Enhanced secretion of tumorigenic effector proteins is a feature of malignant cells. The molecular mechanisms underlying this feature are poorly defined. We identify PITPNC1 as a gene amplified in a large fraction of human breast cancer and overexpressed in metastatic breast, melanoma, and colon cancers. Biochemical, molecular, and cell-biological studies reveal that PITPNC1 promotes malignant secretion by binding Golgi-resident PI4P and localizing RAB1B to the Golgi. RAB1B localization to the Golgi allows for the recruitment of GOLPH3, which facilitates Golgi extension and enhanced vesicular release. PITPNC1-mediated vesicular release drives metastasis by increasing the secretion of pro-invasive and pro-angiogenic mediators HTRA1, MMP1, FAM3C, PDGFA, and ADAM10. We establish PITPNC1 as a PI4P-binding protein that enhances vesicular secretion capacity in malignancy.

Secretion of pro-tumorigenic proteins is a required feature for efficient metastatic colonization. We find that PITPNC1 — a protein that correlates in expression with tumor progression in breast cancer, colon cancer, and melanoma — regulates metastatic secretion. PITPNC1 does this by recruiting the small GTPase RAB1B to the trans-Golgi compartment of the cell by binding to the Golgi-resident lipid PI4P. In the Golgi, the PITPNC1/RAB1B complex enhances metastatic secretion capacity by recruiting the protein GOLPH3 to the Golgi. This enables the enhanced release of vesicles containing effector proteins.

PITPNC1 was previously identified as a gene targeted by miR-126, a metastasis-suppressor microRNA (). The cell-biological function of PITPNC1, however, was not defined. Here we reveal that PITPNC1 is genetically amplified in a large fraction of human breast cancers and that its overexpression significantly correlates with metastatic progression of breast, melanoma, and colon cancers. Through complementary biochemical and in vitro methods, we have found that PITPNC1 binds PI4P — a previously unreported lipid substrate for phosphatidylinositol transfer protein (PITP) domain proteins — and that this interaction localizes PITPNC1 to the Golgi compartment within the cell. Golgi localization of PITPNC1 increases Golgi abundance of the PITPNC1-interacting protein RAB1B. RAB1B is a GTPase that cycles between a guanosine diphosphate (GDP)-bound inactive state bound to GDP-dissociation inhibitor alpha in the cytoplasm and a GTP-bound active state in the Golgi where it is required for Golgi function (). PITPNC1-dependent RAB1B recruitment to the Golgi compartment enhances secretion by cancer cells. This optimized secretory state is accompanied by the extension of the Golgi morphology, which is known to occur in the context of enhanced vesicular release. Conversely, PITPNC1 depletion in malignant cells reduces the secretion of a number of secreted factors.

The steps of the metastatic cascade require precise regulation of multiple cellular phenotypes and extracellular interactions. Understanding the molecular and cell-biological mechanisms by which genes execute these processes is a prerequisite for the development of effective therapies for treating and potentially preventing metastatic disease (). A key feature of metastatic cells is their ability to affect various cell types within the tumor microenvironment through the release of secreted factors (). Yet the mechanisms that govern this phenotype are incompletely understood.

Phosphatidylinositols (PIs) are members of a large family of complex amphiphilic compounds comprised of a glycerol backbone, two fatty acids, a phosphate group, and an inositol head group. While these molecules constitute a minor component of eukaryotic cell membranes, they play key roles as transducers of signals within cells (). PIs can be phosphorylated by various kinases, yielding mono, di-, or tri-phosphorylated products. Specific phosphorylated PIs can be bound within lipid membranes by signal-transducing proteins, can serve as docking sites for the selective recruitment of effector proteins to local cellular membrane compartments, or can be metabolized by specific PI phosphatases (). The significance of this class of molecules to physiology and disease has been well demonstrated through mechanistic studies on phosphatidylinositol 3-kinase (PI3K) and PTEN, which respectively positively and negatively regulate the formation of the tri-phosphorylated PIP3, a central regulator of growth and survival of cells (). While earlier studies implicated PI3K and PTEN in the regulation of oncogenesis, subsequent work has revealed pervasive and fundamental roles for these PI-modifying enzymes in the physiological and pathological functions of many organelle systems ().

Given the individual importance of each of these secreted factors, we next investigated whether they each contributed to PITPNC1-mediated metastatic colonization. Consistent with the cooperative impact of these secreted factors on metastatic progression, we found all five of these genes to be required for the optimized metastatic colonization activity induced by PITPNC1 overexpression ( Figures 7 C–7F). These epistatic experiments reveal PITPNC1 to drive metastasis, in part, through augmenting the cellular release of FAM3C, MMP1, HTRA1, PDGFA, and ADAM10 secreted pro-metastatic proteins.

Our model thus predicts that FAM3C, MMP1, HTRA1, PDGFA, and ADAM10 are pro-metastatic and pro-angiogenic genes that may drive metastasis. To test this, we first depleted each gene in highly metastatic LM2 cells and assessed their ability to invade and to recruit endothelial cells. Both invasion and endothelial recruitment phenotypes by cancer cells were highly dependent on each of these proteins to varying degrees ( Figures 7 A, 7B, and S7 A). This suggests that in vivo-selected metastatic cells are optimized for maximal invasion and endothelial recruitment capacity through the enhanced secretion of multiple factors that cooperate in these processes. Moreover, these findings reveal that these secreted factors similarly affect the movement of both cancer cells and endothelial cells through the extracellular matrix.

These findings suggest that the PITPNC1-RAB1B complex promotes the metastatic potential of cancer cells by enhancing Golgi secretion capacity. To identify the set of secreted proteins that mediate the pro-metastatic effects of PITPNC1, we conducted stable isotope labeling by amino acids in cell culture (SILAC) on media conditioned by control versus PITPNC1-depleted cells. Proteomic analysis revealed six proteins (MMP1, PDGFA, HTRA1, PDGFRL, FAM3C, and ADAM10) that are all known to be expressed by breast cancer cells to be at least 2-fold less abundant in conditioned media from PITPNC1-depleted cells relative to control cells ( Figure 6 C). Western blot analysis of conditioned media confirmed that the secreted protein levels of all these factors except PDGFRL decreased significantly upon PITPNC1 knockdown ( Figures 6 D and S6 C). These effects were not secondary to reduced protein production as no differences in total cellular protein abundance were observed ( Figure 6 E). Conversely, overexpression of PITPNC1 leads to the increased secretion of all five proteins without changing their overall intracellular protein levels ( Figure S6 D and S6E). These findings are consistent with a model wherein these secreted factors are not released from the Golgi upon PITPNC1 depletion as a consequence of impaired PI4P/GOLPH3 function. To further test this, we used MMP1-RFP as a marker for metastatic secretion in a Golgi exit assay. Consistent with our model, PITPNC1 depletion significantly reduced MMP1-RFP release from the TGN ( Figure 6 F).

Multiple steps in the metastatic cascade are orchestrated by the secretion of regulatory proteins from cancer cells in the microenvironment. We have thus far demonstrated that PITPNC1 in concert with RAB1B promotes metastasis by recruiting GOLPH3 to extend the Golgi morphology. Given that PITPNC1 regulates invasion and endothelial recruitment ( Figures 1 G and 1H), which are both mediated by secretion of proteins, that RAB1B is critical for Golgi function (), and that Golgi extension reflects enhanced PI4P/GOLPH3/MYO18A activity to promote vesicular release (), we hypothesized that the pro-metastatic effects of PITPNC1 are mediated through enhanced secretory capacity. This model is also consistent with our previous finding that PITPNC1 acts upstream of secreted IGFBP2 in miR-126-regulated metastasis (). To directly test this, we followed Golgi vesicular release by tracking vesicular PI4P in the cytoplasm in a Golgi exit assay ( Figure S6 A). Interestingly, PITPNC1 depletion significantly reduced the release of PI4P-containing vesicles from the Golgi ( Figure 6 A ). Conversely, PITPNC1 overexpression increased the secretory capacity of the Golgi in a RAB1B-dependent manner in MDA-MB-231 cells ( Figures 6 B and S6 B).

(A) Golgi exit assay analysis of LM2 cells transfected with control or PITPNC1-targeting siRNA. Following a 2-hr incubation at 23°C in the presence of 100 μg/ml cyclohexamide, the cells were returned to 37°C and prepared for immunofluorescence for PI4P (FAPP-PH) and p230 analysis at time 0, 10, and 30 min. The abundance of PI4P-containing vesicles released to the cytoplasm was determined by subtracting Golgi-localized PI4P from the total cellular PI4P signal. All measurements of cytoplasmic PI4P were blinded. n = 10/time point/group. Significance test between groups was performed with Fisher's method.

The above findings reveal a pathway wherein metastatic cancer cells upregulate PITPNC1, which mediates GOLPH3 recruitment to the trans-Golgi. This recruitment extends Golgi morphology and leads to the enhancement of pro-metastatic phenotypes of invasion and endothelial recruitment.

Having demonstrated that PITPNC1 promotes Golgi extension and that Golgi extension correlates with increased metastatic capability, we next sought to examine how the PITPNC1-RAB1B complex regulates Golgi structure. The Golgi structure condensation (decreased cisternae length and increased thickness) we observed upon acute depletion of either PITPNC1 or RAB1B phenocopies depletion of another protein, GOLPH3, which bridges Golgi PI4P and the actomyosin MYO18A to promote vesicle release and associated Golgi extension (). Increased GOLPH3 abundance in the trans-Golgi would therefore allow for increased Golgi extension and vesicular release. To this end, we performed immunofluorescence analysis of GOLPH3 and the trans-Golgi marker p230 in the setting of either PITPNC1 or RAB1B depletion. Indeed, we found that depletion of either PITPNC1 or RAB1B led to an approximate 2-fold reduction in GOLPH3 Golgi abundance, without changing the total cellular GOLPH3 protein content ( Figures 5 A and 5B ; Figure S5 A). Overexpression of PITPNC1 caused an increase in GOLPH3 Golgi levels ( Figures 5 C and S5 B). We then asked if PITPNC1-dependent recruitment of GOLPH3 to the Golgi is required for PITPNC1-mediated phenotypes. We found that PITPNC1-mediated Golgi extension is dependent on Golgi GOLPH3 expression, as depletion of GOLPH3 reversed the extended Golgi phenotype induced by PITPNC1 overexpression by 63% ( Figures 5 D and S5 C–S5E). Furthermore, in accordance with the findings of, we found that depletion of GOLPH3 in malignant cells condensed the Golgi in breast cancer cells ( Figure 5 D). Additional epistasis experiments confirmed that GOLPH3 is functionally downstream of PITPNC1-mediated invasion and endothelial recruitment phenotypes ( Figures 5 E and 5F). As GOLPH3 is recruited to the Golgi through PI4P, we next wondered if the PITPNC1-RAB1B complex recruits GOLPH3 to the Golgi complex through regulation of the local Golgi PI4P abundance. Interestingly, co-immunofluorescence imaging using the trans-Golgi marker p230 with the FAPP1-PH domain and an anti-PI4P antibody revealed that PITPNC1 knockdown significantly reduced trans-Golgi PI4P abundance, while PITPNC1 overexpression significantly increased trans-Golgi PI4P levels ( Figures 5 G, 5H, and S5 F–S5H). This indicates that PI4P abundance in the trans-Golgi is governed by PITPNC1 expression in cancer cells and leads to a model whereby PITPNC1 recruits RAB1B to the Golgi and augments Golgi PI4P levels. Increased Golgi PI4P abundance enhances the binding of GOLPH3 to the Golgi, allowing for Golgi extension and vesicular release. PITPNC1 could enhance Golgi PI4P abundance through multiple mechanisms such as recruitment of a specific PI4K to the Golgi or by enhancing the activity of Golgi-resident PI4Ks. To identify a potential PI4K that could mediate this effect, we performed invasion experiments analyzing the epistatic relationship between PITPNC1 and each of the four PI4Ks. This revealed that only depletion of PI4KIIIalpha abolished PITPNC1-induced invasion, while not affecting baseline invasion capacity ( Figure S5 I). While these data suggest that PI4KIIIalpha could represent a downstream mediator of PITPNC1-enhanced Golgi PI4P abundance, further studies are required to mechanistically establish this relationship.

Box and whiskers plots represent the data in (A–D), (G), and (H) with the upper and lower bars showing minimum and maximum data points. In (E) and (F), error bars represents SEM. See also Figure S5

(G and H) LM2 cells transfected with siRNAs targeting PITPNC1 or a control siRNA (G) or MDA-MB-231 cells overexpressing PITPNC1 or control vector (H) were stained for PI4P using FAPP-PH domain (red), p230 (green), and DAPI (blue). PI4P levels in the trans-Golgi were quantified as mean fluorescence intensity of FAPP1-PH signal in p230-positive regions. n = 50/group.

(E and F) MDA-MB-231 cells were transfected with GOLPH3 siRNA or control siRNA in the setting of control or PITPNC1 overexpression and subjected to the endothelial recruitment (E) and invasion (F) assays. n = 4/group.

(A and B) LM2 cells transfected with either control siRNA or siRNAs targeting PITPNC1 (A) or RAB1B (B) were immunocytochemically stained for endogenous GOLPH3 and p230. GOLPH3 levels in the trans-Golgi were quantified as mean fluorescence intensity of GOLPH3 in p230-positive regions. n = 40/group.

Our findings suggest a critical role for Golgi-localized RAB1B in PITPNC1-mediated metastasis. Previous studies have described a crucial role for RAB1B in maintaining Golgi structure and function (). The Golgi network consists of stacks of membrane-bound structures. Proteins destined for secretion are transported to the trans face of the Golgi network, from which vesicular budding and release occurs (). In actively secreting cells, enhanced vesicular budding results in extension of the TGN, which is “stretched” by tensile forces from actin cytoskeletal proteins interfacing with proteins bound to vesicular membranes mediating vesicular release (). While investigating the phenotypic effect of RAB1B depletion, we noted a clear impact on Golgi morphology. We found that acute depletion of RAB1B led to the condensation of the Golgi ( Figure 4 A , Figure S3 E). We next asked if changes in Golgi structure is a morphological feature that correlates with metastatic capacity in breast, colon, and skin cancers by comparing the Golgi morphology between poorly metastatic parental cell populations and their in vivo-selected highly metastatic derivative sublines (). Interestingly, we found that in these cancers, highly metastatic subpopulations consistently exhibited a more extended Golgi structure compared with their poorly metastatic parental cell populations ( Figure 4 B). This suggests that altered Golgi structure and function may contribute to metastatic capacity. We next investigated whether PITPNC1 regulates such Golgi morphology. Indeed, PITPNC1 depletion caused the Golgi to condense as analyzed by both immunofluorescence and transmission electron microscopy (TEM) in breast cancer cells ( Figures 4 C and 4D). In addition to cisternae length, PITPNC1 depletion resulted in increased cisternae thickness, but did not significantly affect the number of Golgi stacks or the number of Golgi-associated vesicles ( Figures 4 E and S4 A). Conversely, gain-of-function experiments revealed that PITPNC1 expression promotes Golgi cisternae length and reduces cisternae thickness, consistent with PITPNC1 functioning as a metastasis promoter ( Figure S4 B). In addition, the ability of PITPNC1 to extend the Golgi was dependent on its binding to PI4P, as the N88F lipid-binding mutant was unable to alter Golgi structure ( Figures 4 F and S4 C). Consistent with our in vivo metastasis results, PITPNC1 also promoted Golgi extension in melanoma and colon cancer cells, suggesting that PITPNC1 may affect the metastatic phenotypes of these cancer types via its effect on Golgi function ( Figures 4 G and S4 D).

(B) Highly metastatic derivates of the poorly metastatic MDA-MB-231 breast-, Ls714 colon-, and MeWo melanoma cancer cells developed previously () as well as their parental cell populations were analyzed for Golgi extent as in (A). n = 40/group.

(A) LM2 cells transfected with either control or siRNAs targeting RAB1B for 24 hr were immuno-stained for p230 and DAPI. The Golgi extent was quantified as the fraction of the nucleus circumference that was covered by p230-positive Golgi signal. n = 40/group.

These results suggest that PITPNC1 regulates RAB1B Golgi localization to mediate its metastatic effects. To functionally test this relationship, we performed epistasis experiments by assessing the ability of PITPNC1 to promote invasion and endothelial recruitment in the absence of RAB1B. RAB1B depletion completely abrogated the PITPNC1-mediated enhancement of invasion and endothelial recruitment capacity ( Figures 3 I, 3J, S3 E, and S3F). Importantly, the reduction in invasion and endothelial recruitment was more pronounced in PITPNC1 overexpressing cells relative to control cells. These finding are consistent with RAB1B acting as a downstream effector of PITPNC1-mediated metastasis. Collectively, these findings reveal that PITPNC1 interacts with RAB1B in the Golgi to drive metastasis.

To gain further mechanistic insights into the cellular function of PITPNC1, we next sought to identify potential PITPNC1-interacting proteins. To this end, we expressed Flag-tagged PITPNC1 in MDA-MB-231 breast cancer cells and performed immunoprecipitation of the Flag epitope followed by in-solution trypsin digestion and label-free mass spectrometry quantification. This revealed that PITPNC1 binds significantly to six isoforms of the 14-3-3 protein family and to the GTPase RAB1B ( Figure 3 A ). 14-3-3 proteins are scaffolding proteins that bind phosphorylated serine residues. Previously, 14-3-3 was found to bind phosphorylated PITPNC1 serine 274 and serine 299 in a manner that protects PITPNC1 from degradation (). Western blot analysis of immunoprecipitated protein from wild-type and S274/299A mutant PITPNC1 confirmed that the interaction with 14-3-3 is dependent on serine 274 and serine 299 phosphorylation ( Figure 3 B). We next asked if binding to 14-3-3 is required for the pro-metastatic function of PITPNC1 by performing invasion and endothelial recruitments assays with both wild-type and the S274/299A serine mutant of PITPNC1. We found 14-3-3 binding is necessary for PITPNC1-mediated pro-metastatic phenotypes ( Figures 3 C and 3D). As expected, this effect is likely explained by the shorter half-life of the S274/299A mutant ( Figure 3 E). In addition to 14-3-3, we identified the small GTPase RAB1B as a binding partner for PITPNC1 ( Figures 3 A and 3B). To further elucidate the cellular function of the interaction between PITPNC1 and RAB1B, we performed co-localization experiments and found that the vast majority of the overlap between the two proteins is detected in the perinuclear Golgi region ( Figure 3 F). Given our finding that PITPNC1 localization to the Golgi is required for its ability to promote metastasis and that RAB1B regulates Golgi function (), we next investigated whether PITPNC1 promotes metastasis by enhancing the abundance of RAB1B in the Golgi. Immunofluorescence analysis revealed a 1.8-fold reduction of endogenous RAB1B in the trans-Golgi upon depletion of PITPNC1 ( Figures 3 G, S3 A, and S3B) without any accompanying change in total cellular RAB1B protein levels as determined by western blotting ( Figure S3 C). Conversely, overexpression of wild-type PITPNC1 led to a significant increase in Golgi-localized RAB1B ( Figures 3 H and S3 D). Consistent with the effect of PITPNC1 on Golgi localization of RAB1B being dependent on its PI4P-binding capacity, overexpression of the lipid-binding mutant of PITPNC1 minimally affected RAB1B abundance in the Golgi ( Figure 3 H).

In (C), (D), (I), and (J) error bars represents SEM. Box and whiskers plots represent the data in (G) and (H) with the upper and lower bars showing minimum and maximum data points. See also Figure S3

(I and J) Matrigel invasion (I) and endothelial recruitment assay (J) of MDA-MB-231 cells expressing empty control vector or PITPNC1. Forty-eight hours prior to the experiments, both the control and PITPNC1 overexpressing cells were transfected with control siRNA or siRNA targeting RAB1B. n = 4/group.

(G) Golgi levels of endogenous RAB1B in LM2 breast cancer cells treated with control or two independent siRNAs targeting PITPNC1. Areas of the trans-Golgi compartment were defined by anti-p230 (green) immunoreactivity. Analysis of the anti-RAB1B signal intensity (red) in p230-positive areas defined the Golgi-specific RAB1B signal. n = 30/group.

(E) The half-lives of wild-type and S274/299 mutant PITPNC1 in MDA-MB-231 cells were determined by treating cells with 100 μg/ml cyclohexamide over 24 hr and analyzing PITPNC1 abundance in cellular lysates by western blotting at the given time points. The experiment was repeated twice.

(A) Lysates from MDA-MB-231 cells expressing Flag-tagged PITPNC1 or empty control vector were subjected to immunoprecipitation by anti-Flag beads. The eluate was trypsin digested in solution and the liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra were analyzed by label-free quantification. Comparison of eluted proteins by the empty vector (horizontal axis) and PITPNC1-Flag (vertical axis) revealed PITPNC1, several 14-3-3 protein forms and RAB-1B to be significantly different (p < 0.05) between the two samples. n = 3/group.

Having observed lipid binding toward PI4P, we questioned if this activity is required for the cellular localization of PITPNC1. GFP-tagged PITPNC1 displayed diffuse cytoplasmic localization as previously described () but showed most intense staining in perinuclear regions that co-stained with p230-marker of the trans-Golgi network (TGN) ( Figure 2 C). The Golgi is the sorting hub for vesicles destined for the plasma membrane and endosomal compartments, and these functions are dependent on resident PI4P (). Conversely, co-staining of PITPNC1 and PI4P using the PI4P-specific PH domain of FAPP1 confirmed that PITPNC1 accumulates in PI4P-positive areas of the Golgi ( Figures 2 D and S2 B). Previous studies have shown that T59E and N90F mutations of PITPα, another PITP domain family member that binds PI and phosphatidylcholine in vitro, abrogate lipid binding (). Homology analysis of PITPα and PITPNC1 revealed these amino acids to be conserved in PITPNC1 (T58 and N88). We therefore produced recombinant forms of both single amino acid mutations and tested their capacities for binding PI4P. Both mutations abrogated the PI4P-binding activity of PITPNC1 ( Figure 2 E). To determine if PI4P binding mediates Golgi localization of PITPNC1, we expressed Flag-tagged wild-type or N88F lipid-binding mutant PITPNC1 in breast cancer cells and performed immunofluorescence analysis between PITPNC1-Flag and the Golgi marker Giantin. These studies revealed that PITPNC1 localization to the Golgi is dependent upon PI4P binding ( Figures 2 F and S2 C). We then asked if PI4P binding and Golgi localization is required for the metastasis-promoting function of PITPNC1. To this end, we overexpressed either wild-type or the two lipid-binding mutant forms of PITPNC1 in MDA-MB-231 cells and found that wild-type PITPNC1, but neither of the lipid-binding mutants, was sufficient to promote invasion, endothelial recruitment, and metastatic colonization ( Figures 2 G–2I, S2 D, and S2E). Finally, in complementary experiments, we reduced trans-Golgi PI4P levels via expression of the trans-Golgi-localized PI4P phosphatase mutant Sac1K2A and asked if this manipulation would affect PITPNC1's ability to promote metastatic phenotypes ( Figures S2 F and S2G) (). Consistent with a required role for trans-Golgi PI4P to recruit PITPNC1 to the Golgi compartment, reduction of trans-Golgi-localized PI4P by Sac1K2A reduced PITPNC1 localized in the Golgi ( Figure 2 J). Further, PITPNC1 overexpression failed to promote invasion and endothelial recruitment phenotypes following Sac1K2A-mediated Golgi PI4P reduction in MDA-MB-231 cells ( Figures 2 K and 2L). Overall, these findings reveal that the interaction between PI4P and PITPNC1 in the trans-Golgi is required for its ability to promote metastasis.

Cloning and characterization of a novel variant (mM-rdgBbeta1) of mouse M-rdgBs, mammalian homologs of Drosophila retinal degeneration B gene proteins, and its mRNA localization in mouse brain in comparison with other M-rdgBs.

The N-terminal portion of PITPNC1 contains a PITP lipid-binding and transfer domain. To understand the molecular mechanism by which PITPNC1 regulates metastatic colonization, we sought to identify PITPNC1's lipid substrate. We first performed a lipid-overlay assay in which a broad range of lipids were spotted on a membrane and blotted with purified recombinant PITPNC1 protein in order to identify its substrate. Lipid-blot assays revealed that PITPNC1 binds most strongly to PI4P with weaker binding to other PIs containing a single phosphate head group, namely PI3P and PI5P ( Figures 2 A and S2 A). To test these protein-lipid interactions in a more physiological context, we conducted vesicular pull-down experiments. Consistent with the robust lipid binding of PI4P binding by PITPNC1, recombinant PITPNC1 was found to bind to vesicles containing PI4P, but not those containing PI3P, PI5P, PI3,4P2, or phosphatidic acid (PA) ( Figure 2 B).

For (B), (E), (G), (H), (I), (K), and (L) error bars represent SEM. Box and whiskers plots represent the data in (F) and (J) with the upper and lower bars showing minimum and maximum data points. See also Figure S2

(K and L) MDA-MB-231 cells expressing PITPNC1 or control vector and cells expressing PITPNC1 that was transfected with Sac1K2A were subjected to Matrigel invasion assay (K) and the endothelial recruitment assay (L). n = 4/group. NS, not significant.

(J) MDA-MB-231 cells with stable expression of Flag-tagged wild-type PITPNC1 were transfected with mock or Sac1K2A and co-stained with anti-Flag and anti-Giantin. The intensity of Flag immunoreactivity in areas positive for Giantin was considered the Golgi signal. The Golgi signal was normalized to total cellular levels. n = 30/group.

(F) MDA-MB-231 cells with stable expression of Flag-tagged wild-type or N88F mutant PITPNC1 co-stained with anti-Flag and anti-Giantin were analyzed by immunofluorescence microscopy. The intensity of Flag immunoreactivity in areas positive for Giantin was considered as the Golgi signal. The Golgi signal was normalized to total cellular levels to control for differences in expression levels between wild-type and N88F mutant PITPNC1. n = 30/group.

Analysis of genomic copy number data using the dataset derived byrevealed PITPNC1 to be significantly (q = 2.32 × 10) amplified in 46% of 244 human breast cancer cells lines and tumors, with 15.9% of such amplifications being focal in nature ( Figure 1 A ). Specific analysis of the breast cancer subtypes further revealed that triple-negative breast cancer patients (TNBC) have increased levels of amplification relative to non-TNBC ( Figure S1 A). Expression of PITPNC1 was further increased in a large independent collection of human breast cancers relative to normal breast tissues ( Figure S1 B), and protein levels of PITPNC1 are elevated in MDA-MB-231 breast cancer cells compared with human mammary epithelial cells ( Figure S1 C). Furthermore, in vivo-selected highly metastatic breast cancer cell populations LM2 and CN34Lm1a1 () exhibited increased PITPNC1 protein abundance compared with their parental populations (MDA-MB-231 and CN34, Figure S1 C). Interestingly, PITPNC1 knockdown reduced the ability of murine 4T1 as well as human CN34Lm1a1 and HCC1806 breast cancer cells to metastasize in immunocompetent and immunocompromised mice, respectively ( Figures 1 B and S1 D–S1G). These findings establish PITPNC1 as a pro-metastatic protein that is induced in metastatic breast cancer. Similar to breast cancer, we find that PITPNC1 protein levels are elevated in highly metastatic melanoma MeWo-LM2 () and colon cancer LS174T-LvM3 cells () relative to non-cancerous primary melanocytes and colon epithelial cells, respectively ( Figure S1 H). PITPNC1 expression in primary tumors was further significantly correlated with human metastatic progression outcomes in colorectal cancer and melanoma, suggesting that PITPNC1 may act centrally in the metastatic cascade to promote metastasis in multiple cancer types ( Figures 1 C and 1D). Knockdown of PITPNC1 in the highly metastatic colon cancer subline LS174T-LvM3 and the metastatic MeWo-LM2 melanoma line significantly reduced metastatic colonization by 15- and 8-fold, respectively ( Figures 1 E, 1F, and S1 D). To further investigate the mechanism by which PITPNC1 promotes metastasis, we next sought to determine the metastatic phenotype(s) it governs. Specific depletion of PITPNC1 significantly inhibited invasion and endothelial recruitment () by breast, colon, and skin cancer cells, but did not affect in vitro migration or proliferation capacity ( Figures 1 G, 1H, and S1 I–S1N). These findings reveal that the phenotypic effects of PITPNC1 depletion are selective and do not result from reduced cellular viability. Importantly, overexpression of PITPNC1 was sufficient to significantly enhance invasion, endothelial recruitment, and metastatic colonization by MDA-MB-231 breast cancer cells ( Figures 1 I, S1 O–S1R). These findings establish PITPNC1 as a robust mediator of metastasis and pro-metastatic phenotypes in multiple cancer types.

(C) Kaplan-Meier curve representing a metastasis-free survival cohort of colorectal patients (n = 177) as a function of their primary tumor's PITPNC1 expression levels (data from GEO: GSE17536 ). Patients whose primary tumors' PITPNC1 expressions levels were greater or lower than the median for the population were classified as PITPNC1-positive (red) or -negative (blue), respectively.

Discussion

Garner et al., 2012 Garner K.

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Cockcroft S. Phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) binds and transfers phosphatidic acid. Our findings reveal PITPNC1 to be genetically amplified in nearly half of human breast tumors, overexpressed in multiple prevalent cancer types, and its expression levels to correlate with metastatic progression in melanoma, breast, and colon cancers. PITPNC1 promotes metastasis by enhancing vesicular secretion, leading to the augmented cellular release of a set of pro-metastatic proteins comprising MMP1, PDGFA, HTRA1, FAM3C, and ADAM10 ( Figure 7 G). PITPNC1 accomplishes this by increasing the abundance of RAB1B in the Golgi compartment of the cell. Through a complementary set of biochemical and cell-biological experiments, we have identified PI4P as the lipid substrate of PITPNC1. PITPNC1 was originally named Phosphoinositide transfer protein, cytoplasmic 1 based on its homology to the phosphoinositol transfer proteins; however, it is less well characterized than other members of the PITP family. While an in vivo lipid substrate of PITPNC1 has not been previously identified, a recent study demonstrated in vitro binding and transfer of PA by recombinant PITPNC1 (). In addition to our biochemical analyses, the localization of PITPNC1 to the TGN, a compartment that is enriched in PI4P, as well as our SAC1-K2A epistasis experiments, support the conclusion that PI4P is the predominant physiological in vivo substrate for PITPNC1 in the cells studied herein. Our findings expand on the molecular, cellular, and organismal role of the PITP family by suggesting that PITPNC1 acts to coordinate the morphology of the Golgi by the recruitment of RAB1B through binding to Golgi-resident PI4P.

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et al. Overexpression of Golgi phosphoprotein-3 (GOLPH3) in glioblastoma multiforme is associated with worse prognosis. Our findings furthermore identify extended Golgi morphology as a key morphological feature of highly metastatic cells. We find that PITPNC1 in complex with RAB1B regulates Golgi PI4P levels. Enhanced Golgi PI4P levels increase the abundance of the PI4P-binding protein GOLPH3, a key regulator of Golgi morphology. Our work thus identifies GOLPH3 and its function in secretion as a key component of the metastatic cascade. This is corroborated clinically by the association of GOLPH3 overexpression with poor prognosis in multiple cancer types (). By enhancing the levels of PITPNC1, highly metastatic cells are able to target GOLPH3 to its site of action in the trans-Golgi.

3 levels, which are regulated by PI3K and PTEN. PI3K and PTEN constitute two of the most frequently mutated genes in cancer. The signaling pathway downstream of PI(3,4,5)P 3 has also been well documented in human cancer through mechanistic and pathological studies ( Wong et al., 2010 Wong K.K.

Engelman J.A.

Cantley L.C. Targeting the PI3K signaling pathway in cancer. The importance of phosphoinositides in cancer development and progression has previously mainly focused on PI(3,4,5)Plevels, which are regulated by PI3K and PTEN. PI3K and PTEN constitute two of the most frequently mutated genes in cancer. The signaling pathway downstream of PI(3,4,5)Phas also been well documented in human cancer through mechanistic and pathological studies (). However there exist a large number of other phosphoinositides with largely uncharacterized roles in cancer progression. Each of these PIs demonstrates spatial and temporal restriction within the cell, serving as markers of distinct cellular organelles or as platforms for signaling pathways. While previous studies have focused on the phosphorylation state of the phosphoinositides, our studies underscore the importance of phosphoinositide-mediated recruitment of effector proteins to malignant phenotypes such as metastatic invasion and endothelial recruitment.

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Beug H. ILEI: a cytokine essential for EMT, tumor formation, and late events in metastasis in epithelial cells. Our unbiased proteomic analysis of metastatic cells revealed that PITPNC1 depletion impairs secretion of multiple of proteins, many of which have been previously implicated in cancer invasion and angiogenesis. These five secreted factors found to be downstream of PITPNC1 in augmenting metastasis were all found to promote both invasion and endothelial recruitment, indicating that each secreted factor affects both breast cancer cell invasion and endothelial cell migration through interactions with the microenvironment or through activation of receptors expressed by these cell types. Several of these secreted factors have been previously implicated in cancer cell invasion. MMP1, a matrix metalloprotease, has been implicated in the early steps of metastatic progression in multiple cancer types through its role in degradation of the extracellular matrix to facilitate invasion of cancer cells (). Cancer cell-induced remodeling of the microenvironment may also be exploited to facilitate migration of endothelial cells, thereby further enhancing cancer cells' metastatic capacity. Two of the other secreted factors identified are also proteases: ADAM10, another metalloprotease, and HTRA1, a serine protease. These factors may also engage in a role similar to MMP1 by modifying the extracellular environment for cancer cells and endothelial cells to promote metastasis. PDGFA is an established pro-angiogenic factor that has been previously shown to be essential for tumor angiogenesis in multiple solid tumor types, including breast and colon cancers (). While the endothelial role of PDGF proteins is well characterized, breast cancer cells have also been found to upregulate PDGFR (), indicating a potential cell-autonomous role for PDGF proteins in cancer progression. FAM3C, also called ILEI, is the least characterized of the PITPNC1-regulated secreted proteins. While the mechanistic basis of its action is still uncharacterized, FAM3C has been shown to associate with human breast cancer progression ().

PITPNC1's clinical correlation with metastatic outcomes in breast, colon, and melanoma cancers is surprising given the diverse tissue origins of these cancer types. While both breast cancer and melanoma exhibit large tropisms for the lung and brain, colon cancer metastasizes mainly to the liver and to a lesser extent the lung. However, successful metastasis of all these cancers requires invasion, extravasation, and angiogenesis, processes that require secreted factors acting on the microenvironment or on other cell types. PITPNC1's role as a general regulator of secretion highlights the importance of the secretory pathway in the metastatic progression of these cancer types. Given its overexpression in a broad set of human cancers and its regulatory control of a large number of pro-metastatic genes, our findings suggest promise for therapeutic targeting of PITPNC1.