Introduction
Paclitaxel is a powerful chemotherapy for many cancers, such as breast, prostate and ovarian cancers [
1-
5], as well as chemotherapy-refractory cancers such as small cell lung cancer [
6-
8]. Paclitaxel polymerizes tubulin to disrupt normal microtubule dynamics leading to cell death [
4]. Despite preclinical and clinical success, intrinsic or acquired paclitaxel resistance remain a challenge in oncology [
9-
12].
Survivin, a structurally unique member of the inhibitor of apoptosis proteins family (IAP) is involved in cell division and apoptosis [
13-
15]. Survivin is a poor prognostic factor in several tumor types, and is involved in tumor cell resistance to ionizing radiation and chemotherapies for example, paclitaxel [
16-
20]. In fact, survivin expression is induced following paclitaxel exposure in breast cancer cells [
21,
22]. Survivin expression is negatively controlled by the Forkhead box class O (FOXO) transcription activity [
23,
24] and therefore is positively controlled by activated protein kinase B (AKT) [
24-
27].
FOXO proteins play a pivotal role in the regulation of cell cycle arrest, cell death and protection from stress stimuli [
23]. Perturbation of FOXO’s function deregulates cell proliferation and leads to accumulation of DNA damage [
23,
25]. AKT, or mitogen-activated protein kinases (MAPK), phosphorylate FOXOs at specific sites, causing its nuclear exclusion and degradation [
23,
26]. Constitutive AKT activation is frequently correlated with cytoplasmic Forkhead box O3a (FOXO3a) and decreased patient survival in breast cancer and other malignancies [
25,
27,
28]. Drugs like paclitaxel achieve their therapeutic effects through activation of FOXO3a [
29-
31].
Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype lacking estrogen receptor (ER) and progesterone receptor (PR) expression and human epidermal growth factor receptor 2 (HER2) gene amplification. Hence, patients with this subtype lack targeted therapies. TNBC constitutes approximately 20% of all breast cancers in the USA and is overrepresented in young African American women. TNBC tumors have the poorest prognosis and tend to grow and spread to other parts faster than other cancers and often harbor BRCA1 mutations or lack of expression. Although, initially responsive to paclitaxel, TNBCs often recur with chemotherapy-resistant, visceral and brain metastasis.
The
BRCA1 locus product, BRCA1-IRIS, shares 1,365 residues with the full-length product of this locus, the tumor suppressor, BRCA1 [
32,
33]. Despite that, BRCA1-IRIS is a genuine oncogene in breast and ovarian cancers. Indeed, BRCA1-IRIS overexpression induces over-replication [
32], over-proliferation by upregulating Cyclin D1 expression [
34,
35], apoptosis-resistance in human mammary (HME) and ovarian surface (HOSE) epithelial cells by inactivating p53 and/or activating AKT/survivin [
36,
37]. The majority of breast tumors, especially TNBCs express high levels of BRCA1-IRIS associated with increased p-AKT and survivin expression, and lack of BRCA1 expression [
38]. Interestingly, BRCA1-IRIS-overexpressing HME cells when injected in SCID mice mammary fat pads develop invasive TNBCs that also show increased AKT and survivin expression and/or activation and lack BRCA1 expression [
38].
Understanding the various mechanisms leading to paclitaxel resistance may help in the design of novel, more accurate therapies [
12]. Here, we show BRCA1-IRIS overexpression is involved in TNBCs intrinsic and acquired paclitaxel resistance, through, in part, increasing expression and activation of autocrine signaling loops involving epidermal growth factor receptor 1 (EGFR) and epidermal growth factor receptor 3 (ErbB3) that activate AKT leading to FOXO3a degradation and survivin overexpression. BRCA1-IRIS inactivation using a novel inhibitory mimetic peptide reversed these effects and significantly reduced TNBC cells growth, survival and aggressiveness,
in vitro and
in vivo. More importantly, this peptide sensitized established preclinical TNBCs to low paclitaxel concentrations. BRCA1-IRIS inactivation represents a novel and attractive target for TNBCs.
Methods
Cell culture
Generation and maintenance of the immortalized HME cells and its variants, BRCA1-IRIS-overexpressing (HME/IRIS) cell lines, were described earlier [
32,
36]. In brief, immortalized HME cells were generated from mammary epithelial cells purified from tissues isolated from mammary gland reductions (Clontech Laboratories, Mountain View, CA, USA) using standard techniques by a suitable human telomerase reverse transcriptase (TERT)-expressing virus and selection [
32]. Full-length BRCA1-IRIS cDNA was cloned into the pRevTRE plasmid (retrovirus version, Clontech Laboratories) to produce the pRevTRE-IRIS retrovirus. Retroviral particles were generated by transfecting 293T cells with pRevTRE-IRIS together with all necessary packaging plasmids. On days 2 and 3 viral supernatant was collected, pooled, and used as is to infect immortalized HME cells stably expressing p-TetOn plasmids (Clontech Laboratories, with suitable selection). Infected cells were then selected using hygromycine for 2 weeks, and 10 to 15 clones were generated, all referred to HME/IRIS. Ectopic BRCA1-IRIS expression in these clones is induced by exposure to 2 μg/ml of doxycycline (Invitrogen, Carlsbad, CA, USA). BRCA1-IRIS was verified at the beginning and periodically using western blot using mouse anti-human BRCA1-IRIS-specific antibody. While variation between clones exists, in every clone the expression is approximately two- to fivefold above the level in normal immortalized HME cells and resembling that found endogenously expressed in TNBC cell lines. Other cell lines, such as MDA-MB-231 (HTB-26), MDA-MB-468 (HTB-132) and BT-549 (HTB-122) cell lines were from American Type Culture Collection (ATCC) and were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS). All cell lines used in this study were transfected with a retrovirus expressing the luciferase gene.
Antibodies
Rb anti-survivin (#2808), −EGF (ab9695), −EGFR (ab23430), −p-ErbB2 (ab131104), −ErbB3 (ab20161), −FOXO3a (ab47409), −FOXO1 (ab39670), and -p-FOXO (T32, ab26649), or m anti-p-EGFR (Y1173; ab24912), −H2B (ab52484) and -PCNA (ab18197) were from Abcam (Cambridge, MA, USA). Rb anti-ErbB2 (#2165), −Cyclin D1 (#2978), −PTEN (#9188), −AKT (#2938), −p-AKT (S463, #4060), −ERK (#4695), −p-ERK (T202/Y204, #4370), −JNK (#9258), −p-JNK (T183/Y185, #9255), −p38 (#8690), −p-p38 (T180/Y182, #2387), −Bcl2 (#2870), −Bcl-xL (#2762) and -Skp2 (#4358) were from Cell Signaling Technology (Danvers, MA, USA). The m anti-NRG1 (MAB377) was from R&D Systems (Minneapolis, MN, USA). The m anti-nuclear factor kappa B (NF-κB)/p65 (IMG-150A) was from Imgenex (San Diego, CA, USA), the Rb anti-MDM2 (s1357) was from Epitomics (Burlingame, CA, USA) and the m anti-actin (cp01) was from Calbiochem (San Diego, CA, USA). Mouse monoclonal anti-human anti-BRCA1-IRIS was developed in our laboratory.
Small interfering RNA transfections, and small hairpin RNA construction and generation of stable knockdown cell lines
Small interfering RNA (siRNA) transfections and protocol were described previously [
32]. BRCA1-IRIS small hairpin RNA (shRNA) was designed using the ‘shRNA Design Tool’ from the IDT website [
39], (sequence available upon request), inserted between BamHI and EcoR1 sites in pSIREN-RetroQ plasmid (Addgene, Cambridge, MA, USA).
Immunohistochemistry (IHC) staining and scoring
Breast tissue microarrays comprised of normal, ductal carcinoma
in situ (DCIS), invasive and metastatic samples were purchased from US Biomax, Inc. (Rockville, MD, USA). IHC protocols were described earlier [
38]. A semi-quantitative scoring system was used to identify the percentage of tumor cells showing positive staining [
40]. Scoring represents: overall stain intensity and percentage of cancer cells stained in four high magnification fields for each sample. Average overall staining intensity [
41] was valued as percentage of cell stained/field: zero (<1% staining) was considered negative; 1 (1 to 10% staining) was considered weakly stained; 2 (10% to 50% staining) was considered medium stained and 3 (>50% staining) was considered strongly stained. The positive staining scoring method is totally subjective and artifacts such as high background or variable stain deposition can skew the results and the scores for the two categories remain as separate functions and cannot be combined for analysis and comparison [
42].
In vivo tumorigenicity assay
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Mississippi Medical Center. SCID (Jackson Laboratory, Bar Harbor, ME, USA) or Nu/Nu (Harlan Laboratories, Indianapolis, IN, USA) female mice were used. Protocols were previously described [
38].
BRCA1-IRIS inhibitory peptide
A synthetic peptide corresponding to amino acids 1365–1399 of BRCA1-IRIS protein (see [
32] for sequence) conjugated to cell and nuclear penetrating sequence was used.
Cell viability measurement
Cell viability under different experimental conditions was determined using cell counting or MTS assay.
Cell migration assay
μ-Dish (35mm, high Culture-Inserts, ibidi GmbH, Munich, Germany) was used. Inserts surrounded control or BRCA1-IRIS shRNA MDA-MB-231 or MDA-MB-468-expressing cells until confluence. At which time, inserts were removed, floating cells washed and attached cells allowed to migrate for 24 h. A montage of multiple pictures representing the whole well was mounted digitally together and migration calculated from a fixed point. Each experiment was done in triplicate repeated three separate times.
Cell invasion assay
Growth factor-reduced BD matrigel™ invasion chambers (24-well plate, 8.0μm, BD BioCoat™) were used (BD Biosciences, San Jose, CA, USA). Invaded cells were Crystal Violet stained 7 days later, photographed and counted. Each experiment was done in triplicate repeated three separate times.
Mammosphere assay
Ultra-low attachment 6-well plates (Corning Life Sciences, Union City, CA, USA) were used. Every third day, medium was exchanged with one containing treatments for up to 10 days when mammospheres were counted and photographed. Each experiment was done in triplicate repeated three separate times.
In vivo efficacy of BRCA1-IRIS inhibitory peptide
Female Nu/Nu mice (6 to 8 weeks old) were injected with 2 x 106 of MDA-MB-468 cells in the second right and fourth left mammary gland. Mice bearing tumors of approximately 100 mm3 were randomly grouped to receive DMSO (intraperitoneally (i.p.)) + scrambled peptide (10 mg/kg) intratumorally (i.t.), IRIS peptide (10 mg/kg, i.t.), paclitaxel (10 mg/kg, i.p.), or IRIS peptide (5 mg/kg, i.t.) + Taxol (5 mg/kg, i.p.) every third day for four times per experiment. Tumor volume was measured by caliper and is represented as percentage of volume at day 0 of treatment. At the end point, tumors or their remnants were collected, fixed in 10% formalin and histologically or immunohistochemically analyzed.
Discussion
Development of chemotherapy-resistant recurrences plays a major role in breast cancer mortalities [
43]. Paclitaxel promotes apoptosis in tumor cells by inducing a permanent mitotic arrest. However, adaptation that develops in some paclitaxel-treated tumor cells can lead to tumor progression [
45,
46]. BRCA1-IRIS expression is elevated in the majority of breast cancers, including TNBCs [
38]. BRCA1-IRIS-overexpressing tumors show adverse outcomes, progression and metastasis [
38].
Here, we present extensive evidence showing a paclitaxel-resistance-promoting role for BRCA1-IRIS overexpression in TNBC tumor cells,
in vitro and
in vivo. BRCA1-IRIS overexpression drastically diminishes paclitaxel efficacy as evidenced by decreased apoptosis of treated cells
in vitro (Figure
2) and
in vivo (Figure
7). Only following BRCA1-IRIS silencing or inactivation (using a novel BRCA1-IRIS inhibitory peptide) was the efficacy of paclitaxel restored. BRCA1-IRIS mediates this resistance by upregulating expression of survivin through activation of AKT and/or inactivation of FOXO3a
in vitro and
in vivo. In addition, increased expression of other prominent apoptosis-suppressing proteins, such as Bcl-2, Bcl-xL and NF-κB by BRCA1-IRIS could also play a role.
The most troubling observation in the studies described above is the fact that at low concentration, paclitaxel upregulates BRCA1-IRIS expression in normal (see above) as well as low BRCA1-IRIS-expressing breast cancer cells (not shown). This observation may suggest that paclitaxel promotes its own resistance in patients by selecting certain tumor cells to survive and repopulate the tumor by upregulating BRCA1-IRIS in them. More seriously still is the prospective that the generation of new tumor not just recurrent tumor developed after treatment from normal cells. The mechanism responsible for paclitaxel (low concentration)-induced BRCA1-IRIS expression is being investigated. However, overall, these findings indicate that BRCA1-IRIS upregulation is involved in TNBCs intrinsic and acquired paclitaxel resistance and its inhibition can be pursued as a therapeutic option to reverse this resistance in TNBC patients.
The specificity of BRCA1-IRIS-overexpression-induced acquired paclitaxel resistance is shown here by genetic manipulation of BRCA1-IRIS in three aggressive TNBC lines. In addition, we achieved sensitization to lower concentrations of paclitaxel-induced apoptosis, in vitro and in vivo and corresponding reduction in the aforementioned pathways when BRCA1-IRIS activity was reduced in these cell lines using the novel IRIS peptide. This was further supported by the fact that one of the most prominent effects of low paclitaxel concentration-induced resistance in HME cells was BRCA1-IRIS overexpression, which was followed by upregulation of the survival pathways described above. Taken together, these data strongly support the notion that whether intrinsically or acquired following paclitaxel (especially low concentration) treatment, the upregulation in BRCA1-IRIS in TNBC cells is a major obstacle against obtaining major efficacy for paclitaxel, especially in patients with metastatic breast cancer. We therefore propose that inhibiting BRCA1-IRIS expression and/or activity could sensitize these tumors to paclitaxel and perhaps as our data suggest, lower and less toxic concentrations of this chemotherapy.
Our data, especially with the IRIS peptide, seem to suggest that intact AKT is more important for TNBC than intact ERK pathway since prior to promoting cell death in TNBC cells, BRCA1-IRIS silencing or inactivation inactivated the AKT pathway but had no or the opposite effect on ERK pathway. However, we cannot rule out the possibility that to completely eradicate BRCA1-IRIS-overexpressing TNBC cells, ERK1/2 inhibitors must be combined with AKT and/or BRCA1-IRIS inhibitors. It is also possible - since we cannot distinguish between ERK1 or ERK2 activation in these assays - that the two act in a different manner. Interestingly, AKT - and to a lesser extent ERK - inactivation significantly decreased BRCA1-IRIS level in the TNBCs cell lines tested. This implies a feed-forward mechanism is at work in TNBCs (possibly other cell types as well). It is possible that p-AKT enhances BRCA1-IRIS expression, which enhances AKT expression and activation. One of two possibilities might account for this phenomenon. The first is that through silencing BRCA1 (see [
38,
44]), BRCA1-IRIS is able to prevent AKT ubiquitination and degradation as was previously shown. Alternatively, the two events could be unconnected and merely a consequence of other activities in TNBC cells. Whatever the explanation is, a positive feedback mechanism between BRCA1-IRIS and AKT pathways is directly correlated with the BRCA1-IRIS chemotherapy resistance-inducing role in TNBC survival.
Mechanistically, BRCA1-IRIS-dependent paclitaxel resistance could be mediated by pro-survival autocrine signaling loops, such as those shown here, namely EGF/EGFR-ErbB2 and NRG1/ErbB2-ErbB3. Although paclitaxel-mediated increases in the expression of some oncogenes have been previously reported in both human patients with breast cancer [
46] and experimental breast cancer models [
47], this is the first study that analyzed co-expression of BRCA1-IRIS and ErbB family members. This evidence strongly suggests that therapy-induced activation of BRCA1-IRIS pathway promotes tumor cell survival through autocrine signaling loops. However, secondary pathways initiated from tumor stromal cells are also possible [
48]. Indeed, the fact that intrinsic or paclitaxel-acquired upregulation of BRCA1-IRIS induced expression and activation of NF-κB, as evidenced by increased expression and nuclear accumulation of p65 [
44], could lead to, among other effects, transcription and secretion of a plethora of inflammatory cytokines, such as interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor alpha (TNF-α) and monocyte chemotactic protein 1 (MCP-1) that alter the tumor microenvironment through autocrine and paracrine loops [
49]. Many of these cytokines were recently shown to act in autocrine but mostly in paracrine fashion between tumor cells and the surrounding microenvironment. These factors bind on the surface of stromal cells to specific receptors and induce expression of other factors that promote breast cancer cells aggressiveness, in this case in a paracrine manner [
50].
Interestingly, our data also present a novel yet expected conclusion [
51], which suggest that BRCA1-IRIS overexpression, which has been shown earlier to be associated with metastasis and poor survival in invasive ductal breast carcinoma is linked to uncoupling of the AKT-FOXO3a signaling axis. This conclusion has been reached based on the lack of FOXO3a in the nucleus in more aggressive tumors, which is known to overexpress BRCA1-IRIS and activated AKT. Thus it was predicted that survivin expression would be positively correlated with BRCA1-IRIS overexpression in aggressive and drug-resistant tumors. This also can explain the fact that paclitaxel failed to induce cell cycle arrest in BRCA1-IRIS-overexpressing cells, except after BRCA1-IRIS silencing or inactivation since this would require FOXO3a nuclear translocation to activate gene expression of cell cycle inhibitors, such as p21 and p27, both were not expressed in BRCA1-IRIS-overexpressing cells with intrinsically or acquired paclitaxel resistance.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ZB and BP performed the experiments. BC designed experiments and interpreted the data. WeS designed the studies, contributed to the analysis, gave advice on experiments and data analysis and wrote the manuscript. All authors read and approved the final version of this manuscript.