The AKT serine-threonine protein kinases exhibit a wide-spread expression pattern in virtually all human cell types. They are activated downstream of various growth factor receptors, in particular by receptor tyrosine kinases, through PI3 kinase-dependent mechanisms. The three AKT isoforms (AKT1-3) control a number of intracellular processes such as growth, proliferation, metabolism, and cell survival [
23‐
25]. Studies in single, double, and triple knockout mice have shown that the three AKT proteins can have redundant and non-redundant functions in particular cell types [
26‐
28]. Among the three AKT members, only AKT1 has been shown to be crucial for normal mammary gland development. During pregnancy and lactation, this kinase is upregulated in the mammary epithelium where it controls metabolic pathways that regulate milk synthesis and the functional differentiation of the gland [
27,
29]. Immediately following the cessation of lactation and weaning of the offspring, AKT1 mRNA and protein levels decline rapidly to facilitate a swift remodeling of the mammary epithelium [
4,
30]. A sustained expression of hyperactive or wildtype AKT1 is entirely sufficient to delay apoptosis and mammary gland involution [
30‐
32]. The expression and functionality of AKT1 parallels closely the biological functions of prolactin and its downstream signaling mediators. We have demonstrated previously that the activation of AKT1 as well as the total levels of this kinase are dependent on the Janus kinase 2 (JAK2) and active STAT5 [
16]. More recently, we identified a novel role of JAK2/STAT5 signaling in the transcriptional activation of the
Akt1 gene in mice [
4]. Upon binding to the promoter of
Akt1 in a growth factor-dependent manner, STAT5 initiates the transcription of a unique
Akt1 mRNA from a distinct promoter, which was only present in the mammary gland. Using transgenic mice that express hyperactive STAT5 in a ligand-regulatible manner, we demonstrated that gain-of-function of this transcription factor mediates a sustained upregulation of
Akt1 in vivo[
4]. Phenotypically similar to females that overexpress AKT1, the prolonged activation of STAT5 impaired postlactational remodeling of the mammary gland. Collectively, the results of our previous lines of investigation revealed a novel mechanism by which the
Akt1 gene can be transcriptionally regulated from an alternative promoter depending on the developmental state and physiological needs.
Active STAT5 and AKT1 both mediate evasion from apoptosis and self-sufficiency in growth signals, which are hallmarks of cancer. In support of this notion it has been observed that both signal transducers exhibit a deregulated expression and activation in human breast cancers. Moreover, it has been demonstrated that JAK2/STAT5 signaling and AKT1 play essential roles during mammary tumor initiation in various murine cancer models [
7,
8,
33‐
36]. Specifically, upregulation and activation of AKT1 is required to sustain a hypermetabolic state (e.g., “Warburg effect”) that is a unique characteristic of certain cancer cells [
37,
38]. As demonstrated in this report, the majority of luminal- or basal-type mammary tumors showed an increased expression of AKT1 on the protein level and a significant upregulation of the
Akt1m transcript. Similar to the regulation of AKT1 during normal mammary gland development, cancer cells are able to upregulate this serine-threonine kinase on the transcriptional level to meet the specific metabolic needs in the transformed state. Using
in silico analysis, we were able to identify the human ortholog of the murine
Akt1m, but unlike in mice that only express a single
Akt1m mRNA, we cloned four new transcripts in human cells that originated from a previously unidentified, alternative promoter. Since the ATG start codon is located within the downstream exons (i.e., exons 2 or 3 depending on the specific transcript variants), it is evident that all four newly identified mRNAs include the first coding exon and therefore encode the full-length AKT1 kinase. RT-PCR results using the
AKT1m specific primer in the 5′UTR in combination with two reverse primers within downstream coding exons confirmed a correct splicing within the CDS of the
AKT1 mRNA. The four new
AKT1m transcripts were initially cloned from prolactin-responsive T47-D breast cancer cells, but the analyses of a larger panel of breast cancer cells as well as primary tumors show that expression of
AKT1m is not restricted to luminal-type cancer cells. Although their levels are lower, the
AKT1m transcript variants were also detectable in untransformed mammary epithelial cells and normal breast tissue specimens. This suggests that they are not a result of aberrant splicing, which more frequently occurs in transformed cells [
39]. Another distinct characteristic is the presence of
AKT1m in other normal human tissues (i.e., lung, liver, pancreas, and stomach). Although the expression in these organs was typically lower compared to the breast, the
Akt1m was not detected at all in tissues other than the mammary gland in mice using RT-PCR. The notion that the regulation of the alternative
AKT1m promoter might show some species-specific differences is supported by the absence of the STAT5 binding sites in the human locus. In mice, we found two high-probability STAT5 binding sites upstream and immediately downstream of the
Akt1m exon. Using chromatin immunoprecipitation (ChIP) and quantitative PCR on lysates from cultured cells as well as mammary gland tissues, we demonstrated that STAT5 binds to these particular recognition sites in a growth factor-dependent manner to significantly enhance
Akt1m transcription [
4]. Like the activation of milk protein gene promoters, STAT5 seems to confer a tissue-specific expression profile of
Akt1m in conjunction with other transcription factors such as the glucocorticoid receptor in the mouse [
40]. The absence of STAT5 binding sites might account for the lack of mammary gland specificity in humans. However, there are multiple GR binding sites present within the highly conserved orthologous region in the human sequence immediately upstream of the first exon of
AKT1m. More importantly, the confirmed presence of c-MYC on the conserved putative promoter sequences immediately upstream of
AKT1m using ChIP might be indicative of a growth-factor controlled expression of AKT1 depending on metabolic needs. In support of this notion, both AKT1 and c-MYC synergistically promote metabolic reprograming and aerobic glycolysis in cancer cells [
37,
38]. The identification of a novel putative promoter sequence in
AKT1 that is conserved between humans and mice is an interesting finding, but additional work is required to further pinpoint and confirm the functionality of the
AKT1m promoter along with the transcription factors that control its activation.
Regardless of the species-specific nuances in the regulation of
Akt1m, it is evident that the transcriptional regulation of the
Akt1 locus is more complex than previously thought. In mice as well as humans, the
Akt1 gene is transcriptionally upregulated in a subset of cancer cells, and the growth factor-dependent activation activation of this locus occurs through at least two distinct promoters. This is supported by our 5′RACE data and published sequences in GenBank that the
AKT1m-specific untranslated exon is absent in other known mRNA sequences that start at the originally identified promoter located much further upstream [
3]. The latter promoter and associated non-coding exon are very GC-rich, and attempts to generate primer sets to discriminate and to quantify the contribution of the
AKT1m transcripts to the total pool of
AKT1 mRNA messages have been unsuccessful. Interestingly, besides the upregulation of
AKT1m in a subset of human breast cancers, elevated levels of these transcript variants were also found in other diseased tissues (i.e., pneumonia of the lung, and GI stromal tumor). The qRT-PCR assay that we employed might be a simple, yet sensitive, diagnostic tool to assess pathological changes indicative of an altered metabolism that may correlate with a transcriptional upregulation of
AKT1.