured the number of tumour cells that were proliferating or undergoing apoptosis by assaying for Ki67 staining and caspase 3 cleavage, respectively. Although DR tended to decrease the number of Ki67positive cells in the DR-sensitive but not DR-resistant tumours, these effects did not reach statistical significance (data not shown). However, a common apoptotic response to DR was observed in all DR-sensitive, but not DR-resistant tumour xenografts ( Supplementary  Fig. 2c, d), correlating with the presence of nuclear FOXO1 in DRsensitive tumours ( Supplementary Fig. 2a, b). Indeed, whereas PC3 tumours maintained low levels of apoptosis under ad libitum or DR conditions, MDA-MB-435 tumours increased cellular apoptotic rates by 5.6-fold (from 7% to 39%; P 5 0.003) and SW620 tumours showed massive DR-induced apoptosis, which occurred at the edges of tumours (21.3-fold increase, from 2% to 42.5%) and also at their centres (2.9-fold increase, from 30.7% to 88.8%) averaging to a 3.6-fold increase (from 18.4% to 65.6%; P 5 0.0003) in whole tumours ( Supplementary Fig. 2c). In orthotopic MCF10DCIS and MDA-MB-231 xenografted tumours, no obvious apoptosis was noted after 12 days of DR (data not shown). Nevertheless, when these tumours were collected only 2 days after restriction, a significant sixfold increase in apoptosis was observed in the MDA-MB-231 (1.1 to 6.6%; P 5 0.05) but not the MCF10DCIS tumours ( Supplementary Fig. 2d), indicating that in MDA-MB-231 tumours, sensitivity to DR-induced apoptosis occurs at earlier stages of the tumorigenic process. Hence, these analyses show that decreased PI3K signalling in human tumour xenografts correlates with DR-induced apoptosis.

Conversion of a tumour from DR-resistance to DR-sensitivity
The correlation between tumour sensitivity to DR and the activation status of the PI3K pathway led us to consider that constitutive PI3K signalling in tumour cells is sufficient to decrease the sensitivity of tumours to DR. To begin testing this idea, we used two cell lines derived from the DLD-1 colorectal cancer cell line 21 . These cells are isogenic except that one carries a wild-type allele (DLD-WT) and the other has a constitutively active mutant allele (E545K; DLD-mut) of PIK3CA. Consistent with their genetic statuses, DLD-mut, but not DLD-WT cells, maintained high levels of phospho-S473 Akt after serum withdrawal for 1 or 24 h (Fig. 3a). The growth responses of the DLD-mut and the DLD-WT cells in culture to increasing concentrations of insulin or IGF1 (Fig. 3b, c) were reminiscent of those of the cancer lines that form DR-resistant and DR-sensitive tumours, respectively (Fig. 2).
In xenograft studies, the subcutaneous injection of 0.6 3 10 6 DLD-WT or 0.25 3 10 6 DLD-mut cells into NOD/SCID mice yielded tumours of approximately equal volume in 18 days (Fig. 3d). The application of a 14-day DR regimen to xenografted mice 4 days after tumour cell injection resulted in DLD-WT tumours that were 2.7fold smaller in volume than those in ad libitum mice (Fig. 3d). Although DLD-mut tumours grew at a faster rate than DLD-WT tumours, they showed no significant decrease in tumour size after DR (Fig. 3d). There was a significant relocalization of FOXO1 from the cytoplasm to the nucleus in the DLD-WT tumour cells after DR (Fig. 4a). Conversely, FOXO1 remained cytoplasmic in DLD-mut cells regardless of restriction (Fig. 4a). Furthermore, DLD-WT but not DLD-mut tumours had a significant enhancement in caspase 3 cleavage (12.5-fold) after the 14-day DR period (Fig. 4c). Thus, the presence of a PI3K activating mutation in an otherwise DR-sensitive tumour cell line is sufficient to protect tumours formed by these cells from the anti-growth and pro-apoptotic effects of DR.
Because PTEN loss also leads to aberrant PI3K activation 20 , we investigated the role of PTEN in determining tumour sensitivity to DR. We made use of a PTEN-null cell line (U87-MG) that was engineered to induce PTEN expression after the addition of doxycycline 30 . In culture in vitro, doxycycline-induced PTEN expression caused a decrease in phospho-S473 Akt levels in these cells (Fig. 3e,  left panel) and was sufficient to transform the serum-insensitive U87-MG cells (see Fig. 2c) into serum-sensitive cells that responded to serum withdrawal by decreasing levels of phospho-S473 Akt (Fig. 3e, right panel). In contrast to the PTEN-null U87-MG cells (Fig. 2b), PTEN-expressing U87-MG cells increased their net growth rates in response to increasing levels of insulin and IGF1 (Fig. 3f).
In vivo doxycycline administration induced PTEN expression in tumour xenografts formed from U87-MG cells (Supplementary Fig.  3a, b). As expected from restoring the expression of a tumour suppressor, the growth rate of U87-MG tumours decreased significantly in the presence of doxycycline, regardless of diet. Nevertheless, the reexpression of PTEN was sufficient to switch the response of U87-MG tumours from being resistant to DR (Figs 1a and 3g, left panel) to being DR-sensitive (Fig. 3g, right panel). After DR, the number of Ki67-positive cells tended to decrease (by 15%) in PTEN-expressing U87-MG tumours, but without reaching statistical significance (data not shown). In the absence of PTEN, FOXO1 was mostly sequestered in the cytoplasm of U87-MG tumour cells in ad libitum as well as DR mice (Fig. 4b). However, after DR of mice bearing PTEN-expressing U87-MG tumours, FOXO1 relocalized to the nucleus of tumour cells (Fig. 4b), and this correlated with enhanced apoptosis (Fig. 4d), consistent with results obtained for all DR-sensitive tumours (Supplementary Fig. 2 and Fig. 4). Therefore, restoration of PTEN expression in a DR-resistant, PTEN-null cancer cell line is sufficient to convert it to a cell line that forms DR-sensitive tumour xenografts in vivo.

FOXO1 overexpression sensitizes tumours to DR
To investigate the effects of the FOXO factors on the response of tumours to DR, we overexpressed FOXO1 in SW620 cells ( Supplementary Fig. 4a), and analysed the sensitivity to DR of the corresponding tumour xenografts compared to control tumours expressing green fluorescent protein (GFP). A cell line that forms DR-sensitive rather than DR-resistant tumours was used because FOXO1 function is unlikely to be regulated by DR in cancer cells in which Akt is constitutively active. FOXO1 overexpression in SW620 xenograft tumours slightly slowed tumour growth (30% decrease) in the ad libitum feeding condition, probably owing to aberrant entry of exogenous FOXO1 into the nucleus (Supplementary Fig. 4b). Notably, in tumours overexpressing FOXO1, DR caused a greater fold reduction in tumour volume (4.1-fold) than in tumours expressing GFP (2.3-fold) ( Supplementary Fig. 4b). Immunohistochemical analyses showed that after DR, FOXO1 localized to the nucleus in FOXO1-overexpressing as well as control tumours ( Supplementary Fig. 4c), particularly in areas that coincided with enhanced caspase 3 cleavage ( Supplementary Fig. 4d). Under ad libitum conditions the number of apoptotic cells was slightly enhanced in FOXO1-overexpressing tumours compared to the control tumours. However, the DR-induced apoptotic response increased to a greater extent after DR in FOXO1-overexpressing tumours than in control tumours ( Supplementary Fig. 4d). No significant changes in proliferation were detected among the different tumours (data not shown). These findings are consistent with FOXO1 having a crucial role in the anti-tumour effects of DR, but do not preclude the involvement of other PI3K effectors, including other FOXO family members.
A murine PTEN-null prostate cancer is resistant to DR We next examined the effects of DR in two engineered mouse models of cancer. The first model (probasin-Cre; PTEN lox/lox -hereafter Pb-Cre; PTEN L/L )) is driven by PTEN loss in the prostate and recapitulates human prostate cancer progression 31 . The second (KRAS LA2 ; p53-lox-stop-lox/WT-hereafter KRAS LA2 ; p53 LSL/WT ) is driven by KRAS activation as well as p53 heterozygosity and leads to the development of lung adenocarcinoma 32,33 . Compared to the ad libitum condition, DR significantly reduced the size of tumour nodules (42% decrease) in the lungs of 7-week-old KRAS LA2 ; p53 LSL/WT mice (Fig. 5a). Although the lung tumours were of similar pathological grade under both the ad libitum feeding and dietary restriction (Fig. 5b), DR strongly decreased the number of proliferating tumour cells (fourfold; 12.5% to 3.1%; P 5 8 3 10 28 ) without affecting the rates of apoptosis (Fig. 5c, d).
In the prostates of 11-week-old Pb-Cre; PTEN L/L mice, DR did not have any detectable effect on the extent or histological appearance of the prostate intraepithelial neoplasia (Fig. 5e) or on the activation levels of Akt (Fig. 5f). Indeed, neither the proliferative nor the apoptotic indices of the prostate intraepithelial neoplasia were affected by DR (Fig. 5g, h). Similar results were obtained in the prostates of 7-8-weekold Pb-Cre; PTEN L/L mice given the same DR regimen (data not shown). Thus, consistent with the human tumour xenograft studies, engineered mouse tumours without mutations that confer constitutive PI3K signalling seem to be DR-sensitive, whereas those with such mutations are DR-resistant.

Discussion
Dietary restriction has long been known to suppress tumour growth in laboratory rodents 10 . We find that genetic alterations in PIK3CA or PTEN can predict the response of tumours to DR, classifying them into DR-sensitive and DR-resistant tumours. Our results indicate that differential levels of PI3K activation in tumours contribute to their differential sensitivities to DR. It is important to note that we have studied the effects of relatively short-term DR on the growth of tumours at early stages of the tumorigenic process. Thus, at more advanced tumour stages, signalling pathways other than PI3K may have a central involvement in mediating the effects of DR.
It is interesting to consider which of the downstream effectors of PI3K may mediate the responses of tumours to DR that we have observed. Attractive candidates are the FOXO family of transcription factors that regulate proliferation and apoptosis. Reminiscent of the cell-type-and tissue-microenvironment-dependent effects of the FOXO factors on proliferation and apoptosis 34 , we find that DR causes strong suppression of proliferation in the engineered mouse lung cancer model, while having a prominent pro-apoptotic effect in the human tumour xenograft models. After DR, FOXO moves from the cytoplasm to the nucleus only in the cells of DR-sensitive tumours and overexpression of FOXO sensitizes tumours to the anti-growth effects of DR. These results indicate an important role for the FOXO factors but do not exclude potential roles for other PI3K effectors. The activity of signalling molecules that interact at several levels with the PI3K pathway, such as mTOR, AMPK and SIRT1, probably finetune the sensitivities of tumours to DR (see Supplementary Discussion).
Our findings are consistent with the effects of dietary restriction on the growth of early tumours being the result of both systemic changes in the host and signalling events intrinsic to the tumour. These findings should allow for the prediction of the responsiveness of a specific tumour to DR, on the basis of a readily measurable molecular signature, namely the activation status of the PI3K pathway in the tumour cells.

METHODS SUMMARY
Cell lines were obtained from the Karmanos Cancer Institute (MCF10DCIS.com), B. Vogelstein (DLD-WT and DLD-mut), M. M. Georgescu (U87-MG) and the American Type Culture Collection (ATCC) (all others), and grown under conditions described by the providers. SW620 cell lines stably overexpressing human FOXO1 or GFP were generated by infection with lentiviruses expressing the corresponding complementary DNA. Cellular proliferation was measured using the XTT kit (Roche). For immunoblotting, protein lysates were probed with phospho-S473 Akt, Akt or PTEN antibodies (1:1,000, Cell Signaling Technologies). Immunohistochemical detection of PTEN, FOXO1 and cleaved caspase 3 antibodies (1:100, Cell Signaling Technologies) was performed on paraffin-embedded, sliced tumour sections according to the manufacturer's protocols. The PI3K mutations E545K and H1047R were detected by sequencing two PIK3CA fragments amplified by RT-PCR from total RNA extracted from each cell line. All animal studies and procedures were approved by the MIT Institutional Animal Care and Use Committee. Mouse xenografts were generated by injecting tumour cells subcutaneously into male (female for breast cancer cell lines) or orthotopically in the fat pads of female NOD/SCID mice. Five-week-old KRAS LA2 ; p53 LSL/WT mice, obtained from the Jackson laboratory 32,33 , and 5-and 9-week-old Pb-Cre; PTEN L/L (ref. 31) mice were used in the DR studies. DR was achieved by providing individually caged mice a daily portion of a chow diet fortified with vitamins and minerals amounting to 60% of the daily food intake of their ad libitum counterparts. Mice were euthanized at the beginning of the light cycle, after retro-orbital blood withdrawal. Tumours were collected, measured and either flash-frozen in liquid nitrogen or fixed in formalin. Plasma insulin and IGF1 levels were determined by ELISA (CrystalChem and Diagnostic Systems Laboratories, respectively). Data are presented as means 6 s.e.m., and significant P values (# 0.05) were obtained by performing non-paired, two-tailed Student's t-tests to compare two groups.
Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.