Transmembrane and coiled-coil domain family 3 (TMCC3) regulates breast cancer stem cell and AKT activation

Cancer stem cells (CSC) play a pivotal role in cancer metastasis and resistance to therapy. Previously, we compared the phosphoproteomes of breast cancer stem cells (BCSCs) enriched subpopulation and non-BCSCs sorted from breast cancer patient-derived xenograft (PDX), and identified a function unknown protein, transmembrane and coiled-coil domain family 3 (TMCC3) to be a potential enrichment marker for BCSCs. We demonstrated greater expression of TMCC3 in BCSCs than non-BCSCs and higher expression of TMCC3 in metastatic lymph nodes and lungs than in primary tumor of breast cancer PDXs. TMCC3 silencing suppressed mammosphere formation, ALDH activity and cell migration in vitro, along with reduced tumorigenicity and metastasis in vivo. Mechanistically, we found that AKT activation was reduced by TMCC3 silencing, but enhanced by TMCC3 overexpression. We further demonstrated that TMCC3 interacted directly with AKT through its 1-153 a.a. domain by cell-free biochemical assay in vitro and co-immunoprecipitation and interaction domain mapping assays in vivo. Based on domain truncation studies, we showed that the AKT-interacting domain of TMCC3 was essential for TMCC3-induced AKT activation, self-renewal, and metastasis. Clinically, TMCC3 mRNA expression in 202 breast cancer specimens as determined by qRT-PCR assay showed that higher TMCC3 expression correlated with poorer clinical outcome of breast cancer, including early-stage breast cancer. Multivariable analysis identified TMCC3 expression as an independent risk factor for survival. These findings suggest that TMCC3 is crucial for maintenance of BCSCs features through AKT regulation, and TMCC3 expression has independent prognostic significance in breast cancer. Thus, TMCC3 may serve as a new target for therapy directed against CSCs.


Introduction
Breast cancer is the most common cancer among women worldwide. The majority of breast cancer deaths occur as a result of recurrent or metastatic disease rather than from the effects of the primary tumor [1]. The existences of cancer stem cells (CSCs) have been demonstrated in a variety of human cancers, including breast cancer [2,3]. In 2003, Al-Hajj et al. reported that breast cancer stem cells (BCSCs) were enriched in the CD24 − CD44 + subpopulation of breast cancer [2]. Subsequently, Ginestier et al. demonstrated that tumor cells with high aldehyde dehydrogenase activity (ALDH) which mediated the conversion of retinaldehyde to retinoic acid, harbored stem/progenitor properties [1,4,5].
These CSCs possessed the capacity for self-renewal, differentiation, and displayed resistance to chemotherapeutic agents and radiation, which might contribute to tumor relapse years after the clinical remission [6]. Thus, it will be important to understand the molecular characteristics of CSCs that may lead to the development of novel targets for CSC-directed therapy.
Genes encoding putative proteins of the transmembrane and coiled-coil domain (TMCC) family have been found in many organisms. The TMCC family consists of three putative proteins (TMCC1-3) that are conserved from nematode to human [16]. However, the properties and functions of TMCC proteins are little known. It was reported that TMCC1 localizes to the rough ER and is crucial for ER-associated bud fission [16,17]. TMCC2 is a neuronal, ER-located protein, and the interaction between TMCC2 and apoE contributes to amyloid-β protein precursor metabolism in Alzheimer's disease [18]. Impaired function of dementin (Drosophila orthologue of TMCC2) causes neurodegeneration and early death in Drosophila [19]. TMCC3 is first cloned from human normal brain tissue. It is also predicted as an integral membrane protein and localized in ER [17,20]. However, the function of TMCC3 remains elusive. In this study, we provide first evidence for the important roles of TMCC3 in self-renewal, metastasis, and tumorigenicity of BCSCs and AKT activation.

Results
BCSCs express higher levels of TMCC3 protein than non-BCSCs Previously, we established several patient-derived xenograft tumors (PDXs) of breast cancer and identified their enrichment markers for BCSCs by determining the frequency of tumor-initiating cells, after sorting by fluorescence-activated cell sorter. Mice were injected with serial dilution of sorted cells to identify the markers for enrichment of BCSC. The supporting evidence for CD24 − CD44 + as BCSC enrichment marker for BC0145 was published previously [14]. In our previous phosphoproteomic analysis of BCSCs and non-BCSCs sorted from BC0145 PDX, the functions of 21% of 455 phosphoproteins upregulated in BCSCs, including TMCC3, were unknown ( Fig. 1a) [15]. In replicate phosphoproteomic studies, greater phosphorylated TMCC3 at Serine 216 was noted in BCSCs than in non-BCSCs by 1.5 and 3.8 folds (Supplementary Fig. S1a). TMCC3 belongs to the TMCC family that includes TMCC1-3 and contains two coiled-coil domains of the N-terminal region and two transmembrane domains of the C-terminal region ( Supplementary Fig. S1b). Sequence alignment of TMCC3 from various vertebrate species shows several highly conserved regions ( Supplementary Fig. S1c), suggesting the important roles of this protein in the vertebrates.
To confirm the differential expression levels of TMCC3 protein in BCSCs enriched populations and non-BCSCs, we determined BCSC enrichment markers in three breast cancer PDXs. Previously, H2k d− CD24 − CD44 + cell population was shown to be enriched in BCSC of BC0145 PDX [14]. For BC0350R1 and BC0634 PDXs, aldehyde dehydrogenase (H2k d− ALDH h ) activity was characterized as the BCSC enrichment marker base on tumorigenicity assay in vivo (Supplementary Table S1). Using the aboveidentified BCSC enrichment markers, BCSCs and non-BCSCs were sorted from these three PDXs for western blotting. As shown in Fig. 1b, the protein levels of TMCC3 were higher in BCSCs than non-BCSCs by 10.7, 9.8, and 8.9 folds, for BC0145, BC0350R1, and BC0634 tumors, respectively.
To facilitate in vitro investigation of the involvement of TMCC3 in BCSCs, AS-B145 and AS-B634 cells were cultured from sorted H2K d− CD24 − CD44 + cells of BC0145 and H2K d− ALDH h cells of BC0634 PDXs, respectively, for limited passages (≤10 passages) [14,15]. Using these two short-term cultured cells and established breast cancer cell lines, we compared the expression levels of TMCC3 in mammosphere-cultured (Sphere) and monolayer-cultured (2D) cells. As shown in Fig. 1c, TMCC3 protein expression was greater in mammosphere-cultured cells by 1.5, 3.8, 6.0, and 3.3 folds in MCF7, MDA-MB231 (MB231), AS-B145, and AS-B634, respectively. These findings suggest that TMCC3 may play a role in the maintenance of BCSCs.

TMCC3 silencing suppresses tumor growth and tumorigenesis in vivo
We next evaluated the in vivo effects of TMCC3 silencing on the tumorigenicity of AS-B145 and AS-B634. Control shRNA (shControl) and shRNA-TMCC3 (shTMCC3 #B) transduced AS-B145 and AS-B634 were injected into mammary fat pads of NSG female mice. Tumor size was monitored weekly for 6-8 weeks, and tumors were harvested for BCSC population examination and Ki67 staining. As shown in Fig. 2, the photographs (a) and weights (b) of tumors from TMCC3 silenced (shTMCC3) AS-B145 and AS-B634 were obviously smaller than control tumors (shControl) (p < 0.01). The growth rates of TMCC3 silenced tumors were also significantly slower than control tumors (p < 0.0001 for both AS-B145 and AS-B634) (Fig. 2c, d). We also examined the tumor growth of TMCC3 overexpressing MCF7 in vivo. As shown in Fig. 2e, f, TMCC3 overexpressing tumors (Fig. 2f) grew faster than the vector control group (Fig. 2e). Analysis of BCSC cell population (H2k d− CD24 − CD44 + ) in the TMCC3 silenced AS-B145 tumors showed reduction of BCSCs from 45.8% in shControl tumors to 15.1% in shTMCC3 tumors (Fig. 2g). Furthermore, Ki67 expression was significantly reduced from 24.3 ± 1.6% in shControl AS-B634 tumors to 7.2 ± 1.7% in TMCC3 silenced tumors (n = 3, p < 0.01) (Fig. 2h). These findings indicate that TMCC3 is not only important for tumor growth, but also crucial for the maintenance of BCSC population in vivo.
To determine the tumor-initiating capacity (TIC) of TMCC3 silenced AS-B145 and AS-B634, serial dilutions of cells were injected into NSG mice. As shown in Table 1, TIC of AS-B145 and AS-B634 decreased from 1:19,707 to 1:70,682 (p = 0.014) and 1:59 to 1:987 (p < 0.001), respectively, after TMCC3 knockdown. This provides further evidence for the crucial role of TMCC3 in CSC properties in vivo.

TMCC3 is crucial for tumor metastasis in vitro and in vivo
The propensity of CSCs for tumor metastasis is well documented [25,26]. To delineate the involvement of TMCC3 in metastasis of breast cancer, we examined the expression of TMCC3 in metastatic lesions of breast cancer PDXs. Two to three months after injection of BC0145 or BC0634 tumor cells in mammary fat pads of NSG mice, metastatic lesions were observed in ipsilateral lymph nodes and lungs. The lymph nodes and primary tumor of BC0145 PDX were harvested for staining with anti-CD44 and anti-TMCC3 antibodies. As shown in Fig. 3a, 4.1% and 15.3% of cells in the BC0145 primary tumor and metastatic lymph node, respectively, were CD44 + TMCC3 + . The relative fold of CD44 + TMCC3 + subsets in metastatic lymph node was 2.72 ± 0.7, as compared with primary tumor cells (Fig. 3b, n = 5, p = 0.04). In addition, the representative IHC staining for TMCC3 expression of primary tumor, lymph node, and lung-metastatic tissues from one of three BC0634 Fig. 1 TMCC3 is highly expressed in BCSCs and crucial for BCSC maintenance. a Workflow illustration of phosphoproteomic analysis using BCSCs and non-BCSCs of BC0145 PDX tumor to discover function unknown phosphoproteins in BCSCs. b The protein levels of TMCC3 in BCSCs and non-BCSCs FACS-sorted from breast cancer PDX tumors, BC0145, BC0350R1, and BC0634 using H2k d-CD24 − CD44 + (BC0145) and H2k d− ALDH + (BC0350R1 and BC0634), respectively. c The protein levels of TMCC3 in MCF7, MDA-MB231 (MB231), AS-B145, and AS-B634 cultured as monolayer (2D) and mammosphere culture (Sphere) condition, as determined by western blotting. d The representative images of mammosphere in TMCC3 silenced AS-B145 and AS-B634 (left panels). Sphere forming capacities of AS-B145 and AS-B634 transduced with shRNA-TMCC3 (shTM #A and shTM #B) vs shRNA-control (shCtrl) after 7 days culture (right panels). Data represent mean ± SD. **p < 0.01, # p < 0.001 and ## p < 0.0001 (n = 6, t-test). Scale bar = 200 μm. e The representative images of mammospheres and sphere forming capacities of MCF7 overexpressing TMCC3 vs vector control after 7 days culture. Data represent mean ± SD. # p < 0.001 (n = 6, t-test). Scale bar = 200 μm. f, g ALDH activity of TMCC3 silenced AS-B145 and AS-B634 (shTM #A and shTM #B), as compared with their respective controls. ALDH + subpopulation was determined by flow cytometry and the relative folds of ALDH + cells were calculated from three independent experiments (h). Data represent mean ± SEM. ## p < 0.0001 (n = 3, t-test). i CD24 − CD44 + cell population of TMCC3overexpressed MCF7 was determined by flow cytometry, as compared with vector control transduced MCF7. Data represent mean ± SEM. *p = 0.01 (n = 6, t-test).
bearing mice were shown in Fig. 3c. The histoscores (hscore) of TMCC3 expression in tumors as shown in Fig. 3d demonstrated highest TMCC3 expression in lung lesions followed by lymph node and then primary tumors (n = 3, p < 0.05). These findings suggest that TMCC3 may contribute to tumor metastasis.
To examine the role of TMCC3 in tumor metastasis, we next determined whether TMCC3 silencing suppresses the migration of TMCC3 expressing BCSCs in migration assay.
To provide further evidence supporting the notion that TMCC3 is important for metastasis, we examined the impact of TMCC3 silencing on the metastatic frequency of AS-B634. After injection of AS-B634, primary tumor, lymph node, and lung of tumor bearing mice were harvested at 3 months. As shown in Fig. 3g, we found TMCC3 silencing abolished metastasis in vivo. The lymph node and lungmetastatic frequency were 75% (3/4) and 50% (2/4), respectively, in control group, but none in TMCC3 knockdown. These findings support the participation of TMCC3 in tumor metastasis in vitro and in vivo.

TMCC3 is involved in AKT activation in BCSCs
It is well known that AKT plays important roles in the center of diverse signaling cascades and essential to stem cell activation, CSC survival, and self-renewal [27][28][29][30][31][32].
Previously, we demonstrated that IGF-1R/PI3K/AKT/ mTOR pathway was important for BCSC maintenance [14]. To examine a possible linkage of TMCC3 to AKT signaling, we determined AKT phosphorylation in TMCC3 silenced AS-B634. As shown in Fig. 4a, TMCC3 knockdown by shTM #A and #B decreased the relative folds of pAKT S473 /AKT to 0.4 and 0.2, and pAKT T308 / AKT to 0.6 and 0.3 in AS-B634, respectively. On the other hand, relative folds of pAKT S473 and pAKT T308 increased to 1.3 and 1.5 in TMCC3 overexpressing MCF7 upon insulin stimulation, respectively (Fig. 4b). These findings suggest that AKT activation may contribute to the critical role of TMCC3 in maintenance of BCSC properties. We further examined the possibility that TMCC3 may enhance AKT phosphorylation via activation of PI3K/PDK1, the up-stream regulators of AKT. As shown in Fig. 4a, b and Supplementary Fig. S3, the phosphorylation of regulatory subunit p85 (p-p85/p85) of PI3K and PDK1 (pPDK/PDK) did not decrease upon TMCC3 silencing in AS-B634 or increase in TMCC3 overexpressing MCF7. In addition, TMCC3 overexpression in MCF7 did not enhance membrane translocation of catalytic subunit p110-α of PI3K (Fig. 4c). These findings suggested that TMCC3-induced AKT activation likely does not involve PI3K or PDK1 phosphorylation. Recent studies have identified that mTOR complex 1 (mTORC1) and mTORC2 activation correlate to AKT phosphorylation [33][34][35]. To explore the consequence of TMCC3-induced AKT activation, we examined mTORC1 and mTORC2 phosphorylation upon TMCC3 knockdown or overexpression. As shown in Fig. 4d, e, there were no alterations in the activation of mTORC1 (pRaptor/Raptor and p-mTOR/mTOR) and mTORC2 (pRictor/Rictor and p-SIN1/SIN1) in TMCC3 silenced or overexpressing cells. However, the use of phosphoproteins/total proteins ratios to reflect activation status were affected by reduction of total p85 and PDK1 proteins in shTM#A and shTM#B transduced AS-B634 (Fig. 4a), and total Rictor and SIN1 proteins in one of shRNA-TMCC3 transduced cells (Fig.  4d). It should be noted that TMCC3 silencing reduced cell growth ( Supplementary Fig. S4), which may explain the decrease in these signaling proteins. Taken together, these findings suggested that PI3K, PDK1, mTORC1, or mTORC2 signalings are most likely not involved in TMCC3-induced AKT activation.

1-153 a.a. domain of TMCC3 contributes to interaction and activation of AKT
In order to ascertain the regulation of TMCC3 on AKT activation, we pulled down the endogenous TMCC3 in AS-B634 with anti-TMCC3 antibody. As shown in Fig. 5a, AKT was detected in the immunoprecipitation. To confirm the interaction of TMCC3 and AKT, we constructed flag-tagged TMCC3 and HA-tagged AKT for coimmunoprecipitation assay. As shown in Fig. 5b, c, HAtagged AKT was pulled down with flag-tagged TMCC3 using anti-flag antibody, and vice versa.
To decipher the AKT-interacting domain of TMCC3, we constructed flag-tagged full-length (1-477), 1-153, 1-282, 154-282, and 283-416 amino acid (a.a.) domains of TMCC3 for immunoprecipitation assay (Fig. 5d). 293T cells were co-transfected with flag-tagged TMCC3 (full-length or truncated protein) and HA-tagged AKT. After flag protein pull-down using anti-flag antibody, the immunoprecipitates were examined for HA-AKT proteins with anti-HA antibody. As shown in Fig. 5e, AKT was pulled down together with 1-153, 1-282 a.a. domains and full-length TMCC3, but not with 283-416 a.a. domain. However, the expression of 154-282 a.a. domain of TMCC3 was too low to delineate whether it interacted with AKT or not. To provide evidence for direct interaction between TMCC3 and AKT in a cellfree system, luminex immunosandwich assay was performed as illustrated in Fig. 5f [36]. Recombinant 1-158 a.a. domain of TMCC3 protein was generated and coupled onto Luminex beads, which were then incubated with various concentrations of recombinant AKT. After washing, AKT interacting with bead-bound 1-158 a.a. domain of TMCC3 were recognized by the fluorescence labeled anti-AKT antibody. As shown in Fig. 5g, AKT can bind directly with 1-158 a.a. domain of TMCC3 protein in vitro in a concentration dependent manner, with KD of 1.67 ± 0.43 nM. On the other hand, AKT did not bind to bead-bound control Fig. 4 TMCC3-induced AKT activation likely does not involve PI3K/PDK1/mTORC1 or mTORC2 pathway. AS-B634 cells were transduced with shRNA-TMCC3 #A, #B, or shRNA-control for 3 days. MCF7 cells were transiently transfected with TMCC3 in the presence (+) or absence (−) of 10 μg/ml human insulin for 5 min after 12 h serum starvation. Levels of phosphorylated AKT (S473 and T308), p85 of PI3K and PDK1, and total protein in TMCC3 silenced AS-B634 (a) and TMCC3 overexpressing MCF7 (b), were determined by western blotting. Relative intensity was quantified and normalized to non-phospho protein or GAPDH. c Subunit p85 phosphorylation and p110-α membrane translocation were determined in TMCC3 overexpressing MCF7 cells. Integrin β1 and GAPDH served as membrane and cytoplasmic markers, respectively. d, e mTORC1 (Raptor and mTOR) and mTORC2 (Rictor and SIN1) activation were assessed in TMCC3 silenced AS-B634 and TMCC3 overexpressing MCF7 cells.  1-153 a.a.-or 154-477 a.a.-truncated TMCC3. i Sphere numbers were calculated after culture for 7 days. # p < 0.001, ## p < 0.0001 (n = 8, t-test). j The numbers of migrated cells in each group were determined in trans-well migration assay. *p < 0.05, **p < 0.01 (n = 3, t-test).
protein (Puf-A) [37]. These findings support the notion that 1-158 a.a. domain of TMCC3 directly interacted with AKT without other accessory proteins.

Expression of TMCC3 mRNA in breast cancer tissues
To evaluate the clinical relevance of TMCC3 expression, we examined the mRNA levels of TMCC3 in tumor specimens of 202 patients with breast cancer by qRT-PCR. Clinical characteristics and demographic information of these patients were summarized in Supplementary Table S2. The mean age was 54.4 ± 12.2 (range: 30-89). The median follow-up time was 9.55 years (range: 0.30-12.7 years). The relationship between TMCC3 mRNA expression and clinical pathological variables in breast cancer was presented in Supplementary  Table S3. Among 202 patients, 51 patients (25.7%) had disease recurrence. Using Youden index to determine the optimal cutoff values defining high and low expression groups, we found that patients with high expression level of tumor TMCC3 mRNA were at greater risk than those with low expression level for lymph nodes involvement (p = 0.04, OR: 1.89, 95% CI: 1.06-3.42), relapse (p < 0.001, OR: 4.45, 95% CI: 2.27-8.69), and death (p < 0.001, OR: 3.77, 95% CI: 1.96-7.23) (Supplementary Table S3).

Association of TMCC3 expression levels with survival
The relapse-free survival (RFS) and overall survival (OS) of breast cancer patients as analyzed by the Kaplan-Meier method, and a log-rank test showed that patients with low expression level of TMCC3 in tumor had significantly greater RFS (p < 0.001) and OS (p < 0.001) than patients with high expression levels (Fig. 6a, b). We then analyzed the potential prognostic value of TMCC3 expression in 161 patients with early-stage disease (stages 1-2). As shown in Fig. 6c, d, early-stage patients with low expression of TMCC3 in tumor part had significantly greater RFS (p < 0.001) and OS (p < 0.001) than patients with high expression levels. The survival benefit of patients with low expression of TMCC3 was even more striking for earlystage luminal A and B patients (p = 0.0001 for RFS, p < 0.0001 for OS) (Fig. 6e, f). These results demonstrated the adverse impacts of high expression of TMCC3 on clinical outcome of breast cancer, especially for those with early-stage luminal subtypes.
Higher expression level of TMCC3 is an independent prognostic factor for breast cancer  Table  2). Next, to identify the independent variables associated with poor RFS or OS, we selected those important covariates that showed statistical significance in the univariate analysis for multivariable Cox regression analysis in a stepwise manner. As shown in Table 2, age was independent risk factors for RFS. And age and stage were independent risk factors for OS. Notably, the expression level of tumor TMCC3 was an independent risk factor for RFS (HR: 2.81, 95% CI: 1.45-5.45, p = 0.002) and OS (HR: 2.28, 95% CI: 1.31-3.96, p = 0.004). These data indicate that TMCC3 expression in tumor tissue is an important and independent predictor for RFS and OS in breast cancer patients.

Data mining confirms poor prognostic impact of high expression of TMCC3 in breast cancer and other cancers
Using an online DNA microarray database at the ONCO-MINE website, we found higher mRNA level of TMCC3 in cancer part than normal tissue in cervical cancer, prostate cancer, pancreatic cancer, lung cancer, glioblastoma, skin cancer, hepatoma, and thyroid gland papillary carcinoma ( Supplementary Fig. S6a). Using Kaplan-Meier Plotter website to evaluate the clinical significance of TMCC3 in cancer progression, we showed that higher expression of TMCC3 correlated with poor OS of patients with ovarian, lung, and gastric cancer (Supplementary Fig. S6b). These findings lend further support for the crucial roles of TMCC3 in cancer progression, and for developing strategies to target TMCC3 for cancer therapy.

Discussion
In our previous comparative phosphoproteomic analysis of BCSCs and non-BCSCs, we found a function unknown protein, TMCC3 to be expressed at higher level in BCSCs than non-BCSCs [15]. Here, we demonstrated that TMCC3 is crucial for maintaining the characteristics of BCSCs, including self-renewal, differentiation, metastasis, and tumorigenesis. In addition, we showed that TMCC3 could enhance AKT phosphorylation, which is known to promote BCSC properties.
TMCC3 was reported as an ER-anchored protein and localized at the three-way junctions of tubular ER network [20,38]. In mouse developing embryos, TMCC3 was expressed in the mesenchyme of developing tongue, hind brain forming tissues and lung, based on in situhybridization analysis [20]. However, the biological functions of TMCC3 remain enigmatic. Our study has provided the first evidence for the crucial role of TMCC3 in sustaining BCSCs properties as reflected by diminished mammosphere forming capacity, ALDH activity, in vivo metastasis and tumorigenicity upon TMCC3 silencing. In addition, we demonstrated that TMCC3 promoted AKT activation, and TMCC3 directly interacted with AKT through 1-153 a.a. fragment. This AKT-interacting domain of TMCC3 is crucial for TMCC3-induced AKT activation, mammosphere formation, and metastasis.
Dysregulation of AKT pathway is frequently observed in many type of cancers, including breast cancer, and associated with poor outcome [39,40]. AKT activation contributes to self-renewal and differentiation of normal mammary stem cell and BCSCs [41,42]. Mechanistically, we found TMCC3 contributes to self-renewal and metastasis by the direct binding and activation of AKT via 1-153 a.a. domain of TMCC3. In previous reports, TMCC3 was found to bind with 14-3-3 in HEK 293 cells [20]. In our mass spectrometric analysis, we did not find 14-3-3 family proteins as interacting partners in the immunoprecipitates of TMCC3 in MCF7 cells. This suggests that the interacting proteins and regulatory mechanisms of TMCC3 might vary according to the types of cells. Based on our cell-free biochemical studies, recombinant 1-158 a.a. domain of TMCC3 can directly interact with AKT without any accessory proteins. However, whether any other molecular complexes are recruited or involved in TMCC3-induced AKT activation remains to be determined.
AKT plays a center role of tumorigenesis. Upon activation, p110 PIK3CA phosphorylates phosphatidylinositol (3,4)-bisphosphate (PIP2) to form phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Binding of AKT to PIP3 leads to AKT translocation from the cytoplasm to the plasma membrane in close proximity to PDK1, which phosphorylates AKT at T308 [43,44]. The full activation of AKT requires AKT to be phosphorylated at S473 by mTORC2 [34]. Although, TMCC3 activates AKT phosphorylation at both T308 and S473, we have provided experimental evidence that PI3K, PDK1, mTORC1, or mTORC2 pathways are most likely not involved in TMCC3-induced AKT activation. Although AKT is known to be activated by PI3K/ PDK1, several reports show that AKT can be activated independent of PI3K/PDK1 [45][46][47][48][49]. In addition, many studies have demonstrated the existence of complex crosstalks between AKT and multiple cell signaling cascades, which can further promote cancer progression and influence drug sensitivity [50]. Whether TMCC3 participates in such crosstalks awaits further investigation.
Through analysis of proteomics profiling of colorectal cancer and chronic lymphocytic leukemia, TMCC3 protein level was found to be greater in cancer parts than their normal counterparts [51,52]. However, the clinical relevance of TMCC3 was not addressed. Our study is the first to demonstrate the prognostic significance of TMCC3 in breast cancer. More importantly, we found that high TMCC3 expression adversely impacted clinical outcome in earlystage luminal breast cancer. Until now, Oncotype DX, MammaPrint, and PAM50 are the only validated biomarker assays for early-stage luminal breast cancer [53]. However, these tests are quite costly and complex. The expression level of TMCC3, if confirmed to be highly prognostic in future study, will make TMCC3 as a simple and valuable biomarker for early-stage luminal breast cancer.
In view of the adverse impact of high expression of TMCC3 on clinical outcome of breast cancer, which was corroborated by similar prognostic significance of TMCC3 in ovarian, lung and gastric cancers by data mining, and the negligible expression of TMCC3 mRNA in human vital organs (heart, lung, liver, and kidney) based on microarray database at the ONCOMINE, TMCC3 an ideal target for the design of therapeutics to eradicate CSCs in the future.
In conclusion, we provide the first evidence supporting that TMCC3 plays important roles in maintaining BCSCs features, tumor metastasis, and tumorigenicity. TMCC3 enhances AKT activation through direct interaction with AKT. Clinically, high TMCC3 expression is an independent poor prognostic factor in breast cancer, including earlystage breast cancer. These findings support future pursuit of TMCC3 as a target for BCSCs-directed therapeutic agents.

Compliance with ethical standards
Conflict of interest YHW, JY, and ALY are holders of a patent for "Methods for suppressing cancer by inhibition of TMCC3" US patent 9,447,193. The remaining authors (YTC, THH, JTH, MWK, SHW, YH, YJL, SCC, JCY, and JCW) declare no competing financial interests.
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