Brent N. Rexer

Mutations in the phosphatidylinositol 3-kinase pathway: role in tumor progression and therapeutic implications in breast cancer

Mutations in genes that constitute the phosphatidylinositol 3-kinase (PI3K) pathway occur in >70% of breast cancers. Clinical and experimental evidence suggest that PI3K pathway activation promotes resistance to some of the current breast cancer therapies. PI3K is a major signaling hub downstream of human epidermal growth factor receptor (HER)2 and other receptor tyrosine kinases. PI3K activates AKT, serum/glucocorticoid regulated kinase (SGK), phosphoinositide-dependent kinase 1 (PDK1), mammalian target of rapamycin (mTOR), and several other molecules involved in cell cycle progression and survival. In estrogen receptor (ER)+ breast cancer cells, PI3K activation promotes estrogen-dependent and -independent ER transcriptional activity, which, in turn, may contribute to anti-estrogen resistance. Activation of this pathway also confers resistance to HER2-targeted therapies. In experimental models of resistance to anti-estrogens and HER2 inhibitors, pharmacological inhibition of PI3K/AKT/mTOR has been shown to overcome drug resistance. Early clinical data suggest that combined inhibition of either HER2 or ER plus inhibition of the PI3K pathway might be an effective strategy for treatment of respective HER2+ and ER+ breast cancers resistant to standard therapies. Here, we review alterations in the PI3K pathway in breast cancer, their association with therapeutic resistance, and the state of clinical development of PI3K pathway inhibitors.

Overcoming resistance to tyrosine kinase inhibitors: lessons learned from cancer cells treated with EGFR antagonists.

Tyrosine kinase inhibitors (TKIs) are effective anti-cancer therapies but resistance to these agents eventually develops. Several models of resistance to TKIs have been studied including resistance to EGFR inhibitors. Recent studies in EGFR-dependent A431 cells found upregulation of the IGF1R pathway as a mechanism to overcome blockade of EGFR. This was associated with amplification of IGF1R signaling and recovery of downstream PI3K-AKT activation. This work adds to a growing body of cell lines in culture that have provided insights into mechanisms of resistance that can be interrogated in primary tumors in patients. In this review, these model systems and their applicability to human cancers, as well as strategies to identify and overcome resistance to TKIs, are discussed.

Retinoic acid does not affect alveolar septation in adult FVB mice with elastase-induced emphysema.

Background: Administration of all-trans retinoic acid (ATRA) to adult Sprague-Dawley rats with emphysema induced by porcine pancreatic elastase (PPE) reversed the emphysema perhaps by inducing new alveolar formation. Objective: A study was conducted to determine whether ATRA can induce new alveolar septa and reverse the airspace enlargement caused in adult mice by PPE treatment. Methods: 48 FVB mice were divided into 6 groups. Three groups received 15 µg of PPE in 0.1 ml of 0.9% saline and 3 groups received 0.1 ml of saline, intratracheally. Starting at day 22, the mice received 12 daily intraperitoneal injections of cottonseed oil, with or without ATRA (12.5 µg or 50 µg). The mice were killed for study 1 day after the last injection. Results: Measurements of plasma and lung tissue ATRA levels showed statistically significant elevated levels after the 50-µg but not after the 12.5-µg doses of ATRA. In situ hybridization studies of elastin and α1(I) collagen mRNA expression in pulmonary parenchyma as well as in airways and blood vessels showed no effect of ATRA. Airspace size was determined by the mean linear intercept (Lm) method. The Lm of the groups receiving PPE and ATRA (46.2 ± 4.1 µm, mean ± SD) was not significantly different from the group receiving PPE and oil (47.8 ± 6.0 µm). The Lm for groups receiving saline and ATRA (40.6 ± 2.5 µm) were not significantly different from the group receiving saline and oil (41.0 ± 2.7 µm). Comparison of the fixed lung volume data and calculated internal surface area also showed no differences between the control and ATRA-treated groups. Conclusion: ATRA treatment does not affect airspace size or expression of elastin or α1(I) collagen mRNA in adult FVB mice with PPE-induced emphysema.

CRBP suppresses breast cancer cell survival and anchorage-independent growth

We showed earlier that cellular retinol-binding protein (CRBP) expression is downregulated in a subset of human breast cancers. We have now investigated the outcome of ectopic CRBP expression in MTSV1-7 cells, a SV40 T antigen-transformed human breast epithelial cell line devoid of endogenous CRBP expression. We found that: (i) CRBP did not inhibit adherent cell growth but suppressed foci formation in post-confluent cultures and colony formation in soft agar; (ii) this effect was due to CRBP inhibition of cell survival, as demonstrated by viability and TUNEL assays of cells in soft-agar or plated on polyHEMA-coated dishes; (iii) CRBP inhibited protein kinase B/Akt activation in cells in suspension but not in adherent cells and the CRBP suppression of anchorage-independent growth was mimicked by cell treatment with the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002; (iv) CRBP enhanced retinyl ester formation and storage but did not regulate retinoic acid synthesis or retinoic acid receptor activity. Ectopic CRBP-mediated inhibition of anchorage-independent cell survival and colony formation in the absence of significantly altered responses to either retinol or retinoic acid was also documented in T47D human breast cancer cells. In conclusion, the data suggest two novel and linked CRBP functions in mammary epithelial cells: inhibition of the PI3K/Akt survival pathway and suppression of anchorage-independent growth.

Inhibition of PI3K and MEK: It Is All about Combinations and Biomarkers

A small molecule inhibitor of MAP/ERK kinase (MEK) was effective against human breast cancer cells with a basal-like gene expression signature. Antitumor activity was limited by both feedback upregulation of phosphatidylinositol-3 kinase (PI3K)/AKT upon inhibition of MEK as well as loss of the phosphatase PTEN. Therefore, MEK inhibitors should preferably be investigated in combination with PI3K inhibitors in basal-like breast cancers.(Clin Cancer Res 2009;15(14) July 2009).

Optical imaging of metabolism in HER2 overexpressing breast cancer cells

The optical redox ratio (fluorescence intensity of NADH divided by that of FAD), was acquired for a panel of breast cancer cell lines to investigate how overexpression of human epidermal growth factor receptor 2 (HER2) affects tumor cell metabolism, and how tumor metabolism may be altered in response to clinically used HER2-targeted therapies. Confocal fluorescence microscopy was used to acquire NADH and FAD auto-fluorescent images. The optical redox ratio was highest in cells overexpressing HER2 and lowest in triple negative breast cancer (TNBC) cells, which lack HER2, progesterone receptor, and estrogen receptor (ER). The redox ratio in ER-positive/HER2-negative cells was higher than what was seen in TNBC cells, but lower than that in HER2 overexpressing cells. Importantly, inhibition of HER2 using trastuzumab significantly reduced the redox ratio in HER2 overexpressing cells. Furthermore, the combinatorial inhibition of HER2 and ER decreased the redox ratio in ER+/HER2+ breast cancer cells to a greater extent than inhibition of either receptor alone. Interestingly, trastuzumab had little impact upon the redox ratio in a cell line selected for acquired resistance to trastuzumab. Taken together, these data indicate that the optical redox ratio measures changes in tumor metabolism that reflect the oncogenic effects of HER2 activity within the cell, as well as the response of the cell to therapeutic inhibition of HER2. Therefore, optical redox imaging holds the promise of measuring response and resistance to receptor-targeted breast cancer therapies in real time, which could potentially impact clinical decisions and improve patient outcome.

Optimal targeting of HER2-PI3K signaling in breast cancer: Mechanistic insights and clinical implications

The combination of a PI3K inhibitor with trastuzumab has been shown to be effective at overcoming trastuzumab resistance in models of HER2(+) breast cancer by inhibiting HER2-PI3K-FOXO-survivin signaling. In this review the potential clinical implications of these findings are discussed.

A Novel Short-Chain Alcohol Dehydrogenase from Rats with Retinol Dehydrogenase Activity, Cyclically Expressed in Uterine Epithelium

Abstract Retinoic acid is necessary for the maintenance of many lining epithelia of the body, such as the epithelium of the luminal surface of the uterus. Administration of estrogen to prepubertal rats induces in these epithelial cells the ability to synthesize retinoic acid from retinol, coincident with the appearance of cellular retinoic acid-binding protein, type two, which is normally present in these cells only at estrus in the mature, cycling animal. Here, we report the isolation, from a cDNA library prepared from uterine mRNA collected at the estrous stage and from a rat mammary adenocarcinoma cell line, of a cDNA that encodes a novel retinol dehydrogenase. A member of the short-chain alcohol dehydrogenase family, the encoded enzyme was capable of metabolizing retinol to retinal when expressed in cells after transfection of its cDNA. When cotransfected with the cDNA of human aldehyde 6, a known retinaldehyde dehydrogenase, the transfected cells synthesized retinoic acid from retinol. Immunohistochemical analysis revealed that the protein was present in the uterine lining epithelium of the mature animal only at estrus, coincident with the presence of cellular retinol-binding protein and cellular retinoic acid-binding protein, type two. Consequently, this novel short-chain alcohol dehydrogenase is an excellent candidate for the retinol dehydrogenase that catalyzes the first step in retinoic acid biosynthesis that occurs in uterine epithelial cells.

Rictor/mTORC2 Drives Progression and Therapeutic Resistance of HER2-Amplified Breast Cancers

HER2 overexpression drives Akt signaling and cell survival and HER2-enriched breast tumors have a poor outcome when Akt is upregulated. Akt is activated by phosphorylation at T308 via PI3K and S473 via mTORC2. The importance of PI3K-activated Akt signaling is well documented in HER2-amplified breast cancer models, but the significance of mTORC2-activated Akt signaling in this setting remains uncertain. We report here that the mTORC2 obligate cofactor Rictor is enriched in HER2-amplified samples, correlating with increased phosphorylation at S473 on Akt. In invasive breast cancer specimens, Rictor expression was upregulated significantly compared with nonmalignant tissues. In a HER2/Neu mouse model of breast cancer, genetic ablation of Rictor decreased cell survival and phosphorylation at S473 on Akt, delaying tumor latency, penetrance, and burden. In HER2-amplified cells, exposure to an mTORC1/2 dual kinase inhibitor decreased Akt-dependent cell survival, including in cells resistant to lapatinib, where cytotoxicity could be restored. We replicated these findings by silencing Rictor in breast cancer cell lines, but not silencing the mTORC1 cofactor Raptor (RPTOR). Taken together, our findings establish that Rictor/mTORC2 signaling drives Akt-dependent tumor progression in HER2-amplified breast cancers, rationalizing clinical investigation of dual mTORC1/2 kinase inhibitors and developing mTORC2-specific inhibitors for use in this setting. Cancer Res; 76(16); 4752-64. ©2016 AACR.

Evaluating HER2 amplification status and acquired drug resistance in breast cancer cells using Raman spectroscopy

Abstract. The overexpression of human epidermal growth factor receptor 2 (HER2) is associated with increased breast cancer recurrence and worse prognosis. Effective treatments such as trastuzumab and lapatinib for patients with HER2 overexpression target the blockade of HER2 signaling activities but are often limited by the emergence of acquired drug resistance. This study applied Raman spectroscopy to differentially identify the amplification status of HER2 in cells and to characterize the biochemical composition of lapatinib resistant and sensitive HER2+ breast cancer cells in response to the drug. Raman spectra from BT474 (HER2+ breast cancer cell), MCF-10A (HER2− control), and HER2+ MCF-10A (HER2+ control) were analyzed using lasso and elastic-net regularized generalized linear models (glmnet) for multivariate statistical analysis and were discriminated to groups of different HER2 expression status with an overall 99% sensitivity and specificity. Enhanced lipid content and decreased proteome were observed in HER2+ cells. With lapatinib treatment, lapatinib-resistant breast cancer cells demonstrated sustained lipogenesis compared with the sensitive cells.