Expression of pp32 gene family members in breast cancer.

The pp32 gene family consists of at least three closely related members, pp32, pp32r1 and pp32r2. In spite of a high degree of identity at the nucleotide level, pp32 functionally behaves as a tumor suppressor where as pp32r1 and pp32r2 are pro-oncogenic. The purpose of this pilot study was to determine pp32-related expression and whether alternative gene use among the pp32 family members occurred in human breast cancer. As a first step, in situ hybridization with a riboprobe capable of hybridizing with all the three members showed abundant pp32-related mRNA in benign ducts and acini and in infiltrating ductal carcinomas. A total of 100/102 cases were positive. Further, a detailed molecular analysis by RT-PCR, cloning, and sequencing was performed in five frozen infiltrating breast carcinomas and matched benign breast tissues. Oncogenic pp32r1 (5/5) and pp32r2 (3/5) expression was observed in carcinomas where as benign breast tissues expressed pp32. 4/5 carcinomas continued to express pp32 but one was devoid of pp32 expression. These results suggest that alternative expression of pp32 family members may be common in human breast cancer and the analysis of the profile of pp32-related expression might be helpful in understanding the role of these genes in breast cancer pathogenesis.

Tumor suppression and potentiation by manipulation of pp32 expression.

Alternative use of genes of the closely-related pp32 family is a common occurrence in human prostate cancer. pp32r1 and pp32r2, the oncogenic members of the pp32 family, are expressed in prostatic adenocarcinoma, while adjacent benign prostate continues to express pp32. This study focuses upon the role of pp32 in tumor suppression. We demonstrate that antisense inhibition of pp32 in NIH3T3 cells leads to a variety of phenotypic changes associated with transformation including reduced serum dependence and loss of contact inhibition. NIH3T3 cells with antisense-inhibited pp32 are not tumorigenic, but are markedly more susceptible to oncogenic stimuli such as ras. In contrast, constitutive expression of pp32 abolishes ras mediated transformation in vitro and tumorigenesis in vivo. These data demonstrate, from the functional aspect, that pp32 acts as a tumor suppressor. Furthermore, inactivation of pp32 function through alternative gene use may be a critical event in tumor evolution and progression.

In situ hybridization-theory and practice.

In situ hybridization is a technique to determine and localize target nucleic acids in morphologically preserved tissue sections. Recent advances in methods have greatly increased the sensitivity of the technique, and it is currently possible to detect extremely few copies of any given target sequence with nonisotopic methods. In this teaching review, we integrate theoretical background, technical considerations, and guidelines for usage for this important component of molecular diagnosis.

Identification of sequences required for inhibition of oncogene-mediated transformation by pp32.

Oncogenic potential in prostate cancer is modulated in part by alternative use of genes of the pp32 family. This family includes the tumor suppressor pp32, expressed in normal tissue, and the pro-oncogenic genes pp32r1 and pp32r2 that are found principally in neoplastic cells. At the protein level, pp32, pp32r1, and pp32r2 are approximately 90% identical, yet they subsume opposite functions. In this study, we identify the region of pp32 associated with the ability to inhibit oncogene-mediated transformation in a rat embryo fibroblast system, an in vitro correlate of tumor-suppressive activity. Deletion and truncation analysis define a region spanning pp32 amino acids 150-174 as absolutely required for inhibition of transformed foci elicited by RAS and MYC. Comparison of pp32 with the pp32r1 sequence by moving averages of sequence identity reveals divergence over this region; pp32r2 also differs in this region through truncation after pp32 amino acid 131. The deletion experiments and the experiments of nature therefore converge to demonstrate that tumor-suppressive functions of pp32 reside in amino acids 150-174. Identification of this minimal tumor-suppressive region should help elaborate the pathways and mechanisms through which pp32 family members exert their functions.

Modulation of oncogenic potential by alternative gene use in human prostate cancer.

Only a small percentage of primary prostate cancers have genetic changes. In contrast, nearly 90% of clinically significant human prostate cancers seems to express high levels of the nuclear phosphoprotein pp32 by in situ hybridization. Because pp32 inhibits oncogene-mediated transformation, we investigated its paradoxical expression in cancer by comparing the sequence and function of pp32 species from paired benign prostate tissue and adjacent prostatic carcinoma from three patients. Here we demonstrate that pp32 is expressed in benign prostatic tissue, but pp32r1 and pp32r2, closely-related genes located on different chromosomes, are expressed in prostate cancer. Although pp32 is a tumor suppressor, pp32r1 and pp32r2 are tumorigenic. Alternative use of the pp32, pp32r1 and pp32r2 genes may modulate the oncogenic potential of human prostate cancer.

Fatty acid synthase expression in endometrial carcinoma: correlation with cell proliferation and hormone receptors.

BACKGROUND: Fatty acid synthase (FAS), a biosynthetic enzyme, normally functions in the liver to convert dietary carbohydrate to fat, but it is minimally expressed in most other normal adult tissues. FAS is expressed at markedly elevated levels in subsets of human breast, ovarian, and prostate carcinomas that are associated with poor prognoses. During the menstrual cycle, the expression of FAS in the human endometrium is closely linked to the expression of the proliferation antigen Ki-67, estrogen receptor (ER), and progesterone receptor (PR). METHODS: This study reports the expression patterns of these antigens in 35 endometrial carcinomas as determined by immunohistochemical analysis. RESULTS: All cases demonstrated a close direct correlation between FAS and Ki-67 expression. Average FAS expression levels were correlated with tumor grade. Twenty-five carcinomas that were positive for ER and PR showed close correlation in expression of FAS, Ki-67, and hormone receptors. Individual tumors displayed varying degrees of heterogeneity of expression. A few well-differentiated carcinomas showed very low expression of all four antigens, similar to the antigenic profile of secretory endometrium. Nine high grade carcinomas that were negative for ER and PR also showed close correlation in expression of FAS and Ki-67 with uniformly high expression. CONCLUSIONS: These data suggest the following hypothesis: In hormone-dependent endometrial cells, FAS expression is part of the estrogen-driven cellular response that leads to proliferation; however, its linkage to proliferation is such that FAS expression is maintained in proliferating cells in endometrial carcinomas that acquire hormone independence. The use of these four antibodies as a panel may increase the diagnostic utility of ER and PR immunohistochemistry for tumor classification and prediction of the responsiveness of tumors to hormonal therapy.

Novel nuclear phosphoprotein pp32 is highly expressed in intermediate- and high-grade prostate cancer.

BACKGROUND: pp32 is a differentiation-regulated nuclear phosphoprotein that is highly expressed in many cancers, but is restricted to self-renewing and long-lived normal cell populations. During murine embryogenesis, pp32 is expressed in primitive cell populations, diminishing as tissues terminally differentiate. Functionally, pp32 confers resistance to programmed cell death and, paradoxically, inhibits transformation mediated in vitro by a broad range of oncogenes, suggesting that pp32 is a multifunctional molecule with potentially complex activities in cancer. METHODS: We studied pp32 expression in prostatic adenocarcinomas and benign prostatic hyperplasia by in situ hybridization. RESULTS: In benign prostatic tissues, moderate pp32 expression occurs only in the basal cells. This study found elevated pp32 expression in 98% (54/55) of prostatic adenocarcinomas of Gleason score > or = 5 (P < 0.0001). CONCLUSIONS: These results suggest that pp32 may be diagnostically useful and may contribute mechanistically to prostate tumor development. In comparison to other molecular alterations, increased pp32 expression is one of the most frequent events in primary prostate cancer.

Elevated expression of fatty acid synthase and fatty acid synthetic activity in colorectal neoplasia.

Expression of the primary enzyme catalyzing the synthesis of fatty acids, ie, fatty acid synthase (FAS), and ex vivo fatty acid synthetic activity were examined in colorectal epithelium and neoplasms, including the relationship to tumor progression and prognosis. Immunohistochemistry for FAS showed only faint staining of native colorectal mucosa, but increased expression was found in all sporadic adenomas (n = 18), adenomas associated with familial adenomatous polyposis (n = 7), hyperplastic polyps (n = 3), dysplasias arising in ulcerative colitis (n = 17), and colorectal carcinomas (n = 130) including 11 with contiguous adenomas. The intensity of staining was strong in 53% of carcinomas, intermediate in 38%, and weak in 9%. Activity of the fatty acid synthetic pathway measured by labeling of six surgical specimens with [U-14C]acetate was 2- to 7-fold higher in colorectal carcinomas than adjacent native mucosa (P = 0.006) and 6- to 16-fold higher than serosal fat (P = 0.01). Activity correlated with immunohistochemical expression (Spearman's rank correlation coefficient = 0.85; P < 0.001). There was no statistically significant association between patient survival and FAS staining intensity of carcinomas. Our study shows that FAS is expressed in all colorectal neoplasms and there is a concomitant increase in fatty acid synthesis. FAS may therefore represent a potential therapeutic target.

Expression of fatty acid synthase is closely linked to proliferation and stromal decidualization in cycling endometrium.

Estrogen-driven proliferative phase growth is the most rapid physiological proliferative process that occurs in the adult. The tissue growth that occurs during this phase of the menstrual cycle requires incorporation of a substantial quantity of fatty acid into the structural lipids of cell membranes. Fatty acid synthase (FAS) is the major biosynthetic enzyme required for de novo synthesis of fatty acids. In this immunohistochemical study, we have observed that human endometrium displays distinct patterns of FAS expression in the proliferative and secretory phases of the normal menstrual cycle. Proliferative endometrial glands and stroma show high FAS expression that closely correlates with expression of Ki-67, estrogen and progesterone receptors, supporting the view that FAS expression plays a role in cellular proliferation in response to estrogen. FAS expression declines during early to midsecretory phase, then reappears in decidualized stromal cells in late secretory phase as well as in the decidua of pregnancy. The second wave of FAS expression correlates with progesterone-receptor localization in the decidual cells, a finding suggesting a second induction of FAS expression in the endometrium, associated with differentiation, that may be regulated by progesterone.

Enzymes of the fatty acid synthesis pathway are highly expressed in in situ breast carcinoma.

Expression of high levels of fatty acid synthase (FAS), an important enzyme in fatty acid synthesis, has been identified in a wide variety of human carcinomas. In breast and prostate carcinoma, FAS expression appears to be associated with aggressive disease. Recent biochemical studies have demonstrated that FAS expression in cancer cells connotes activation of the entire fatty acid synthesis pathway leading to the production of palmitic acid. Here, we explore the immunohistochemical expression of FAS and human acetyl-CoA carboxylase (HACC), the rate-limiting enzyme in fatty acid synthesis, in breast cancer progression from histologically normal breast through the development of in situ duct and lobular carcinoma to infiltrating carcinoma. Both FAS and the Mr 275,000 isoform of HACC are expressed in a small subset of cells in normal breast lobules and terminal ducts. Upon development of either in situ duct or lobular carcinoma, FAS and both isoforms of HACC are expressed at higher levels and in a majority of the cells. These findings suggest that expression of the enzymes of fatty acid synthesis are frequently altered early in the progression of human breast carcinoma.

Structure of pp32, an acidic nuclear protein which inhibits oncogene-induced formation of transformed foci.

pp32 is a nuclear protein found highly expressed in normal tissues in those cells capable of self-renewal and in neoplastic cells. We report the cloning of cDNAs encoding human and murine pp32. The clones encode a 28.6-kDa protein; approximately two-thirds of the N-terminal predicts an amphipathic alpha helix containing two possible nuclear localization signals and a potential leucine zipper motif. The C-terminal third is exceptionally acidic, comprised of approximately 70% aspartic and glutamic acid residues; the predicted pI of human pp32 is 3.81. Human and murine pp32 cDNAs are 88% identical; the predicted proteins are 89% identical and 95% similar. Although the structure of pp32 is suggestive of a transcription factor, pp32 did not significantly modulate transcription of a reporter construct when fused to the Gal4 DNA-binding domain. In contrast, in cotransfection experiments, pp32 inhibited the ability of a broad assortment of oncogene pairs to transform rat embryo fibroblasts, including ras + myc, ras + jun, ras + E1a, ras + mutant p53, and E6 + E7. In related experiments, pp32 inhibited the ability of Rat 1a-myc cells to grow in soft agar, whereas it failed to affect ras-induced focus formation in NIH3T3 cells. These results suggest that pp32 may play a key role in self-renewing cell populations where it may act in the nucleus to limit their sensitivity to transformation.

pp32 overexpression induces nuclear pleomorphism in rat prostatic carcinoma cells.

Nuclear pleomorphism is an important diagnostic factor in tumour pathology. Traditionally, nuclear pleomorphism is evaluated qualitatively or semiquantitatively, often as a component of tumour grade; the molecular basis of nuclear pleomorphism, however, remains unclear. In this study, we investigated the quantitative effects on nuclear morphology of overexpressing pp32, a recently described nuclear phosphoprotein highly expressed in self-renewing and neoplastic cell populations. Assessment of Feulgen-stained transfected and control lines of AT3.1, a rat prostatic carcinoma cell line, using a computerized Cellular Image Analysis System (BD CAS-200) showed that stable overexpression of human pp32 in AT3.1 cells is accompanied by marked increases in the coefficient of variation of nuclear shape, nuclear size and chromatin textures but not in DNA content. In contrast, stable transfection with control vector, with ras, or with bcl-2 failed to affect nuclear morphology. Cell cycle analysis further showed that pp32-related increases in variation of nuclear structure manifested principally in G1. These studies suggest that pp32 plays a role either directly or indirectly in the control of nuclear shape of G1 cells.

Inhibition of fatty acid synthesis induces programmed cell death in human breast cancer cells.

One of the key limiting factors in the treatment of advanced stage human epithelial malignancies is the lack of new, selective molecular targets for antineoplastic therapy. A substantial subset of human breast, ovarian, endometrial, colorectal, and prostatic cancers express elevated levels of fatty acid synthase, the major enzyme required for endogenous fatty acid biosynthesis, and carcinoma lines are growth inhibited by cerulenin, a noncompetitive inhibitor of fatty acid synthase. We have shown previously that the difference in fatty acid biosynthesis between cancer and normal cells is an exploitable target for metabolic inhibitors in the in vitro setting and in vivo in a human ovarian carcinoma xenograft in nude mice. Here, we report that cerulenin treatment of human breast cancer cells inhibits fatty acid synthesis within 6 h after exposure, that loss of clonogenic capacity occurs within the same interval, and that DNA fragmentation and morphological changes characteristic of apoptosis ensue.

Inhibition of fatty acid synthesis delays disease progression in a xenograft model of ovarian cancer.

One of the key limiting factors in the treatment of advanced stage human epithelial malignancies is the lack of selective molecular targets for antineoplastic therapy. A substantial subset of human ovarian, endometrial, breast, colorectal, and prostatic cancers exhibit increased endogenous fatty acid biosynthesis and overexpress certain enzymes in the pathway. Cell lines derived from these tumors use endogenously synthesized fatty acids for cellular functions, whereas normal cells and tissues appear to utilize dietary lipids preferentially. We have previously shown that the difference in fatty acid biosynthesis between cancer and normal cells is an exploitable target for metabolic inhibitors in vitro. Here, we report observations in vivo using the i.p. model of the multiply drug-resistant OVCAR-3 human ovarian carcinoma in nude mice which demonstrate that: (a) fatty acid synthase overexpression in OVCAR-3 is comparable to levels in primary human tumors assessed by immunohistochemistry; (b) fatty acid synthetic activity of OVCAR-3 is comparably elevated in vitro and in vivo and is 4 to >20-fold higher than normal murine tissues; (c) treatment with the specific fatty acid synthase inhibitor, cerulenin, markedly reduces tumor cell fatty acid biosynthesis in vivo; (d) fatty acid synthase inhibition produces regression of established ascites tumor; and (e) treatment with cerulenin causes reduction in ascites incidence, delay in onset of ascites, and significantly increased survival (P<0.04).

Fatty acid synthase (FAS): a target for cytotoxic antimetabolites in HL60 promyelocytic leukemia cells.

Many human cancers express elevated levels of fatty acid synthase (FAS), with correspondingly increased fatty acid synthesis and abnormal fatty acid utilization. Recent studies have shown that the FAS inhibitor, cerulenin, is selectively cytotoxic to cell lines derived from human malignancies, suggesting that those carcinoma cells are dependent upon endogenous fatty acid synthesis for growth. These data further suggest that the fatty acid synthesis pathway is a potential target for chemotherapy development. The present studies demonstrate that cerulenin cytotoxicity is mediated by fatty acid pathway inhibition. Proliferating HL60 promyelocytic leukemia cells express high levels of FAS mRNA and protein and synthesize fatty acid predominantly for membrane phospholipid. Following exposure to 12-O-tetradecanoylphorbol-13-acetate, the FAS expression in HL60 cells is abolished, fatty acid synthesis diminishes, and the cells become insensitive to cerulenin while acquiring a differentiated, macrophage-like phenotype. HL60 cells adapted to growth in serum- and fatty acid-free medium show a dose-dependent sensitivity to cerulenin, which is reversed by palmitate, the major product of FAS, indicating that cerulenin cytotoxicity is mediated through fatty acid starvation. Cells grown in the presence of exogenous fatty acid partially downmodulate FAS expression and increase mean cell volume (phospholipid mass/cell) but retain their sensitivity to cerulenin, which is reversed by 3-fold excess oleate supplementation. These results demonstrate that malignant cells can retain dependence on endogenous fatty acid synthesis and sensitivity to FAS inhibitors in the presence of physiological fatty acid levels and thus support the notion that FAS inhibitors may be useful in treating cancer in vivo.

Simplified high-sensitivity sequencing of a major histocompatibility complex class I-associated immunoreactive peptide using matrix-assisted laser desorption/ionization mass spectrometry.

Cytotoxic T cells (CTL) are known to recognize small peptide fragments of cytoplasmic proteins bound to major histocompatibility complex (MHC) class I molecules on cell surfaces. Recent work indicates that tumor antigens are processed and presented in a manner similar to viral antigens. Identification of the peptides recognized by tumor-specific CTL would provide valuable information about their parent proteins, as well as allowing for the development of recombinant antigen-specific tumor vaccines. While highly represented MHC-bound peptides have been routinely purified by reversed-phase HPLC for Edman degradation sequencing, identification and sequencing of infrequent peptides that represent the biologically relevant targets of tumor-specific CTL have proved elusive. We have combined matrix-assisted laser desorption/ionization mass spectrometry with on-slide exopeptidase digestion to successfully identify and directly sequence a model tumor-specific peptide antigen derived from an integrated viral gene. The enhanced sensitivity of this technique (femtomolar range) allows for the sequencing of specific MHC-bound peptides derived from as few as 1 x 10(9) cells.

Fatty acid synthesis: a potential selective target for antineoplastic therapy.

OA-519 is a prognostic molecule found in tumor cells from breast cancer patients with markedly worsened prognosis. We purified OA-519 from human breast carcinoma cells, obtained its peptide sequence, and unambiguously identified it as fatty acid synthase through sequence homology and enzymology. Tumor fatty acid synthase is an approximately 270-kDa polypeptide which specifically abolished immunostaining of human breast cancers by anti-OA-519 antibodies. Tumor fatty acid synthase oxidized NADPH in a malonyl-CoA-dependent fashion and synthesized fatty acids composed of 80% palmitate, 10% myristate, and 10% stearate from acetyl-CoA, malonyl-CoA, and NADPH with a specific activity of 624 nmol of NADPH oxidized per min per mg. Tumor cell lines with elevated fatty acid synthase showed commensurate increases in incorporation of [U-14C]acetate into acylglycerols demonstrating that fatty acid synthase increases occur in the context of overall increases in endogenous fatty acid synthesis. Cerulenin inhibited acylglycerol synthesis in tumor cells and fibroblast controls in a dose-dependent fashion and also caused a growth inhibition which generally paralleled the level of endogenous fatty acid synthesis. Supraphysiologic levels of palmitate, 14 microM in dimethyl sulfoxide, significantly reversed the growth inhibition caused by cerulenin at concentrations of up to 5 micrograms/ml, indicating that cerulenin-mediated growth inhibition was due to fatty acid synthase inhibition.

A novel M(r) 32,000 nuclear phosphoprotein is selectively expressed in cells competent for self-renewal.

We investigated the association between expression of a novel M(r) 32,000 nuclear phosphoprotein (pp32) and cell proliferation in vivo using the well-characterized physiological model of androgen-dependent regeneration of prostate in orchiectomized rats, pp32 is expressed at high levels in neoplastic cell lines and in certain anatomically defined stem cell compartments of normal human tissues such as intestinal crypt epithelial cells. Immunohistochemistry and in situ hybridization were used to monitor pp32 expression in rat ventral prostatic epithelium following castration and androgen restoration. Castrated rats retained only 6% of prostate wet weight compared to intact controls but were capable of complete gland restoration upon androgen replacement. In intact controls, pp32 expression localized to small acini at the periphery of the gland and to rare basal cells in the central regions. Ten days following castration, there was a 3,5-fold enrichment in the frequency of pp32-positive cells with greater than 56% of remaining epithelial cells expressing pp32 protein. In situ hybridization showed that all remaining epithelial cells contained pp32 mRNA. Upon testosterone replacement, pp32 expression and localization returned to that of intact controls. In order to determine the association between pp32 expression and cell division, DNA synthesis was monitored by bromodeoxyuridine incorporation during prostate involution and regeneration. Bromodeoxyuridine incorporation peaked 3 days after androgen replacement and occurred diffusely throughout the gland. Thus, pp32-positive cells are anatomically distinguishable from the population of terminally differentiating cells undergoing rapid expansion. Preliminary immunohistochemical studies of human prostatic neoplasia demonstrated increased expression of pp32 in human prostatic adenocarcinoma and prostatic intraepithelial neoplasia compared to benign prostatic hypertrophy and normal human prostate. The highest degree of expression occurred in the higher Gleason grades and prostatic intraepithelial neoplasia. This work suggests that pp32 is a nuclear protein which has a selective but presently undefined role in cells competent for self-renewal.

Expression of oncogenic antigen 519 (OA-519) in prostate cancer is a potential prognostic indicator.

Predicting the prognosis of patients with prostate cancer is a clinically important problem. Previous studies have indicated that the expression of haptoglobin-related protein epitopes in samples of breast cancer in early stages was associated with earlier relapses and higher risk for tumor recurrence. Oncogenic antigen 519 (OA-519) is the new marker designation for molecules expressing haptoglobin-related protein epitopes. The objective of this immunohistochemical study was to examine OA-519 expression in prostate cancer samples and its relationship to the established prognostic indicators of tumor grade, tumor volume, and clinical stage. Forty-two consecutive tissue samples of prostate adenocarcinoma were examined using an affinity-purified anti-OA-519 antibody. Twenty specimens (48%) tested positive, whereas 22 (52%) tested negative. No staining was observed in normal or hyperplastic prostate tissue. Staining occurred in 6 of 9 (67%) grade III, 14 of 23 (61%) grade II, and in none of 10 (0%) grade I cases (I vs. II and/or III: Fisher exact test, P less than 0.006). Twenty-three of the 42 samples were transurethral resection specimens with cancer; 11 (48%) of these tested positive. The mean percentage of tissue chips with tumor, a measure of tumor volume, was significantly higher in the positive group (57%) than in the negative group (15%) (P = 0.004). The proportion of positively stained cases increased with advancing clinical stage, with 25% of Stage A cases expressing OA-519, and 46%, 67%, and 64% of Stages B, C, and D, respectively, expressing OA-519. OA-519 expression correlates with higher tumor grades, larger tumors, and possibly with advanced stage, and thus, it is potentially of prognostic value in prostate cancer.

Expression of haptoglobin-related protein in primary and metastatic breast cancers. A longitudinal study of 48 fatal tumors.

The ability to establish a prognosis for patients with early breast cancer is an important clinical issue. Recent studies have shown that antibodies to haptoglobin-related protein (Hpr) may be useful in stratifying early patients with breast cancer according to their relative risks of recurrence. Nearly 30% of early breast cancers express proteins bearing Hpr epitopes. Hpr-positive breast cancers are more likely to recur after primary resection and are associated with shorter disease-free intervals. This immunohistochemical study examines temporal changes in Hpr expression during the course of disease in 48 patients with fatal breast carcinoma. Thirty-seven primary tumors (77%) were Hpr positive. Ten of the 11 initially negative tumors (91%) were Hpr positive at the time of recurrence. In contrast, only 10 of the 37 initially positive tumors (27%) were Hpr negative with relapse. Of 18 axillary nodes that were examined, 16 (89%) were Hpr positive; all four lymph nodal metastases in patients with initially negative primary tumors were Hpr positive. The authors conclude that the acquisition of Hpr expression parallels increased malignant potential and that Hpr expression, once acquired, tends to remain a permanent characteristic of any given mammary tumor.