The PI3K/Akt/mTOR pathway stimulates protein synthesis, angiogenesis, and tumor proliferation and is an important target in multiple malignancies. Its activation has been implicated in resistance to endocrine agents, chemotherapy, and HER2-directed agents. The combination of PI3K/Akt/mTOR pathway inhibition with traditional or targeted agents may overcome this resistance.

Everolimus is approved for the treatment of advanced hormone receptor-positive breast cancer and is currently being evaluated in other breast cancer subtypes. Ongoing studies are also assessing biomarkers that will help define the patients who will derive the most benefit.

The PI3K (phosphoinositide-3-kinase)/Akt (protein kinase B)/mTOR (mechanistic target of rapamycin) pathway responds to nutrient availability and growth factor tyrosine kinases. It plays a significant role in stimulating protein synthesis, angiogenesis and tumor proliferation, and inhibiting apoptosis.

The pathway starts with the PI3K unit, which comprises two subunits, the regulatory (p85) and the catalytic (p110) subunit; each has different isoforms and is encoded by its corresponding genes. The regulatory subunit is encoded by 

 and the p110α, p110β, and p110δ subunits are encoded by 

 PI3K leads to the phosphorylation of Akt, a serine/threonine kinase, which activates the mechanistic (or mammalian) target of rapamycin (mTOR) by inhibiting the tuberous sclerosis complex proteins 1/2 (TSC1/2).

 mTOR is a serine/threonine protein kinase that consists of complexes mTORC1 (mTOR Complex 1) and mTORC2 (mTOR Complex 2). mTORC1 is the main target of rapamycin analogs, while more recent data suggest that mTORC2 is also inhibited with sufficient dosing and exposure (Figure 1).

Everolimus is approved by the FDA (Food and Drug Administration) for the treatment of hormone receptor (HR)-positive breast cancer in postmenopausal women who had previously experienced disease progression or recurrence with a non-steroidal aromatase inhibitor, based on the BOLERO-2 trial.

This review will focus on the current data of mTOR inhibitors in breast cancer. However, as the role of mTOR inhibition in breast cancer is evolving, ongoing studies are evaluating biomarkers that will identify the patients who will benefit.

The Role of mTOR Pathway in Breast Cancer

Aberrations of the PI3K/Akt/mTOR pathway have been implicated in the pathogenesis and growth of multiple malignancies. PIK3CA is the most common mutation found in breast cancer and is thought be oncogenic.

The mTOR pathway has been implicated in trastuzumab resistance in HER2-overexpressing breast cancer.

 HER2-targeted therapy resistance has been associated with loss of PTEN, PI3K phosphorylation, and activation of mTOR.

 mTOR inhibition can sensitize HER2-overexpressing breast cancer to HER2-directed therapy.

 In HR-positive breast cancer, the PI3K/Akt/mTOR signaling pathway can stimulate ERα (Estrogen Receptor Alpha) in both an estrogen-dependent and estrogen-independent manner

; thus, activation of the mTOR pathway appears to be involved in endocrine resistance.

 Preclinical data has demonstrated that inhibition of mTOR can restore hormone sensitivity, promote cell cycle arrest, and induce apoptosis when used in combination with endocrine therapy

 In triple negative breast cancer models, mTOR was also found to be activated and to mediate invasiveness and metastasis.

In conclusion, preclinical data demonstrate that activation of the mTOR pathway plays a role in the pathogenesis, promotion of invasiveness, and the development of resistance in various subtypes of breast cancer; this supports the further evaluation of mTOR inhibition in breast cancer, specifically in cancers that are resistant to standard therapies.

Multiple clinical studies have evaluated the role of mTOR inhibition in breast cancer. Several landmark trials have shown its benefit especially in the HR-positive setting. Phase 3 clinical data have led to the approval of everolimus with exemestane in postmenopausal women with HR-positive, HER2-negative metastatic breast cancer.

A phase 3 study that evaluated temsirolimus in combination with letrozole as a first-line treatment for HR-positive tumors (HORIZON) failed to show a significant improvement in progression-free survival (PFS) with mTOR inhibition, which was the primary endpoint and was closed for futility. The median PFS (9 and 8.9 months in the temsirolimus and placebo arm, respectively), objective response rate (ORR) (27% in both treatment groups), and clinical benefit rate (CBR) (17% and 19%, respectively) were similar in the temsirolimus and placebo arms, while more side effects were experienced by patients receiving temsirolimus.

The BOLERO-2 trial was a randomized, phase 3 trial of everolimus or placebo in combination with exemestane in postmenopausal women with advanced or metastatic HR-positive breast cancer; the eligible patients had disease relapse or progression on prior non-steroidal aromatase inhibitors (AI), such as anastrozole or letrozole.

 An interim analysis revealed that everolimus significantly improved the primary endpoint of PFS. The local assessment showed a PFS of 6.9 months in the treatment arm, versus 2.8 months in the placebo arm (HR = .43, 

 <.001); the central assessment showed a PFS of 10.6 months versus 4.1 months, respectively (HR = .36,

An updated analysis with 18-month median follow-up showed continued benefit of mTOR inhibition versus placebo, 7.8 months versus 3.2 months, respectively (HR = .45, 

 <.0001) by investigator analysis, and 11 months versus 4.1 months, respectively (HR = .38, 

 The benefit was seen regardless of the presence of visceral metastases.

 Although there were fewer deaths in the everolimus arm, compared to the placebo arm, the difference was not statistically significant.

 More adverse events were reported in the treatment arm and additionally, more patients in the everolimus arm discontinued treatment.

 The biomarker analysis of BOLERO-2 reported that the efficacy of everolimus was independent of the genetic status of the tumors.

The TAMRAD trial was an open label, phase 2 study that evaluated everolimus plus tamoxifen in patients with HR-positive metastatic breast cancer. The CBR at 6 months, which was the primary endpoint, was significantly better with mTOR inhibition (CBR = 61% with everolimus versus 42% with tamoxifen alone,

 = .045). Time to progression and risk of death were also superior in the everolimus group.

A neoadjuvant study by Baselga, et al evaluated everolimus with letrozole in postmenopausal women with operable HR-positive breast tumors.

 Patients received letrozole and everolimus or letrozole and placebo for 4 months prior to definitive surgery. The primary endpoint, clinical response by palpation, was found to be significantly improved in the everolimus group. Everolimus also had a more significant effect on Ki67 down regulation. Tumors that harbor mutations in the PIK3CA exon 9 helical domain showed a relatively small antiproliferative response to letrozole alone but had a good response to the combination of everolimus and letrozole.

The HORIZON study data and the data reported with everolimus clearly contrast as noted above. This can be partly explained by the different patient population enrolled in these studies. Patients treated in the HORIZON trial were treated as first line for metastatic disease. About 60% were naïve to endocrine therapy.

The BOLERO-2 trial required prior exposure to a non-steroidal aromatase inhibitor and also required progression or recurrence during, within 1 month of metastatic treatment, or within 12 months of adjuvant treatment with these agents, thus implying aromatase inhibitor (AI) resistance.

The TAMRAD trial also showed that the mTOR inhibition benefitted patients with AI-resistant disease. Other possible explanations for the difference in results is the difference between everolimus and temsirolimus, agents with routes of administration, dosing and schedule. Finally, tumor biology and the presence of activating mutations might alter the efficacy of those agents, however not all studies have correlative data in order to draw a conclusion on tumor differences between trials.

The possible role of PI3K pathway in mediating resistance to HER2-directed therapy was reported in a recent correlative study evaluating 504 tumor samples from patients that participated in the neoadjuvant GeparQuattro, GeparQuinto, and GeparSixto studies.

 mutations in this HER2 amplified patient cohort was 21.4%. The presence of the mutation was associated with a significantly lower pathologic complete response (pCR) rate (19.4% versus 32.8% wild-type, 

 = .008). This was also shown in the HR-positive cohort (11.3% versus 27.5% wild-type, 

 = .011). Clinical outcomes were not significantly different based on the 

 mutation status. These data are supported by the biomarker results of the Cleopatra study, which reported that median PFS was longer in patients with wild-type 

In early phase trials that accrued patients with HER2 amplified trastuzumab-resistant advanced breast cancer, the addition of everolimus to trastuzumab-based chemotherapy resulted in promising outcomes.

 The overall response rate (ORR) was noted to be 44% when everolimus was added to paclitaxel plus trastuzumab and 19% when added to vinorelbine plus trastuzumab in 2 phase 1 trials.

 A phase 2 study of everolimus in combination with paclitaxel and trastuzumab demonstrated an ORR of 21.8%, a CBR of 36.4%, a median PFS of 5.5 months, and an estimated overall survival (OS) of 18.1 months.

BOLERO-3 was a phase 3 study that evaluated everolimus (5 mg/day orally) in patients with advanced or metastatic HER2 overexpressing breast cancers resistant to trastuzumab.

 Trastuzumab resistance was defined as having a recurrence within 12 months of adjuvant therapy or within five weeks of starting trastuzumab for metastatic disease. All patients had also been treated with a taxane and about half of the patients had HR-positive cancers. Enrolled patients received everolimus or placebo, plus vinorelbine and trastuzumab.

The trial showed that everolimus improved the PFS by local review, which was the primary endpoint, from 5.78 to 7 months, with an HR of 0.78 (

 A central review and an adjudicated review, both of which were done retrospectively, adjusted the HR to 0.88 (95% CI 0.71—1.07) and 0.85 (95% CI 0.70–1.03), respectively. Subset analysis revealed that the positive signal with the addition of everolimus was noted in patients with HR-negative cancers, but not in HR-positive cancers, though this finding is only hypothesis-generating at this time. OS data from BOLERO-3 are currently still immature.

BOLERO-1 randomized patients with advanced HER2 overexpressing cancer to first-line paclitaxel, trastuzumab, and everolimus (10 mg/day orally), or placebo. The trial did not reach its primary endpoint of investigator-assessed improvement in PFS, even though there was a trend towards improved outcomes in patients with HR-negative cancer, this did not reach statistical significance.

The above studies indicate the need to further investigate the role of mTOR inhibition in HER2 amplified breast cancer, and especially in HR negative disease, a subset of patients where results are more promising.

Several clinical trials have evaluated mTOR inhibition in the triple negative setting.

A phase 2 neoadjuvant study in patients with triple negative breast cancer (TNBC) compared weekly paclitaxel for 12 weeks followed by FEC (fluouracil/epirubicin/cyclophosphamide), to the combination of everolimus (30 mg orally once a week) and paclitaxel, followed by FEC. The combination of paclitaxel and everolimus in this trial was well tolerated and more than 85% of patients completed treatment. Grade 3-4 toxicities included anemia, neutropenia, rash, and gastrointestinal side effects. However, this study used a weekly regimen of everolimus as opposed to the daily 5-10 mg oral regimen used in other studies. At 12 weeks, the response rates between the non-everolimus versus the everolimus arm were 29.6% versus 47.8% (

 = .075), respectively, favoring everolimus, however the pCR rates were similar at 25.9% versus 30.4% (

 = .76), respectively. The downregulation of mTOR by protein expression after 48 hours of treatment did not correlate with the response rates in the everolimus group.

The GeparQuinto trial combined everolimus with paclitaxel in TNBC patients in the neoadjuvant setting and found no difference in disease-free survival (DFS) (HR 0.997; 

A phase 2 study of neoadjuvant paclitaxel and cisplatin for twelve weeks with or without everolimus did not show any benefit in clinical response with the addition of everolimus and no significant improvement in pCR.

A phase 2 trial of carboplatin with daily oral everolimus in patients with TNBC demonstrated a 6-month CBR of 36% (95% CI 21.1 to 57.4%). Median PFS was 3 months (95% CI 1.6 — 4.6 months) and OS was 16.6 months (95% CI 7.3 months to not reached).50 These data demonstrate that the combination of everolimus with cytotoxic chemotherapy is possible and safe and may be worth exploring in future trials.

The most common adverse events encountered with everolimus are stomatitis, rash, fatigue, diarrhea, cough, and hyperglycemia.

 Stomatitis is one of the most common side effects, seen early during the course of treatment; it is very rarely life threatening and usually resolves with dose interruptions and reductions and supportive care.

Ongoing studies are evaluating steroid-based mouthwashes and pastes as a means to prevent everolimus-associated stomatitis. Fatigue was reported in about one third of patients in the BOLERO-2 trial and was easily managed with dose changes.

 Non-infectious pneumonitis is a less common side effect, seen in up to 20% of patients; it can be life threatening and therefore early detection and intervention are critical. Hyperglycemia and lipid abnormalities can be seen and managed supportively but can be clinically significant in elderly patients with risk factors for cardiac events.

Dose interruptions or reductions were required in 62% of patients receiving everolimus versus 12% in the placebo arm in the BOLERO-2 trial; additionally, the discontinuation rate was 26% in the treatment arm versus 9% in the placebo.

The addition of everolimus to endocrine therapy is costly,

 however, the cost of managing side effects of everolimus appears to be lower on average when compared to managing side effects of commonly used chemotherapy agents.

Breast cancer is a heterogeneous disease and the identification of subsets of patients who will benefit from mTOR inhibition is essential, since the addition of PI3K/Akt/mTOR inhibitors may be associated with increased costs and toxicity.

Preclinical studies have demonstrated that elevated levels of phosphorylated S6 kinase (S6K), phosphorylated Akt, GSK3β, TSC2, and loss of inositol polyphosphate 4-phosphatase (INPP4B) can predict response to inhibitors of the PI3K/Akt/mTOR pathway.

 Recently, it was reported that the presence of amplifications of AURKA and HER2 were associated with in vitro sensitivity to everolimus while 

 mutations, has also been associated with response.

However, clinical studies have failed to confirm this observation. Correlative studies from the TAMRAD trial revealed that 

 mutations, PTEN, and pAKT did not have any affect in the response to mTOR inhibition; however, everolimus was more effective in patients with low PI3K expression, low LKB1, and high phospho-4E binding protein (p4-EBP1).

The BOLERO-2 trial failed to find biomarkers predictive of benefit from everolimus.

A clinical study by Ellard et al did not demonstrate an association between PTEN, HER2 and Akt levels and benefit from everolimus.

Resistance to mTOR inhibition is a great concern and can limit the use of mTOR inhibitors in clinical practice.

Multiple mechanisms can lead to resistance, including the increased activity of mTORC2 and insulin-like growth factor receptor-1 due to the inhibition of mTORC1, thus leading to the induction of Akt and Ser 473.

Other mechanisms include upregulation of the ERK/MAPK pathway, activation of the PIM kinases and MYC, PDK1 activation, dysregulation of p27 and production of VEGF.

The co-inhibition of PI3K/Akt and mTORC2 along with mTORC1 or the use of dual inhibitors can overcome these mechanisms of resistance. Several compounds with these properties are undergoing studies to evaluate their efficacy and safety in various settings. In addition, targeting angiogenesis and the mechanisms of autophagy and apoptosis may help overcome resistance.

Aberrant PI3K/Akt/mTOR activation by receptor tyrosine kinases (RTKs), ligand binding, or pathway mutations, are oncogenic and lead to cell survival and proliferation. Inhibitors of the mTOR pathway have a proven role in the management of multiple malignancies; in breast cancer, mTOR inhibitors may have a role in overcoming resistance to HER2-directed and endocrine therapy in breast cancer.

Everolimus is currently FDA approved for the treatment of HR-positive advanced breast cancer in postmenopausal women but is also the subject of undergoing studies in HER2-positive and triple negative disease.

As the role of mTOR inhibitors in breast cancer evolves, future efforts will focus not only on the evaluation of the efficacy of these agents but on identifying the patients who are more likely to benefit.

Elisavet Paplomata, MD, Winship Cancer Institute of Emory University, Georgia Cancer Center for Excellence at Grady Memorial Hospital. Ruth O’Regan, MD, division of Hematology and Medical Oncology, University of Wisconsin.

 Ruth M O’Regan, MD, Chief, division of Hematology and Medical Oncology, University of Wisconsin, 600 Highland Ave, K4/542 CSC, Madison, WI 53792; phone: 608-262-9638. E-mail: roregan@medicine.wisc.edu

 The authors report no conflicts of interest.

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Articles

The role of mTOR inhibition in breast cancer is evolving, and ongoing studies are evaluating biomarkers that will identify the patients who will benefit.  

Ruth Oâ€™Regan, MD

Articles

mTOR Biology and the Growing Role for mTOR Inhibitors in the Management of Breast Cancer