The prognostic impact of Akt isoforms, PI3K and PTEN related to female steroid hormone receptors in soft tissue sarcomas

Soft tissue sarcomas (STS) are malignant tumors arising from extraskeletal connective tissues. They are heterogeneous neoplasms, consisting of more than 50 subtypes, and comprise less than 1% of adult malignancies [1, 2]. Approximately 50% of the STS patients will succumb to their disease because of metastasis or local progression [3]. The prognostic factors determining tumor evolution and ultimately patients' fate include tumor grade, size, location, depth, histological entity, positive resection margins and presence of local relapse [4–10]. In addition, an array of recurrent gene aberrations are found to be prognostic and predictive biomarkers in STSs [11–13].

Akt is a serine/threonine protein kinase that exists in three possible isoforms, including Akt1, Akt2, and Akt3. Akt can be activated by phosphorylation at threonine ³⁰⁸ or at serine ⁴⁷³ for Akt1 or homologous sites for Akt2 and Akt3 by phosphatases which along with Akt isoforms, belong to the phosphoinositide 3-kinase (PI3K)/Akt pathway. The PI3K/Akt pathway has been linked to an extraordinarily diverse group of cellular functions, including cell growth, proliferation, differentiation, motility, survival, intracellular trafficking and angiogenesis [14]. Both PI3K and Akt isoforms have been implicated as major players in many types of cancer [15–17].

The PI3K/Akt pathway seems to be more often deregulated in cancer than any other pathway [18]. However, in the literature there is disagreement regarding the prognostic impact of Akt expression. While the majority of studies agree that Akt expression overtly indicates a poor prognosis [19–21], there are several studies showing the opposite effect [22, 23]. Expressions of PI3K/Akt pathway components have rarely been investigated in STSs and there are almost no studies devoted to their prognostic value [24].

Different physiological function of the Akt family kinases implies that the expression of its isoforms may also have different prognostic impact in cancer. The significance of this variation for the survival of the STS patients is not well investigated and it is not clear whether the site of phosphorylation and the pattern of expression can play prognostic roles.

In previous studies, we have shown the prognostic value of female steroid hormone receptors in STSs, both alone and in the coexpression with TGF-β and fascin [25, 26]. Such prognostic impact is not surprising, since both ER and PgR regulate growth and cell differentiation upon ligand-dependent and ligand-independent activation and are in essence growth factors. In this context we wanted to explore the correlations between female hormone receptors and the members of PI3K/Akt signaling pathway. To our knowledge, these correlations have not been described previously.

In this study, we investigate the prognostic impact of all isoforms of Akt (phosphorylated at threonine ³⁰⁸ and Akt phosphorylated at serine ⁴⁷³, non-phosphorylated Akt2, and total Akt3), PI3K, PTEN, ER and PgR in 249 non-GIST STS patients. GIST cases were excluded from the study since patients with this subtype of sarcoma receive a specific treatment regimen which resulted in significantly better survival.

In this large-scale retrospective study we have investigated the prognostic impact of a set of biomarkers belonging to the Akt-PI3K signaling pathway in non-GIST STS patients, both separately and in relation to gender. Further, we have also elucidated the coexpression of these markers and the female hormone receptors ER and PgR. These proteins participate in a diversity of processes in physiological and pathological conditions, especially in cancer development and progression [14]. p-Akt Thr³⁰⁸, Akt2 and PI3K showed significant unfavorable influence on survival of the whole cohort of patients in univariate analyses and, in addition, high expression of PI3K was a significant independent negative prognostic factor. p-Akt Thr³⁰⁸ expression had a strong unfavorable impact among men, but was not significant in women. p-Akt Ser⁴⁷³ expression had strong adverse impact in women but was not significant in men or in the whole cohort. PgR-/p-Akt Ser⁴⁷³+ phenotype showed less favorable impact in women, but was the most favorable one in men. To our knowledge, this is the first prognostic evaluation of these biomarkers in non-GIST STSs.

Akt, aka protein kinase B, is a serine/threonine protein kinase. Currently, three mammalian isoforms (Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ) have been identified. They are encoded by different genes and have different tissue distribution [29].

In a healthy organism, Akt1 is a key signaling protein in the cellular pathways that result in skeletal muscle hypertrophy, and general tissue growth [30]. Akt can be phosphorylated by its two activating kinases, phosphoinositide dependent kinase 1 (PDK1) - at threonine ³⁰⁸, and mammalian target of rapamycin complex 2 (mTORC2), previously putatively named PDK2, - at serine ⁴⁷³. Both mTORC2 and PDK1 are products of the PI3K pathway. Activated Akt can activate or deactivate its multiple substrates, including mammalian target of rapamycin (mTOR), bcl-2 family member BAD, transcription factor forkhead homolog 1 in rhabdomyosarcoma (FKHR), Mdm2 protein, glycogen synthase kinase 3 (GSK3) and many others, via its kinase activity [31, 32].

Akt1 is involved in cellular survival pathways by inhibiting apoptotic processes. Since it thereby promotes cell survival, Akt1 has been regarded as a major factor in many types of cancer [15–17]. The majority of studies agree that high expression of Akt by tumor cells indicates a poor prognosis [19–21]. However, in a recent study by Baba et al., phosphorylated Akt expression was reported to have a favorable impact on DSS in 717 colorectal cancer patients [22]. Similar results were obtained by Mori et al. in a study devoted to Akt expression in endometrial carcinoma [23]. This discrepancy can probably be explained by the site of Akt phosphorylation. Both studies utilized antibodies against p-Akt Ser⁴⁷³, while the articles describing negative influence of Akt are based on p-Akt Thr³⁰⁸ expression [20, 33, 34]. Al-Saad et al. [33] has recently compared the prognostic impact of Akt phosphorylated on both sites and demonstrated that expression of p-Akt Thr³⁰⁸, unlike p-Akt Ser⁴⁷³, negatively influenced prognosis in patients with non-small cell lung cancer.

For the whole cohort we also found that p-Akt Thr³⁰⁸ expression was associated with a shorter STS survival in univariate analyses, while p-Akt Ser⁴⁷³ expression had no significant value. However, calculated separately for each gender, high expression of p-Akt Thr³⁰⁸ was a negative prognostic factor particularly for men, in contrast to p-Akt Ser⁴⁷³, which appeared to be a negative prognosticator exclusively for female patients. This prompted us to further investigate this phenomenon by studying of co-expression profiles of both types of activated Akt with female steroid hormone receptors. In our previous works we have shown that ER and PgR expression possess variable prognostic significance depending of gender both per se[25] and in co-expression with TGF-β and fascin [26]. ERβ was shown to activate PI3K/Akt signalling pathway [35]. Tsai et al. demonstrated an activation of Akt by estrogen in ER negative breast cancer cell culture [36]. In the present study, the prognostic diversity of these factors in men and women was enhanced in the co-expression profiles: male patients with STSs expressing simultaneously p-Akt Thr³⁰⁸ and PgR had statistically significant minimal survival rate. For women, the ER-/p-Akt Ser⁴⁷³+ expression profile was the most unfavorable phenotype.

Taking into consideration a possible distortion of the results by gender-related sarcomas (i.e. leiomyosarcoma in uterus) we have attempted to exclude these sarcomas and recalculate all analyses. There were no considerable differences in the results by exclusion of gender-related sarcomas comparing to those obtained for whole cohort (data not shown).

Akt2 is an important molecule in the insulin signaling pathway, but in Akt1 deficient mice it is also proved to substitute, at least partly, the role of Akt1 in growth and proliferation [37]. We found Akt2 expression to be associated with significantly shorter DSS in univariate analysis. This might be explained by the extra-endocrine function of Akt2. The role of Akt3 is less clear, it appears to be predominantly expressed in the central nervous system [29]. In this study, we failed to demonstrate any association of Akt3 with the survival of STS patients.

PI3K is, via PDK1 and mTORC2 dependent activation, an upstream regulator of all Akt isoforms, and plays an important role in the PI3K/Akt pathway. Its high expression has been implicated as an adverse prognostic factor in many types of cancer [38–40]. In STS, we observed that PI3K expression was an independent significant indicator of shorter DSS. Not surprisingly, the co-expression of PI3K with both ER and PgR showed multiple independent negative impacts on survival in STS patients with the phenotypes ER-/PI3K+ in women and PgR+/PI3K+ in men being the least favorable.

The tumor suppressor gene PTEN negatively regulates the PI3K/Akt signaling pathway. It is a proapoptotic and antineoplastic factor and shown to be a favorable prognosticator in cancer patients [41]. In our study, we failed to find any statistical difference in survival between patients having PTEN-positive and PTEN-negative STSs.

In our material, all investigated factors showed three distinct patterns of expression; nuclear, cytoplasmic and combined nuclear and cytoplasmic. Little is known about the prognostic value of such subcellular stratification. Le Page et al. reported that nuclear Akt-1 and Akt-2 expression were significantly correlated with favorable outcome in 63 prostate cancer patients, while cytoplasmic Akt-1 expression was correlated with a higher risk of postoperative prostate-specific antigen (PSA) recurrence and shorter PSA recurrence interval [42]. In the present study, we were able to find such dependence only for nuclear p-Akt Thr³⁰⁸ expression, which proved to be prognostically favorable compared to cytoplasmic and especially mixed cytoplasmic and nuclear location.

We have characterized the occurrence and distribution of several proteins belonging to PI3K/Akt signaling pathway in STS patients with respect to tumor aggressiveness and DSS. Our findings are largely in agreement with the results of a number of studies that have investigated the roles of these markers in other, especially epithelial, tumors. Nevertheless, the diverse prognostic values depending on the site of Akt phosphorylation and on the co-expression with female steroid hormones have not been described earlier.

Our findings may help to identify subgroups of patients with aggressive tumors requiring adjuvant therapy which, due to relationship of the PI3K/Akt pathway components with female steroid hormone receptor proteins, could include specific endocrine treatment. Moreover, since the investigated biomarkers belong to the family of serine-threonine kinases, which are comprehended as "drugable"[18], they may represent molecular targets for personalized, small-molecule targeted therapy. This currently is a hotspot of oncological research, and it was shown effect of such Akt targeted agents on several subtypes of sarcomas in vitro [43, 44].