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The Akt/mTOR Pathway and Estrogen Receptor Phosphorylations: a crosstalk with potential to predict tamoxifen resistance in breast cancer
Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Estrogen receptor α content is the primary breast cancer biomarker distinguishing the patients responsive from the non-responsive to endocrine treatments. Tamoxifen is an estrogen competitor with large potential to treat breast cancer patients and prolongs time to recurrence. Despite the estrogen receptor positivity and tamoxifen treatment, many women face recurrence of the disease. An important mechanism of resistance to endocrine treatments is upregulated growth factor signaling, and the subsequent effect on the estrogen receptor, rendering an active receptor that stimulates cell proliferation or reduced estrogen-receptor dependence.

This thesis concerns the investigation of biomarkers, as a complement to the existing markers, for determining optimal treatment for patients with primary invasive breast cancer. Randomized patient tumor materials were used in order to measure variations in gene copies, proteins, and protein phosphorylations and to further relate these variations to time-to-recurrence. Endocrine untreated groups within the patient tumor sets gave us the opportunity to study the prognostic potential of selected markers and to compare tamoxifen-treated patients with endocrine untreated, thus obtaining a treatment-predictive value of each marker or marker combination.

In endocrine-dependent cancer the 11q13 chromosomal region is frequently amplified, harboring the genes encoding the cell cycle stimulator cyclin D1 and the estrogen receptor phosphorylating kinase Pak1, respectively. Amplification of the genes was associated with reduced time-torecurrence, indicating a prognostic value, whereas PAK1 gene amplification predicted reduced response to tamoxifen treatment. Moreover, the protein expression of Pak1 tended to predict treatment response, which led to the investigation of this protein in a larger cohort. Together with one of its targets, the estrogen receptor phosphorylation at serine 305, Pak1 predicted reduced response to tamoxifen treatment when detected in the nucleus of tumor cells, suggesting activation of this pathway as a mechanism for tamoxifen-treatment resistance. The estrogen receptor is phosphorylated by several growth factor stimulated kinases. The role of serine-167 phosphorylation has been debated, with inconsistent results. To study the biomarker value of this site the upstream activity of Akt, mTOR, and the S6 kinases were analyzed individually and in combinations. As a prognostic factor, serine 167 indicated an improved breast cancer survival, and as a treatment predictive factor we could not detect a significant value of serine 167 as a single marker. However, in combination with serine 305, and Akt/mTOR-pathway activation, the response to tamoxifen treatment was reduced. The mTOR effector protein S6K1 was found to be associated with HER2 positivity and a worse prognosis. In the group of patients with S6K1 accumulation in the tumor cell nuclei, treatment did not prolong time-to-recurrence, similarly as observed with expression of active S6 kinases. In vitro, a simultaneous knockdown of the S6 kinases in estrogen receptor-positive breast cancer cells resulted in G1 arrest, and tamoxifen-induced G1 arrest was in part S6 kinase dependent.

The results presented herein suggest biomarkers that would improve treatment decisions in the clinic, specifically for estrogen receptor-positive breast cancer and tamoxifen treatment but in a broader perspective, also for other endocrine treatments and targeted treatments.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2013. , 71 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1379
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-100903DOI: 10.3384/diss.diva-100903ISBN: 978-91-7519-515-5 (print)OAI: oai:DiVA.org:liu-100903DiVA: diva2:664277
Public defence
2013-12-18, Nils-Holgersalen, ing. 71, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2013-11-14 Created: 2013-11-14 Last updated: 2013-12-12Bibliographically approved
List of papers
1. Amplification of CCND1 and PAK1 as predictors of recurrence and tamoxifen resistance in postmenopausal breast cancer.
Open this publication in new window or tab >>Amplification of CCND1 and PAK1 as predictors of recurrence and tamoxifen resistance in postmenopausal breast cancer.
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2007 (English)In: Oncogene, ISSN 0950-9232, Vol. 26, no 49, 6997-7005 p.Article in journal (Refereed) Published
Abstract [en]

The 11q13 region is amplified in approximately 15% of all breast tumors. Situated in this region are the cyclin D1 gene (CCND1) and the p-21-activated kinase 1 (PAK1) gene. Both genes encode proteins shown to activate the estrogen receptor (ER), leading to transcription of CCND1 and other ER-responsive genes. Here, we investigate the prognostic and treatment predictive role of CCND1 and PAK1 gene amplification in postmenopausal breast cancer patients randomized to tamoxifen treatment or no adjuvant treatment. Amplification of CCND1 and PAK1, assessed by real-time PCR, was observed in 12.5 and 9.3%, respectively. Amplification of PAK1 was seen in 37% of the CCND1-amplified tumors, indicating coamplification (P<0.001). In ER-positive patients, amplification of at least one of the genes indicated a reduced recurrence-free survival (P=0.025). When response to tamoxifen treatment was analysed, patients with PAK1 amplification showed decreased benefit from the drug (ER+; relative risk ratio (RR)=1.62; 95% confidence interval (CI), 0.47-5.55) compared to patients without amplification (ER+; RR=0.53; 95% CI, 0.32-0.88). This was not evident for CCND1 amplification. We show that PAK1 may be a predictor of tamoxifen resistance and furthermore, we do not discard PAK1 as a potential candidate oncogene in the 11q13 amplicon. In addition, we show that high pak1 protein levels may predict tamoxifen insensitivity.

Keyword
Cyclin D1, pak1, drug resistance, breast cancer, real-time PCR
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-17456 (URN)10.1038/sj.onc.1210506 (DOI)17486065 (PubMedID)
Available from: 2009-03-25 Created: 2009-03-25 Last updated: 2013-11-14Bibliographically approved
2. Estrogen Receptor-alpha Phosphorylation at Serine 305, Nuclear p21-Activated Kinase 1 Expression, and Response to Tamoxifen in Postmenopausal Breast Cancer
Open this publication in new window or tab >>Estrogen Receptor-alpha Phosphorylation at Serine 305, Nuclear p21-Activated Kinase 1 Expression, and Response to Tamoxifen in Postmenopausal Breast Cancer
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2010 (English)In: Clinical Cancer Research, ISSN 1078-0432, E-ISSN 1557-3265, Vol. 16, no 5, 1624-1633 p.Article in journal (Refereed) Published
Abstract [en]

Purpose: In vitro, p21-activated kinase 1 (Pak1) phosphorylates the serine 305 residue of the estrogen receptor alpha (ER alpha) and influences the response of breast cancer cells to tamoxifen. We investigated the influence of Pak1 and pER alpha(ser305) on breast cancer prognosis and results of tamoxifen therapy. Experimental Design: We examined Pak1 and pER alpha(ser305) protein by immunohistochemistry in a series of 912 tumors from node-negative breast cancer patients randomized to tamoxifen or no adjuvant endocrine treatment. Results: Cytoplasmic Pak1 correlated to large tumors and ER negativity, whereas nuclear Pak1 and pER alpha(ser305) correlated to small tumors and ER positivity. Nuclear expression of Pak1 and pER alpha(ser305) predicted reduced response to tamoxifen in patients with ER alpha-positive tumors (tamoxifen versus no tamoxifen: hazard ratio (HR), 1.33; 95% confidence interval (95% CI), 0.42-4.2; P = 0.63), whereas patients lacking this combination benefitted significantly from tamoxifen (HR, 0.43; 95% CI, 0.30-0.62; P less than 0.0001). Similar nonsignificant trends were detected in analyses of the proteins separately. Pak1 in the cytoplasm was an independent prognostic marker, indicating increased recurrence rate (HR, 1.79; 95% CI, 1.17-2.74; P = 0.0068) and breast cancer mortality (HR, 1.98; 95% CI, 1.14-3.46; P = 0.016) for patients randomized to no adjuvant treatment. Conclusion: Our results suggest that patients with tumors expressing Pak1 and pER alpha(ser305) in combination are a group in which tamoxifen treatment is insufficient. In addition, the pathway may be of interest as a drug target in breast cancer. Furthermore, the findings support previous studies showing that Pak1 has differential roles in the cytoplasm and the nucleus.

Place, publisher, year, edition, pages
American Association for Cancer Research, Inc., 2010
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-58377 (URN)10.1158/1078-0432.CCR-09-1733 (DOI)000278545500030 ()
Note
Original Publication: Josefine Bostner, Lambert Skoog, Tommy Fornander, Bo Nordenskjöld and Olle Stål, Estrogen Receptor-alpha Phosphorylation at Serine 305, Nuclear p21-Activated Kinase 1 Expression, and Response to Tamoxifen in Postmenopausal Breast Cancer, 2010, Clinical Cancer Research, (16), 5, 1624-1633. http://dx.doi.org/10.1158/1078-0432.CCR-09-1733 Copyright: American Association for Cancer Research, Inc. http://www.aacr.org/ Available from: 2010-08-13 Created: 2010-08-11 Last updated: 2017-12-12
3. Activation of Akt, mTOR, and the estrogen receptor as a signature to predict tamoxifen treatment benefit
Open this publication in new window or tab >>Activation of Akt, mTOR, and the estrogen receptor as a signature to predict tamoxifen treatment benefit
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2013 (English)In: Breast Cancer Research and Treatment, ISSN 0167-6806, E-ISSN 1573-7217, Vol. 137, no 2, 397-406 p.Article in journal (Refereed) Published
Abstract [en]

The frequent alterations of the PI3K/Akt/mTOR-growth signaling pathway are proposed mechanisms for resistance to endocrine therapy in breast cancer, partly through regulation of estrogen receptor alpha (ER) activity. Reliable biomarkers for treatment prediction are required for improved individualized treatment. We performed a retrospective immunohistochemical analysis of primary tumors from 912 postmenopausal patients with node-negative breast cancer, randomized to either tamoxifen or no adjuvant treatment. Phosphorylated (p) Akt-serine (s) 473, p-mTOR-s2448, and ER phosphorylations-s167 and -s305 were evaluated as potential biomarkers of prognosis and tamoxifen treatment efficacy. High expression of p-mTOR indicated a reduced response to tamoxifen, most pronounced in the ER+/progesterone receptor (PgR) + subgroup (tamoxifen vs. no tamoxifen: hazard ratio (HR), 0.86; 95 % confidence interval (CI), 0.31-2.38; P = 0.78), whereas low p-mTOR expression predicted tamoxifen benefit (HR, 0.29; 95 % CI, 0.18-0.49; P = 0.000002). In addition, nuclear p-Akt-s473 as well as p-ER at -s167 and/or -s305 showed interaction with tamoxifen efficacy with borderline statistical significance. A combination score of positive pathway markers including p-Akt, p-mTOR, and p-ER showed significant association with tamoxifen benefit (test for interaction; P = 0.029). Cross-talk between growth signaling pathways and ER-signaling has been proposed to affect tamoxifen response in hormone receptor-positive breast cancer. The results support this hypothesis, as an overactive pathway was significantly associated with reduced response to tamoxifen. A clinical pre-treatment test for cross-talk markers would be a step toward individualized adjuvant endocrine treatment with or without the addition of PI3K/Akt/mTOR pathway inhibitors.

Place, publisher, year, edition, pages
Springer Verlag (Germany), 2013
Keyword
mTOR, Akt, Estrogen receptor phosphorylation, Tamoxifen resistance, Immunohistochemistry
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-88458 (URN)10.1007/s10549-012-2376-y (DOI)000313201100007 ()
Note

Funding Agencies|Swedish Cancer Society||Swedish Research Council||King Gustaf V Jubilee Fund||

Available from: 2013-02-07 Created: 2013-02-07 Last updated: 2017-12-06
4. S6 kinase signaling and tamoxifen response in breast cancer cells and in two randomized breast cancer cohorts
Open this publication in new window or tab >>S6 kinase signaling and tamoxifen response in breast cancer cells and in two randomized breast cancer cohorts
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2013 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Detecting signals in the mammalian target of rapamycin (mTOR), and the estrogen receptor (ER) pathways for prediction of treatment response may be a future clinical tool in primary breast cancer. Here, we investigated the validity and value of the mTOR targets p70-S6 kinase (S6K) 1 and 2 as biomarkers for tamoxifen sensitivity in vitro and in two independent tamoxifen randomized postmenopausal breast cancer cohorts. In addition, the prognostic value of the S6Ks was evaluated. A simultaneous knockdown of the S6Ks in ER-positive breast cancer cells resulted in G1 arrest, and tamoxifen-induced G1 arrest was in part S6K1+S6K2 dependent, suggesting separate roles in proliferation and in tamoxifen response. We found S6K1 to correlate with HER2 and cytoplasmic Akt activity, whereas S6K2 and phosphorylated S6K were closer connected with ER positivity, low proliferation and nucleic p-Akt. Treatment prediction and prognosis were evaluated by immunohistochemical staining. Nuclear accumulation of S6K1 was indicative of a reduced tamoxifen treatment effect, compared with a significant benefit from tamoxifen treatment in patients without tumor S6K1 nuclear accumulation. Patients with a combination of S6K1 nuclear accumulation and S6K2 cytoplasmic accumulation in the tumor cells had no tamoxifen benefit. Also, S6K1 and S6K2 activation, indicated by p-S6K-t389 expression, was associated with low benefit from tamoxifen compared with untreated patients. In addition, high protein expression of S6K1, independent of localization, predicted worse prognosis. This was not evident for variations in S6K2 or p-S6K-t389 expression.

In conclusion, the mTOR targeted kinases S6K1 and S6K2 interfere with proliferation and response to tamoxifen. Monitoring their activity andintracellular localization may provide biomarkers for breast cancer treatment, allowing for identification of a group of patients less likely tobenefit from tamoxifen and thus in need of an alternative or additional treatment.

Keyword
pS6K, S6K1, S6K2, mTOR, Akt, estrogen receptor, endocrine treatment
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-100902 (URN)
Available from: 2013-11-14 Created: 2013-11-14 Last updated: 2013-11-14Bibliographically approved

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