Effect of p32 knockdown on growth and survival of tumor cells in vitro. (A) Proliferation of MDA-MB-435 and MDA-MB-231 p32 knockdown (kd) and control cells under high (25 mM)- and low (2.5 mM)-glucose conditions. Average cell number at each time point was determined by counting cells in triplicate wells of three p32 knockdown and control clones (P < 0.001) of MDA-MB-435 cells or a pool of clones of MDA-MB-231 cells. The right panel shows the color of media of three control and p32 kd MDA-MB-435 clones after 6 days in 25 mM or 2.5 mM glucose. (B, left) Microscopic analysis of p32 knockdown and control MDA-MB-435 cells after 3 days in medium containing the indicated glucose concentrations. (Right) Cell death was quantified by fluorescence-activated cell sorter (FACS) analysis of cells that bind FITC-annexin V (P < 0.05).

Rescue of the p32 knockdown (kd) effect by restored p32 expression. (A, left) Immunoblot analysis of a parental p32 kd clone and single clones derived from it that express p32 from a cDNA resistant to p32 shRNA silencing (clones [Cl] 3, 8, and 14) or that were transfected with empty vector (clones 9, 10, and 18). A clone expressing control shRNA (control) was used to detect the endogenous level of p32. (Middle) Restoration of culture medium pH by reintroduction of p32. (Right) Graphs showing lactate production and glucose consumption in control, p32 kd, and p32-restored (p32 kd + p32) clones. (B) Western blot analysis was performed on whole-cell lysates prepared from control cells, parental p32-deficient cells, and single clones with restored (clones 3 and 8) or not restored (clones 9 and 10) p32 expression. Equivalent amounts of proteins were immunoblotted with several OXPHOS complex subunits and β-actin as the loading control. (C) Growth rate of cells with restored p32 expression versus control and p32 knockdown cells. Three single clones with restored p32 expression were tested.

Tumor growth properties and metastatic potential of p32-overexpressing cells. (A) Production of cell lines stably overexpressing p32. The indicated cell lines were transfected with a p32 cDNA-containing vector or an empty vector. MDA-MB-435 cells, which contain the highest levels of endogenous p32 among the cell lines tested, yielded no cultures with stable expression of p32 above the endogenous basal level. (B) Overexpression of p32 does not affect the expression levels of OXPHOS proteins. (C) Macroscopic appearance and volumes as a function of time of MDA-MB-231-luc-D3H2LN tumors derived from cells which stably express control or p32 shRNA or empty vector or p32 cDNA. P was <0.001 (control versus p32 kd tumors). (D) Metastatic potential of MDA-MB-231-luc-D3H2LN vector/p32 cells. (Left) Bioluminescence imaging of intracardially injected, luciferase-expressing MDA-MB-231 D3H2LN cells. Results were obtained 4 weeks after injection. Data depicted were derived from 3 independent experiments using at least 5 animals for each cell line injected. P was <0.05.

Inhibition of complex I activity by rotenone treatment mimics the p32 knockdown phenotype. (A) Control and p32 knockdown (kd) MDA-MB-435 cells were treated with 100 nM rotenone for 4 days. Complex I activity was significantly reduced in both cell lines. Complex I activity is significantly lower in the p32 knockdown cells than in control cells and is almost completely eliminated in the rotenone-treated cells. The dose of rotenone used did not affect cell survival but led to cell medium acidification by enhanced lactate production (middle) accompanied by a reduced cell proliferation rate (right). (B) To investigate the effect of rotenone on tumor growth, rotenone was incorporated into micelles. Both forms of rotenone were equally effective in reducing complex I activity, increasing lactate production and reducing cell growth. Bars indicate averages ± SD of at least three independent experiments, each performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Mice bearing MDA-MB-435 tumor xenografts were treated daily with 1 mg/kg of body weight micellar rotenone or an equal amount of micelles only for 20 days. The growth of p32 knockdown tumors was monitored in parallel for comparison. *, P < 0.05 (micelles versus micellar-rotenone-treated control tumors).

Cytoplasmic p32 is unstable. (A) Schematic representation and cellular localization (images on the right) of p32 cDNA constructs and their protein products. The first 73 amino acids (aa) at the N terminus of wild-type (wt) p32 are required for mitochondrial localization of p32. Within cells p32 is present as the mature form (aa 74 to 282) after cleavage of the mitochondrial localization signal. Constructs encoding aa 74 to 282 produce only mature p32, which is unable to localize to mitochondria. Addition of an N-terminal HA tag to the full-length cDNA construct also prevents the import of p32 to mitochondria and the cleavage to the mature form. (B) The indicated p32 cDNA constructs were transiently transfected into 293 cells. At 24 to 48 h posttransfection, cell lysates were collected and the expression of p32 was analyzed by immunoblotting. Constructs encoding p32 protein unable to localize within the mitochondria are expressed at significantly lower levels than constructs encoding wt p32. Introduction of a Kozak sequence upstream of the mature p32 (aa 74 to 282) cDNA did not enhance the expression level. A green fluorescent protein-encoding construct was cotransfected to check transfection efficiency. α, anti. (C) Treatment of the transfected cells with the proteasome inhibitor MG132 (20 μM for 16 h) significantly increased the levels of cytoplasmic p32, indicating that unprotected cytoplasmic p32 is rapidly degraded.

p32 is involved in mitochondrial protein synthesis. (A) Quantitative PCR (QPCR) of the indicated gene transcripts from three single clones of control and p32 knockdown (kd) MDA-MB-435 cells. (B) Control and p32 knockdown MDA-MB-435 cells were labeled with [³⁵S]methionine in the presence or absence of emetine and/or cloramphenicol (Chloram). Total proteins separated by SDS-PAGE and transferred on nitrocellulose membrane were visualized by Ponceau staining (top), while newly synthesized, ³⁵S-labeled proteins were visualized by phosphorimager (middle). p32 protein was detected by Western blotting. β-Actin was used as a loading control. (C) Control and p32 knockdown MDA-MB-435 cells were labeled with [³⁵S]methionine in the presence or absence of emetine and subsequently fractionated in mitochondrial and cytoplasmic fractions. (Top) Ponceau staining of cytoplasmic and mitochondrial fractions resolved by SDS-PAGE. The ³⁵S radiograph (middle) shows newly synthesized proteins. The quality of the fractionation was checked by Western blotting with tubulin and porin, which are cytoplasmic and mitochondrial markers, respectively. (D) Identification of p32-associated proteins via phylogenic analysis. Phylogenic profiling of p32 and “eukaryote-only” MitoCarta proteins across 42 eukaryotic species is shown. Red squares indicate homology of a mouse mitochondrial protein (row) with a protein in a eukaryotic species (column). The graphs represent the mitochondrial proteins most closely related to p32, as obtained by the following clustering techniques: first (top) graph, dissimilarity, Euclidean distance; linkage rule, McQuitty's criteria; second graph, dissimilarity, Euclidean distance; linkage rule, average linkage; third graph, dissimilarity, Euclidean distance; linkage rule, complete linkage; fourth graph, dissimilarity, Manhattan distance; linkage rule, average linkage; fifth graph, dissimilarity, Manhattan distance; linkage rule, McQuitty's criteria.

Knockdown of p32 in MDA-MB-435 tumor cells. (A, top left) Immunoblot analysis on whole-cell lysates from three MDA-MB-435 clones (Cl) stably expressing shRNA for p32 (p32 knockdown [Kd] clones 1, 2, and 3) and three clones expressing a base mismatch control shRNA (control clones 4, 6, and 7). (Top right) Acidification of the culture media in p32 knockdown clones, as indicated by the color change of the phenol red indicator in the media to orange/yellow. (Bottom) Lactate production and glucose consumption 4 days after cell seeding, calculated as described in Materials and Methods and shown relative to control (P < 0.001). (B) Cellular ATP from lysates of p32 knockdown and control cells grown for 4 days in media with the indicated glucose concentrations. The ATP present in each lysate was normalized for the ATP production of control clones grown in 25 mM glucose. The results are the averages (±SD) of three independent experiments performed with three p32 Kd and three control clones. *, P < 0.05; ***, P < 0.001. (C) Oxygen consumption. Shown are the values for p32 knockdown clones relative to control clones. The results come from three independent experiments (±SD) performed in triplicate. ***, P < 0.001; *, P < 0.05. (D) Confocal analysis of p32 localization in cells. p32 knockdown and control cells were stained for p32 and anti-cytochrome c. The panels on the right are high-magnification images of the white-framed areas in the merge panels.

p32 knockdown (kd) results in decreased protein levels of mitochondrial respiratory complexes I, III, IV, and V. (A, left) Representative BN-PAGE of dodecylmaltoside-solubilized mitochondria from control and p32 knockdown MDA-MB-435 cells. (Right) Subunits of the native complexes resolved by 1D electrophoresis were separated by SDS-PAGE. Arrows mark spots with the highest degree of downregulation upon p32 knockdown, and they have been identified by mass spectrometry (Table 1). The gel is representative of at least three independent BN-PAGE experiments. (B) Lysates from control and p32 knockdown MDA-MB-435, MDA-MB-231, and MCF10-CA1a cells were probes for various respiratory complex subunits. Porin antibody was used to test the effect of p32 knockdown on a non-OXPHOS mitochondrial protein. Actin was used as loading control. The intensities of the bands from 3 or 4 independent Western blots from MDA-MB-435 lysates were quantified by Image J software and expressed relative to controls (graph). (C) Assays of complex I and IV activity from MDA-MB-435 control/p32 knockdown lysates. Complex I activity in p32-deficient cells is expressed as % relative to control cells, and the bars represent the ranges of three independent experiments. The activity of complex IV was determined by following the oxidation of cytochrome c by control and p32 knockdown cell lysates. Lysis buffer only was used as the background reference measurement. The graph is representative of three independent assays, each performed in duplicate and with three different lysate concentrations. OD, optical density.

Effect of p32 knockdown (kd) on central carbon metabolism. The relative flux through the Embden-Meyerhof pathway (A, left) and the pentose phosphate pathway (A, right) and into the TCA cycle (B) was determined by [U-¹³C]glucose tracking and isotopomer analysis. Results are reported as the SD of triplicate integrations. (A) The ¹³C labeling pattern of alanine was used to determine glycolysis and PPP. Examples of labeling patterns are shown for glucose and pyruvate, where the open circles represent ¹²C and the filled circles represent ¹³C. Flux of glucose through glycolysis produces pyruvate that is either uniformly ¹²C or ¹³C labeled, while the rearrangements of the pentose phosphate pathway result in the loss of label at C-3. (B) Effect of p32 knockdown on TCA cycle entry and flux. Pyruvate can enter the TCA cycle through either pyruvate dehydrogenase (PDH) or pyruvate carboxylase (PC), which results in different patterns of glutamate labeling (gray circles represent either ¹²C or ¹³C). Determining the ratio of ¹³C at C-4 versus C-3 yields the ratio of PDH activity to PC activity. Knockdown of p32 results in a shift of pyruvate flux from PDH to PC (right graph). The relative level of succinate in each cell line was determined by integrating the peak volumes of succinate carbons C-3 and C-4 compared to glutamate carbons C-3 and C-4. Succinate pool size increased sevenfold following p32 knockdown (left graph). (C) Quantification of PDH enzymatic activity from control and p32 knockdown MDA-MB-435 cell lysates. The results are representative of four independent experiments and are expressed as percentages of PDH activity (±SD), with PDH activity in control cells as 100%. P was <0.001.

Tumor growth properties and metastatic potential of p32 knockdown and p32-overexpressing cells. (A) Expression levels of the indicated OXPHOS subunits, complex I and IV activity, and total cellular ATP in normal human mammary cells stably overexpressing p32 or an empty vector. The graphs indicate averages relative to empty-vector-expressing cells (±SD) of three independent experiments. P was <0.05. (B) Immunoblotting from lysates of the indicated tumor cell lines. Mouse muscle lysate was included as a control of adult tissue negative for the expression of the PKM2 isoform. Total cell lysates were probed with polyclonal anti-p32, polyclonal anti-PKM2, and antiactin as a loading control. (C) Tumors were grown from three MDA-MB-435 p32 knockdown (kd) and control clones (6 mice per clone) in nude mice. (Left) Control tumors are homogenous in size, while p32 kd tumors are either significantly smaller than the control cell tumors or swollen and hemorrhagic. The lower panel shows an example of a knockdown cell tumor with extensive necrosis accompanied by hemorrhage. (Right) Average tumor volume as a function of time (±SEM; P < 0.001). (D) BrdU incorporation in tumor cells. Mice were administered a pulse of BrdU 24 h prior to sacrifice. The graph indicates the number of cells per field that scored positive for BrdU staining. The data were derived by counting via Image J software the BrdU-positive cells in 4 random fields per tumor (n = 14 tumors per group). P was <0.01. (E) Hematoxylin and eosin staining of tumors derived from p32 kd and control cell clones. Pink areas in p32 kd tumors are indicative of extensive necrosis. The upper images were taken with a 10× magnification, and the lower images correspond to the indicated framed areas at 200× magnification. The percentage of pink/necrotic areas was calculated via Image J software (P < 0.001). (F) Tumor growth properties of control cells, p32-deficient MDA-MB-435 cells, and two single clones derived from p32 knockdown cells by reintroducing p32 shRNA-resistant p32 cDNA.