Early phase trials targeting the T-cell inhibitory molecule PD-L1 have shown clinical efficacy in cancer. This study was undertaken to determine whether PD-L1 is overexpressed in triple-negative breast cancer (TNBC) and to investigate the loss of the phosphatase and tensin homolog (PTEN) as a mechanism of PD-L1 regulation. The Cancer Genome Atlas (TCGA) RNA sequencing data showed significantly greater expression of the PD-L1 gene in TNBC (n=120) compared to non-TNBC (n=716) (P<0.001). Breast tumor tissue microarrays were evaluated for PD-L1 expression which was present in 19% (20 of 105) TNBC specimens. PD-L1+ tumors had greater CD8+ T-cell infiltrate than PD-L1− tumors (688 cells/mm versus 263 cells/mm; P<0.0001). To determine the effect of PTEN loss on PD-L1 expression, stable cell lines were generated using PTEN shRNA. PTEN knockdown led to significantly higher cell-surface PD-L1 expression and PD-L1 transcripts, suggesting transcriptional regulation. Moreover, PI3K pathway inhibition using the AKT inhibitor MK-2206 or rapamycin resulted in decreased PD-L1 expression, further linking PTEN and PI3K signaling to PD-L1 regulation. Co-culture experiments were performed to determine the functional effect of altered PD-L1 expression. Increased PD-L1 cell surface expression by tumor cells induced by PTEN loss led to decreased T cell proliferation and increased apoptosis. PD-L1 is expressed in 20% of TNBC, suggesting PD-L1 as a therapeutic target in TNBC. Since PTEN loss is one mechanism regulating PD-L1 expression, agents targeting the PI3K pathway may increase the antitumor adaptive immune responses.

Purpose We sought to determine whether PI3K pathway mutation or activation state and rapamycin-induced feedback-loop activation of Akt is associated with rapamycin sensitivity or resistance. Experimental Design Cancer cell lines were tested for rapamycin-sensitivity, Akt phosphorylation and mTOR target inhibition. Mice injected with breast or neuroendocrine cancer cells and patients with neuroendocrine tumor (NET) were treated with rapalogs, and Akt phosphorylation was assessed. Results 31 cell lines were rapamycin-sensitive (RS) and 12 were relatively rapamycin-resistant (RR; IC50>100 nM). Cells with PIK3CA and/or PTEN mutations were more likely to be RS (p=0.0123). Akt phosphorylation (S473 and T308) was significantly higher in RS cells (p<0.0001). Rapamycin led to a significantly greater pathway inhibition and greater increase in p-Akt T308 (p<0.0001) and p-Akt S473 (p=0.0009) in RS cells. Rapamycin and everolimus significantly increased Akt phosphorylation but inhibited growth in an in vivo NET model (BON). In patients with NETs treated with everolimus and octreotide, progression-free survival correlated with p-Akt T308 in pretreatment (R=0.4762, p=0.0533) and on-treatment tumor biopsies (R=0.6041, p=0.0102). Patients who had a documented partial response were more likely to have an increase in p-Akt T308 with treatment compared to non-responders (p=0.0146). Conclusion PIK3CA/PTEN genomic aberrations and high p-Akt levels are associated with rapamycin sensitivity in vitro. Rapamycin-mediated Akt activation is greater in RS cells, with a similar observation in patients with clinical responses on exploratory biomarker analysis; thus feedback-loop activation of Akt is not a marker of resistance but rather may function as an indicator of rapamycin activity.

Purpose We tested the hypothesis that allosteric Akt inhibitor MK-2206 inhibits tumor growth, and that PTEN/PIK3CA mutations confer MK-2206 sensitivity. Experimental Design MK-2206 effects on cell signaling were assessed in vitro and in vivo. Its antitumor efficacy was assessed in vitro in a panel of cancer cell lines with differing PIK3CA and PTEN status. Its in vivo efficacy was tested as a single agent and in combination with paclitaxel. Results MK-2206 inhibited Akt signaling and cell-cycle progression, and increased apoptosis in a dose-dependent manner in breast cancer cell lines. Cell lines with PTEN or PIK3CA mutations were significantly more sensitive to MK-2206; however, several lines with PTEN/PIK3CA mutations were MK-2206 resistant. siRNA knockdown of PTEN in breast cancer cells increased Akt phosphorylation concordant with increased MK-2206 sensitivity. Stable transfection of PIK3CA E545K or H1047R mutant plasmids into normal-like MCF10A breast cells enhanced MK-2206 sensitivity. Cell lines that were less sensitive to MK-2206 had lower ratios of Akt1/Akt2 and had less growth inhibition with Akt siRNA knockdown. In PTEN-mutant ZR75-1 breast cancer xenografts, MK-2206 treatment inhibited Akt signaling, cell proliferation, and tumor growth. In vitro, MK-2206 showed a synergistic interaction with paclitaxel in MK-2206–sensitive cell lines, and this combination had significantly greater antitumor efficacy than either agent alone in vivo. Conclusions MK-2206 has antitumor activity alone and in combination with chemotherapy. This activity may be greater in tumors with PTEN loss or PIK3CA mutation, providing a strategy for patient enrichment in clinical trials.

The mammalian target of rapamycin (mTOR) signaling pathway has emerged as a promising target for cancer therapy. Rapamycin inhibits mTOR activity but induces upstream signaling, leading to Akt activation, potentially limiting antitumor activity. Octreotide, a somatostatin analog, decreases phosphatidylinositol-3-kinase/Akt signaling in some models, and thus theoretically may enhance rapamycin's antitumor activity. The aim of this study was to determine the antitumor activity of rapamycin and octreotide as single agents and in combination in neuroendocrine tumors. In carcinoid cell lines BON-1 and NCI-H727, cell proliferation was significantly inhibited by rapamycin in vitro, although rapamycin treatment did lead to Akt phosphorylation. Octreotide had limited antiproliferative effects alone, and did not demonstrate synergistic or additive interactions with rapamycin. Furthermore, octreotide did not overcome rapamycin-induced Akt phosphorylation. In vivo, rapamycin alone caused significant tumor suppression. Octreotide alone did not inhibit in vivo tumor growth and did not enhance rapamycin-mediated growth inhibition. In conclusion, rapamycin causes significant growth inhibition in carcinoid tumor cell lines in vitro and in vivo, thus mTOR is a promising therapeutic target for neuroendocrine tumors. Octreotide does not enhance the efficacy of rapamycin's antiproliferative effects in the models tested, and does not inhibit rapamycin-mediated feedback activation of Akt. Further study is needed in order to determine whether octreotide or other somatostatin analogs enhance the efficacy of mTOR inhibitors in other models.

Synopsis This article presents an overview of the PI3K/Akt/mTOR signaling pathway. As a central regulator of cell growth, protein translation, survival, and metabolism, activation of this signaling pathway contributes to the pathogenesis of many tumor types. Biochemical and genetic aberrations of this pathway observed in various cancer types will be explored. Lastly, pathway inhibitors both in development and already FDA-approved will be discussed.

Background The PI3K/AKT pathway is activated through PIK3CA or AKT1 mutations and PTEN loss in breast cancer. We conducted a phase II trial with an allosteric AKT inhibitor MK-2206 in patients with advanced breast cancer who had tumors with PIK3CA/AKT1 mutations and/or PTEN loss/mutation. Methods The primary endpoint was objective response rate (ORR). Secondary endpoints were 6-month progression-free survival (6 m PFS), predictive and pharmacodynamic markers, safety, and tolerability. Patients had pre-treatment and on-treatment biopsies as well as collection of peripheral blood mononuclear cells (PBMC) and platelet-rich plasma (PRP). Next-generation sequencing, immunohistochemistry, and reverse phase protein arrays (RPPA) were performed. Results Twenty-seven patients received MK-2206. Eighteen patients were enrolled into the PIK3CA/AKT1 mutation arm (cohort A): 13 had PIK3CA mutations, four had AKT1 mutations, and one had a PIK3CA mutation as well as PTEN loss. ORR and 6 m PFS were both 5.6% (1/18), with one patient with HR+ breast cancer and a PIK3CA E542K mutation experiencing a partial response (on treatment for 36 weeks). Nine patients were enrolled on the PTEN loss/mutation arm (cohort B). ORR was 0% and 6 m PFS was 11% (1/9), observed in a patient with triple-negative breast cancer and PTEN loss. The study was stopped early due to futility. The most common adverse events were fatigue (48%) and rash (44%). On pre-treatment biopsy, PIK3CA and AKT1 mutation status was concordant with archival tissue testing. However, two patients with PTEN loss based on archival testing had PTEN expression on the pre-treatment biopsy. MK-2206 treatment was associated with a significant decline in pAKT S473 and pAKT T308 and PI3K activation score in PBMC and PRPs, but not in tumor biopsies. By IHC, there was no significant decrease in median pAKT S473 or Ki-67 staining, but a drop was observed in both responders. Conclusions MK-2206 monotherapy had limited clinical activity in advanced breast cancer patients selected for PIK3CA/AKT1 or PTEN mutations or PTEN loss. This may, in part, be due to inadequate target inhibition at tolerable doses in heavily pre-treated patients with pathway activation, as well as tumor heterogeneity and evolution in markers such as PTEN conferring challenges in patient selection. Trial registration ClinicalTrials.gov, NCT01277757 . Registered 13 January 2011. Electronic supplementary material The online version of this article (10.1186/s13058-019-1154-8) contains supplementary material, which is available to authorized users.

Stearoyl-CoA desaturase 1 (SCD1) is an essential regulator of fatty acid synthesis. We have previously shown that overexpression of SCD1 increases the growth of breast cancer cell lines. The purpose of this study was to determine the relationship between SCD1 expression level and clinical-pathologic characteristics and survival of patients with breast cancer. Fine-needle aspirates were collected from the primary tumors of 250 patients with stage I–III breast cancer. Demographic and clinical characteristics including patient age, ethnicity, and menopausal status and tumor clinical stage, grade, and subtype were reviewed. SCD1 expression was analyzed using reverse-phase protein arrays. Samples were divided into high or low SCD1 expression levels based on a cut-off determined from martingale residual plots and regression tree analysis. SCD1 levels were significantly higher in tumors from patients >50 years old compared to patients ≤50 years old and were lower in triple-negative (estrogen/progesterone receptor-negative and human epidermal growth factor receptor-2-negative) breast cancers than other tumor subtypes. After adjusting for patient age, tumor subtype, tumor grade, and clinical stage, we found that patients with primary breast cancers expressing high SCD1 levels had significantly shorter relapse-free survival (RFS) (P = 0.014) and overall survival (OS) (P = 0.039) in multivariable analysis. We conclude that SCD1 expression varies by breast cancer subtype and that high levels of SCD1 expression are associated with significantly shorter RFS and OS in multivariable analysis. Future studies are needed to define the role of SCD1 in the malignant phenotype of breast cancer and to evaluate the potential for SCD1 as a therapeutic target.

Mammalian target of rapamycin (mTOR) signaling is a central regulator of protein translation, cell growth, and metabolism. Alterations of the mTOR signaling pathway are common in cancer, making mTOR a promising therapeutic target. In clinical trials, rapamycin analogs have shown modest response rates for most cancer types, including breast cancer. Therefore, there is an urgent need to better understand the mechanism of action of rapamycin to improve patient selection and to monitor pathway inhibition. To identify novel pharmacodynamic markers of rapamycin activity, we carried out transcriptional profiling of total and polysome-associated RNA in three breast cancer cell lines representing different subtypes. In all three cell lines, we found that rapamycin significantly decreased polysome-associated mRNA for stearoylCoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis. Activators of mTOR increased SCD1 protein expression, whereas rapamycin, LY294002, and BEZ235 decreased SCD1 protein expression. Rapamycin decreased total SCD1 RNA expression without inducing a significant decline in its relative polysomal recruitment (polysome/total ratio). Rapamycin did not alter SCD1 mRNA stability. Instead, rapamycin inhibited SCD1 promoter activity and decreased expression of mature transcription factor sterol regulatory element binding protein 1 (SREBP1). Eukaryotic initiation factor 4E (eIF4E) small interfering RNA (siRNA) decreased both SCD1 and SREBP1 expression, suggesting that SCD1 may be regulated through the mTOR/eIF4E-binding protein 1 axis. Furthermore, SCD1 siRNA knockdown inhibited breast cancer cell growth, whereas overexpression increased growth. Taken together these findings show that rapamycin decreases SCD1 expression, establishing an important link between cell signaling and cancer cell fatty acid synthesis and growth. Mol Cancer Ther; 9(10); 2770-84. ©2010 AACR.