The PI3K/AKT/mTOR network plays a key regulatory function in cell survival, proliferation, migration, metabolism, angiogenesis, and apoptosis 
. Genetic aberrations such as loss of PTEN
as in SUM-149 cells used herein make this pathway one of the most commonly disrupted in human breast cancer. The common activation of the PI3K pathway in breast cancer has led to the development of compounds targeting the downstream effector, mTOR. The influences of other oncogenic pathways such as MAPK on the PI3K pathway and the known feedback mechanisms of activation have prompted the testing and development of compounds with broader effect at multiple levels to obtain a more potent antitumor activity and possibly a meaningful clinical effect. Our results show that Reishi exhibits these properties, as it affects the expression of various proteins of the PI3K/AKT/mTOR pathway.
We previously reported that treatment of inflammatory breast cancer cells with a commercially available extract consisting of 13% polysaccharides, 6% triterpenes and 1% cracked spores of Ganoderma lucidum
(Reishi) for 24 hours resulted in viability and invasion inhibition, tumor spheroid disruption, apoptosis induction and downregulation of key genes and proteins important in IBC 
. Herein, we investigated Reishi’s effects at early timepoints using the triple negative, PTEN
null, IBC cell line SUM-149 as an in vitro
model, and for longer treatment times using an in vivo
mouse model, focusing on mTORC1 downstream signaling effectors.
We show that Reishi modulatory effects begin to occur as early as 3 hours post-treatment at the level of mRNA expression/abundance, where 74/84 (88%) of the genes were downregulated and 19/84 (23%) were significantly downregulated in SUM-149 cells. Moreover, Reishi was found to compromise the protein expression of mTORC1 effectors. We show that the activity of mTOR through surrogate Ser(P)-2481 phosphorylation is reduced in Reishi treated cells, an effect not seen in total mTOR levels. Studies showed that mTOR Ser(P)-2481 is activated in a wortmannin-sensitive manner in both mTORC1 and mTORC2 complexes, demonstrating a requirement for PI3K in mTORC1 and mTOR2 autophosphorylation. Moreover, the level of mTORC1-associated mTOR Ser(P)-2481 correlates positively with the extent of mTORC1 signaling 
. Herein we show that Reishi reduces PI3KR1 and PI3KR2
gene expression, which code for PI3K regulatory subunits α and β. In addition, we show that Reishi extract also affects the expression of the best-characterized mTORC1 substrates, p70S6K and 4E-BP1. One of the consequences of Reishi’s modulatory effect is protein synthesis inhibition, which we also demonstrate to act by inhibiting cap-dependent translation (reduced eIF4F complex levels), and globally by reducing by half the levels of protein synthesis in IBC cells but not in non-cancerous mammary epithelial cells. Studies silencing eIF4G in SUM-149 IBC cells show reduced eIF4G protein levels by at least 90%, compared with control cells, but with only a slight reduction in overall protein synthesis (15%), no effect on cell viability, and only slightly impaired cell proliferation 
. Therefore, our results suggest that Reishi’s anti-cancer effect is not exclusively through eIF4G downregulation, but likely occurs through an inhibitory combination of signaling pathways that include PI3K and mTOR pathways and on levels/activity of eIF4F complex proteins.
Reishi significantly upregulated JUN
by 1.7 and 1.4 fold, respectively. However, these transcription factors are regulated at various levels including transcriptionally, via mRNA stability, post-translational modifications such as phosphorylation and by protein turnover. Active Fos proteins dimerize with Jun proteins to form Activator Protein-1 (AP-1), a transcription factor that binds to TRE/AP-1 elements and activates transcription of genes such as cyclin D1, which herein we show is modulated by Reishi treatment. Moreover, studies show that the same Reishi extract as the one used in the current study inhibits AP-1 and NF- κB transcriptional activation in MDA-MB-231 breast cancer cells 
. Contrary to Jiang et al. results, where they show that Reishi inhibits Akt expression in MDA-MB-231 breast cancer cells 
our study shows that in IBC cells, the same Reishi extract reduces AKT1
gene levels, but not Akt protein expression or phosphorylation. Independently, Reishi inhibitory effects on mTOR signaling and IBC progression are evident.
Our in vivo
studies show that Reishi treated mice have statistically significantly reduced tumor growth and tumor weight. Previous studies have shown that individual components from Reishi, such as specific polysaccharides or triterpenes inhibit invasion and metastasis in various xenograft models 
. However, our study is the first to show that an extract containing a combination of polysaccharides and triterpenes derived from the Reishi mushroom has anti-tumor growth effects in a very aggressive type of cancer. Even though the concentration of Reishi extract that was required to demonstrate a significant difference in tumor growth is higher than the current concentration suggested for humans, the concentration used was not toxic to the mice. Herein, we show that Reishi-treated mice displayed a disparate effect on tumor growth starting at week eight of treatment, and by the end of the 13th
week of the study the difference in tumor growth was almost 60%. The lag time in tumor growth observed for the first eight weeks was seen in both vehicle and Reishi treated animals. This is due to the unique phenotype of IBC, where SUM-149 cells tend to form a “diffuse” tumor during the first weeks post-inoculation. This phenomenon was reported in a similar study where caliper measurements could not be initiated until 30 days following SUM-149 cell inoculation at the mammary fat pad of SCID mice 
. This tumorigenic effect also resembles IBC tumor formation in women, where most present without a palpable mass at the time of diagnosis and then proliferates at high rates 
. At the end of the study, mouse tumors were excised and weighed; showing that Reishi treated tumors weighed 45% less than vehicle controls. Necrotic centers were observed equally in some vehicle and Reshi treated mice (data not shown). Reishi treated SUM-149 tumors showed a decrease in the levels of the Ki-67 cell proliferation and Vimentin mesenchymal markers compared with those from the control treatment. We previously reported that although E-cadherin expression is compromised by Reishi treatment in SUM-149 cells, β-catenin does not translocate into the nucleus 
. In this study, we confirm that although E-cadherin expression is reduced with Reishi treatment in vivo, epithelial to mesenchymal transition does not occur as shown by lower Vimentin levels in the tumors, and neither do we see a subsequent increase in proliferation due to a potential β-catenin translocation into the nucleus, as demonstrated by lower proliferation rates shown by reduced Ki-67 tumor levels.
The anti-tumor effects of Reishi at the molecular level depicted here may be the result of different compounds within the Reishi extract that are affecting and targeting various signaling pathways simultaneously. We show that Reishi treated tumors reduce the expression at both the gene and protein level of important molecules in the PI3K/AKT/mTOR and MAPK signaling pathways. Specifically, Reishi affects mTOR levels and therefore, activity. This result is in contrast to that seen in vitro
. However, our in vitro
studies presented here show Reishi treatment at early timepoints, thus it will be interesting to study the effects of Reishi on mTOR activation at the cellular level at longer exposure times to determine if it mimics the in vivo effects. In IBC SUM-149 cells, the PI3K/AKT/mTOR signaling pathway is elevated, due to the lack of PTEN
. Herein we demonstrated that Reishi downregulates the expression of molecules involved in this pathway, effectively circumventing the PTEN
null effect. Moreover, our study shows that Reishi reduced the activation of the parallel Ras/Raf kinase/extracellular signal-regulated (ERK) pathway via p-ERK 1/2 (Thr 202/Tyr 204) and reduced Ras levels. ERK1/2 synergizes with the mTOR pathway in the activation of p70S6K, the downstream effector of p-p70S6K in the form of p-S6 expression 
, and on the phosphorylation inactivation of 4E-BP1 
, which we also find to be downregulated by Reishi. Moreover, there seems to be an impact of convergent signaling on cell cycle progression as evidenced by a decrease in the Ki-67 proliferation marker.
In this study we also show that in vivo E-cadherin and p120-catenin downregulation occur together with reduced levels of eIF4G, confirming and extending our previous findings 
. Here we show that downregulation of these proteins contribute to reduced tumor growth. Similar results were found by Silvera and collaborators where eIF4G silencing results in less p120-catenin mRNA translation and subsequent E-cadherin cytoplasmic re-localization, disrupting tumor spheroid formation that is necessary for IBC invasion 
. Based on our findings, we conclude that Reishi is an anti-cancer agent that selectively affects gene and protein expression and therefore, activity of molecules involved on cancer cells and shows tumor inhibitory effects. This action can be correlated with reduced levels of key signaling pathways that ultimately increase cancer cell growth, proliferation and survival. To date, effects of Reishi extract have not been tested on IBC in vivo models or patients. Studies are being conducted in vivo to test the efficacy of Reishi in IBC using this SCID mouse model in combination with conventional therapy and in vitro
at longer exposure times. Therefore, our findings suggest that Reishi extract could be used as a novel anticancer therapeutic for IBC patients.