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Identification of Piwil2-like (PL2L) proteins that promote tumorigenesis.

Ye Y, Yin DT, Chen L, Zhou Q, Shen R, He G, Yan Q, Tong Z, Issekutz AC, Shapiro CL, Barsky SH, Lin H, Li JJ, Gao JX - PLoS ONE (2010)

Bottom Line: Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers.Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB.These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Ohio State University Medical Center, Columbus, Ohio, United States of America.

ABSTRACT
PIWIL2, a member of PIWI/AGO gene family, is expressed in the germline stem cells (GSCs) of testis for gametogenesis but not in adult somatic and stem cells. It has been implicated to play an important role in tumor development. We have previously reported that precancerous stem cells (pCSCs) constitutively express Piwil2 transcripts to promote their proliferation. Here we show that these transcripts de facto represent Piwil2-like (PL2L) proteins. We have identified several PL2L proteins including PL2L80, PL2L60, PL2L50 and PL2L40, using combined methods of Gene-Exon-Mapping Reverse Transcription Polymerase Chain Reaction (GEM RT-PCR), bioinformatics and a group of novel monoclonal antibodies. Among them, PL2L60 rather than Piwil2 and other PL2L proteins is predominantly expressed in various types of human and mouse tumor cells. It promotes tumor cell survival and proliferation in vitro through up-regulation of Stat3 and Bcl2 gene expressions, the cell cycle entry from G(0/1) into S-phase, and the nuclear expression of NF-κB, which contribute to the tumorigenicity of tumor cells in vivo. Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers. Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB. These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction. The identification of PL2L proteins provides a novel insight into the mechanisms of cancer development as well as a novel bridge linking cancer diagnostics and anticancer drug development.

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PL2L proteins rather than Piwil2 are predominantly expressed in human primary and metastatic cancers in association with NF-κB.A, Characterizing mouse mAbs to Piwil2-peptide shared by Piwil2 and PL2L proteins of humans and mice. The supernatants of mAbs (clone Kao1, Kao2, and Kao3; 1∶10 dilution) were used to Western-blot mouse testicular lysates and human colon cancer cell line SW480 lysates. Kao1 specifically reacted with Piwil2 (∼110 kDa) but not with PL2L proteins in the testis. The faint bands detected by mAb Kao1 in SW480 reflected non-specific binding of secondary antibody, because (a) similar bands were observed in the absence of primary antibody (no mAb control); (b) no corresponding bands were observed in the testicular lysates; and (c) they were not matched with known PL2L protein bands blotted by Kao2 and Kao3 mAbs. Note that Kao2 and Kao3 reacted with Piwil2, PL2L80, PL2L60, PL2L50 and PL2L40, and the different intensity between them reflected their variable amounts in the relevant samples. B, PL2L proteins but not Piwil2 are predominantly expressed in primary breast and cervical cancers. TMAs of breast cancer (n = 300) and cervical cancer (n = 100) or regular sections (breast cancer: n = 3; cervical cancer: n = 5) were stained with mAb to Piwil2 (Kao1) or PL2L proteins (Kao2 or Kao3). Shown are representative micrographs of Piwil2 (B1 & B3) and PL2L proteins expression (B2 & B4) in the primary breast (B1 & B2) and cervical cancers (B3 & B4). Arrows in B1 and B3 indicate the Piwil2-expressing (mAb Kao1+) apoptotic or apoptosing cells enlarged in the insets. The inset in B2 shows a tumor cell enriched with heterochromatin was faintly stained by mAb Kao2, while other two cells enriched with euchromatin were strongly stained by mAb Kao2 in cytoplasm but faintly in nuclei. The tumor cells in the inset were enlarged from those indicated by a red arrow in B2. In addition, green arrows in B2 indicate the C-N pattern of mAb Kao2+ cells. Magnification of the micrographs: B1 and B4: x 75; B3, x150, and B2, x300. C, PL2L proteins but not Piwil2 are expressed in all metastatic tumor cells. Shown are representative micrographs of Piwil2 (C1 & C3) and PL2L proteins expression (C2 & C4) in the metastatic cancer cells in tissue stroma (C1 & C2) and in lymph nodes (C3 & C4). Arrows in C1 indicate Piwil2 (mAb Kao1)-negative metastatic tumor cells in the stroma; and arrows in C3 indicate apoptosing tumor cells expressing Piwil2 (mAb Kao1) in the lymph node. Magnification of micrographs: C1, x150; C2, C3 and C4: x400. D, Co-expression of PL2L proteins (mAb Kao2) with NF-κB in tumor cells. Shown are representative micrographs of single and double color IHC staining of serial sections of breast cancer. The serial sections were prepared from an invasive ductal carcinoma infiltrated by inflammatory cells. The serial sections were stained by mouse mAb Kao2 alone (D1: brown; x150), rabbit mAb to p65 alone (D2: pink; x150) or mAb Kao2 followed by mAb to p65 (D3: brown and pink; 150). Note that the infiltrated inflammatory cells are negative for PL2L proteins as shown in D1 but positive for p65 as shown in D2. The box in D3 is projected to D4 (x600). Arrows in D4 indicate the tumor cells doubly stained by mAb Kao2 and mAb to p65 in the nuclei. E, Summarized data of IHC stained TMAs of breast cancer (n = 300) and cervical cancer (n = 100). Shown are the percentages of breast and cervical cancers, respectively, expressing Piwil2 or PL2L proteins.
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pone-0013406-g008: PL2L proteins rather than Piwil2 are predominantly expressed in human primary and metastatic cancers in association with NF-κB.A, Characterizing mouse mAbs to Piwil2-peptide shared by Piwil2 and PL2L proteins of humans and mice. The supernatants of mAbs (clone Kao1, Kao2, and Kao3; 1∶10 dilution) were used to Western-blot mouse testicular lysates and human colon cancer cell line SW480 lysates. Kao1 specifically reacted with Piwil2 (∼110 kDa) but not with PL2L proteins in the testis. The faint bands detected by mAb Kao1 in SW480 reflected non-specific binding of secondary antibody, because (a) similar bands were observed in the absence of primary antibody (no mAb control); (b) no corresponding bands were observed in the testicular lysates; and (c) they were not matched with known PL2L protein bands blotted by Kao2 and Kao3 mAbs. Note that Kao2 and Kao3 reacted with Piwil2, PL2L80, PL2L60, PL2L50 and PL2L40, and the different intensity between them reflected their variable amounts in the relevant samples. B, PL2L proteins but not Piwil2 are predominantly expressed in primary breast and cervical cancers. TMAs of breast cancer (n = 300) and cervical cancer (n = 100) or regular sections (breast cancer: n = 3; cervical cancer: n = 5) were stained with mAb to Piwil2 (Kao1) or PL2L proteins (Kao2 or Kao3). Shown are representative micrographs of Piwil2 (B1 & B3) and PL2L proteins expression (B2 & B4) in the primary breast (B1 & B2) and cervical cancers (B3 & B4). Arrows in B1 and B3 indicate the Piwil2-expressing (mAb Kao1+) apoptotic or apoptosing cells enlarged in the insets. The inset in B2 shows a tumor cell enriched with heterochromatin was faintly stained by mAb Kao2, while other two cells enriched with euchromatin were strongly stained by mAb Kao2 in cytoplasm but faintly in nuclei. The tumor cells in the inset were enlarged from those indicated by a red arrow in B2. In addition, green arrows in B2 indicate the C-N pattern of mAb Kao2+ cells. Magnification of the micrographs: B1 and B4: x 75; B3, x150, and B2, x300. C, PL2L proteins but not Piwil2 are expressed in all metastatic tumor cells. Shown are representative micrographs of Piwil2 (C1 & C3) and PL2L proteins expression (C2 & C4) in the metastatic cancer cells in tissue stroma (C1 & C2) and in lymph nodes (C3 & C4). Arrows in C1 indicate Piwil2 (mAb Kao1)-negative metastatic tumor cells in the stroma; and arrows in C3 indicate apoptosing tumor cells expressing Piwil2 (mAb Kao1) in the lymph node. Magnification of micrographs: C1, x150; C2, C3 and C4: x400. D, Co-expression of PL2L proteins (mAb Kao2) with NF-κB in tumor cells. Shown are representative micrographs of single and double color IHC staining of serial sections of breast cancer. The serial sections were prepared from an invasive ductal carcinoma infiltrated by inflammatory cells. The serial sections were stained by mouse mAb Kao2 alone (D1: brown; x150), rabbit mAb to p65 alone (D2: pink; x150) or mAb Kao2 followed by mAb to p65 (D3: brown and pink; 150). Note that the infiltrated inflammatory cells are negative for PL2L proteins as shown in D1 but positive for p65 as shown in D2. The box in D3 is projected to D4 (x600). Arrows in D4 indicate the tumor cells doubly stained by mAb Kao2 and mAb to p65 in the nuclei. E, Summarized data of IHC stained TMAs of breast cancer (n = 300) and cervical cancer (n = 100). Shown are the percentages of breast and cervical cancers, respectively, expressing Piwil2 or PL2L proteins.

Mentions: To determine whether PL2L60 was also predominantly expressed in native cancers in addition to tumor cell lines, we attempted to generate murine monoclonal antibody (mAb) to Piwil2 and PL2L60 or other PL2L proteins. Because polyclonal Piwil2-peptide specific antibody recognizes both Piwil2 and PL2L proteins (Fig. 3A & B), we used the same strategy to generate mAbs to Piwil2 and PL2L proteins. Although we could not obtain a mAb exclusively detecting PL2L60, we have established one mAb specifically recognizing Piwil2 (clone Kao1) and two mAbs recognizing both Piwil2 and PL2L proteins (clones Kao2 and Kao3). As shown in Fig. 8A, the mAb Kao1 reacted with ∼110 kDa proteins in the murine testis, but not any proteins in the cancer cell lysates from human colorectal cancer cell line SW480 [48]. In contrast, mAbs Kao2 and Kao3 reacted strongly with ∼110 kDa and∼40 kDa proteins in the murine testis and ∼60 kDa and ∼50 kDa proteins in the cancer cell lysates. In addition, mAbs Kao2 and Kao3 reacted weakly with ∼80 kDa, ∼60 kDa and ∼50 kDa proteins in the testis of mice, and ∼110 kDa and ∼80 kDa proteins of the cancer cell lysates (Fig. 8A). The different strength of the proteins bands likely reflected different levels of the proteins in the murine testis and human cancer cell line. Thus, mAbs Kao2 and Kao3 can recognize Piwil2, PL2L80, PL2L60, PL2L50 and PL2L40, similarly to the proteins recognized by Piwil2 peptide-specific pAb, except for PL2L40 (Fig. 3B). Again, PL2L60 was predominantly expressed in the colon cancer cell lines as detected by mAbs Kao2 and Kao3 (Fig. 8A).


Identification of Piwil2-like (PL2L) proteins that promote tumorigenesis.

Ye Y, Yin DT, Chen L, Zhou Q, Shen R, He G, Yan Q, Tong Z, Issekutz AC, Shapiro CL, Barsky SH, Lin H, Li JJ, Gao JX - PLoS ONE (2010)

PL2L proteins rather than Piwil2 are predominantly expressed in human primary and metastatic cancers in association with NF-κB.A, Characterizing mouse mAbs to Piwil2-peptide shared by Piwil2 and PL2L proteins of humans and mice. The supernatants of mAbs (clone Kao1, Kao2, and Kao3; 1∶10 dilution) were used to Western-blot mouse testicular lysates and human colon cancer cell line SW480 lysates. Kao1 specifically reacted with Piwil2 (∼110 kDa) but not with PL2L proteins in the testis. The faint bands detected by mAb Kao1 in SW480 reflected non-specific binding of secondary antibody, because (a) similar bands were observed in the absence of primary antibody (no mAb control); (b) no corresponding bands were observed in the testicular lysates; and (c) they were not matched with known PL2L protein bands blotted by Kao2 and Kao3 mAbs. Note that Kao2 and Kao3 reacted with Piwil2, PL2L80, PL2L60, PL2L50 and PL2L40, and the different intensity between them reflected their variable amounts in the relevant samples. B, PL2L proteins but not Piwil2 are predominantly expressed in primary breast and cervical cancers. TMAs of breast cancer (n = 300) and cervical cancer (n = 100) or regular sections (breast cancer: n = 3; cervical cancer: n = 5) were stained with mAb to Piwil2 (Kao1) or PL2L proteins (Kao2 or Kao3). Shown are representative micrographs of Piwil2 (B1 & B3) and PL2L proteins expression (B2 & B4) in the primary breast (B1 & B2) and cervical cancers (B3 & B4). Arrows in B1 and B3 indicate the Piwil2-expressing (mAb Kao1+) apoptotic or apoptosing cells enlarged in the insets. The inset in B2 shows a tumor cell enriched with heterochromatin was faintly stained by mAb Kao2, while other two cells enriched with euchromatin were strongly stained by mAb Kao2 in cytoplasm but faintly in nuclei. The tumor cells in the inset were enlarged from those indicated by a red arrow in B2. In addition, green arrows in B2 indicate the C-N pattern of mAb Kao2+ cells. Magnification of the micrographs: B1 and B4: x 75; B3, x150, and B2, x300. C, PL2L proteins but not Piwil2 are expressed in all metastatic tumor cells. Shown are representative micrographs of Piwil2 (C1 & C3) and PL2L proteins expression (C2 & C4) in the metastatic cancer cells in tissue stroma (C1 & C2) and in lymph nodes (C3 & C4). Arrows in C1 indicate Piwil2 (mAb Kao1)-negative metastatic tumor cells in the stroma; and arrows in C3 indicate apoptosing tumor cells expressing Piwil2 (mAb Kao1) in the lymph node. Magnification of micrographs: C1, x150; C2, C3 and C4: x400. D, Co-expression of PL2L proteins (mAb Kao2) with NF-κB in tumor cells. Shown are representative micrographs of single and double color IHC staining of serial sections of breast cancer. The serial sections were prepared from an invasive ductal carcinoma infiltrated by inflammatory cells. The serial sections were stained by mouse mAb Kao2 alone (D1: brown; x150), rabbit mAb to p65 alone (D2: pink; x150) or mAb Kao2 followed by mAb to p65 (D3: brown and pink; 150). Note that the infiltrated inflammatory cells are negative for PL2L proteins as shown in D1 but positive for p65 as shown in D2. The box in D3 is projected to D4 (x600). Arrows in D4 indicate the tumor cells doubly stained by mAb Kao2 and mAb to p65 in the nuclei. E, Summarized data of IHC stained TMAs of breast cancer (n = 300) and cervical cancer (n = 100). Shown are the percentages of breast and cervical cancers, respectively, expressing Piwil2 or PL2L proteins.
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pone-0013406-g008: PL2L proteins rather than Piwil2 are predominantly expressed in human primary and metastatic cancers in association with NF-κB.A, Characterizing mouse mAbs to Piwil2-peptide shared by Piwil2 and PL2L proteins of humans and mice. The supernatants of mAbs (clone Kao1, Kao2, and Kao3; 1∶10 dilution) were used to Western-blot mouse testicular lysates and human colon cancer cell line SW480 lysates. Kao1 specifically reacted with Piwil2 (∼110 kDa) but not with PL2L proteins in the testis. The faint bands detected by mAb Kao1 in SW480 reflected non-specific binding of secondary antibody, because (a) similar bands were observed in the absence of primary antibody (no mAb control); (b) no corresponding bands were observed in the testicular lysates; and (c) they were not matched with known PL2L protein bands blotted by Kao2 and Kao3 mAbs. Note that Kao2 and Kao3 reacted with Piwil2, PL2L80, PL2L60, PL2L50 and PL2L40, and the different intensity between them reflected their variable amounts in the relevant samples. B, PL2L proteins but not Piwil2 are predominantly expressed in primary breast and cervical cancers. TMAs of breast cancer (n = 300) and cervical cancer (n = 100) or regular sections (breast cancer: n = 3; cervical cancer: n = 5) were stained with mAb to Piwil2 (Kao1) or PL2L proteins (Kao2 or Kao3). Shown are representative micrographs of Piwil2 (B1 & B3) and PL2L proteins expression (B2 & B4) in the primary breast (B1 & B2) and cervical cancers (B3 & B4). Arrows in B1 and B3 indicate the Piwil2-expressing (mAb Kao1+) apoptotic or apoptosing cells enlarged in the insets. The inset in B2 shows a tumor cell enriched with heterochromatin was faintly stained by mAb Kao2, while other two cells enriched with euchromatin were strongly stained by mAb Kao2 in cytoplasm but faintly in nuclei. The tumor cells in the inset were enlarged from those indicated by a red arrow in B2. In addition, green arrows in B2 indicate the C-N pattern of mAb Kao2+ cells. Magnification of the micrographs: B1 and B4: x 75; B3, x150, and B2, x300. C, PL2L proteins but not Piwil2 are expressed in all metastatic tumor cells. Shown are representative micrographs of Piwil2 (C1 & C3) and PL2L proteins expression (C2 & C4) in the metastatic cancer cells in tissue stroma (C1 & C2) and in lymph nodes (C3 & C4). Arrows in C1 indicate Piwil2 (mAb Kao1)-negative metastatic tumor cells in the stroma; and arrows in C3 indicate apoptosing tumor cells expressing Piwil2 (mAb Kao1) in the lymph node. Magnification of micrographs: C1, x150; C2, C3 and C4: x400. D, Co-expression of PL2L proteins (mAb Kao2) with NF-κB in tumor cells. Shown are representative micrographs of single and double color IHC staining of serial sections of breast cancer. The serial sections were prepared from an invasive ductal carcinoma infiltrated by inflammatory cells. The serial sections were stained by mouse mAb Kao2 alone (D1: brown; x150), rabbit mAb to p65 alone (D2: pink; x150) or mAb Kao2 followed by mAb to p65 (D3: brown and pink; 150). Note that the infiltrated inflammatory cells are negative for PL2L proteins as shown in D1 but positive for p65 as shown in D2. The box in D3 is projected to D4 (x600). Arrows in D4 indicate the tumor cells doubly stained by mAb Kao2 and mAb to p65 in the nuclei. E, Summarized data of IHC stained TMAs of breast cancer (n = 300) and cervical cancer (n = 100). Shown are the percentages of breast and cervical cancers, respectively, expressing Piwil2 or PL2L proteins.
Mentions: To determine whether PL2L60 was also predominantly expressed in native cancers in addition to tumor cell lines, we attempted to generate murine monoclonal antibody (mAb) to Piwil2 and PL2L60 or other PL2L proteins. Because polyclonal Piwil2-peptide specific antibody recognizes both Piwil2 and PL2L proteins (Fig. 3A & B), we used the same strategy to generate mAbs to Piwil2 and PL2L proteins. Although we could not obtain a mAb exclusively detecting PL2L60, we have established one mAb specifically recognizing Piwil2 (clone Kao1) and two mAbs recognizing both Piwil2 and PL2L proteins (clones Kao2 and Kao3). As shown in Fig. 8A, the mAb Kao1 reacted with ∼110 kDa proteins in the murine testis, but not any proteins in the cancer cell lysates from human colorectal cancer cell line SW480 [48]. In contrast, mAbs Kao2 and Kao3 reacted strongly with ∼110 kDa and∼40 kDa proteins in the murine testis and ∼60 kDa and ∼50 kDa proteins in the cancer cell lysates. In addition, mAbs Kao2 and Kao3 reacted weakly with ∼80 kDa, ∼60 kDa and ∼50 kDa proteins in the testis of mice, and ∼110 kDa and ∼80 kDa proteins of the cancer cell lysates (Fig. 8A). The different strength of the proteins bands likely reflected different levels of the proteins in the murine testis and human cancer cell line. Thus, mAbs Kao2 and Kao3 can recognize Piwil2, PL2L80, PL2L60, PL2L50 and PL2L40, similarly to the proteins recognized by Piwil2 peptide-specific pAb, except for PL2L40 (Fig. 3B). Again, PL2L60 was predominantly expressed in the colon cancer cell lines as detected by mAbs Kao2 and Kao3 (Fig. 8A).

Bottom Line: Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers.Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB.These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Ohio State University Medical Center, Columbus, Ohio, United States of America.

ABSTRACT
PIWIL2, a member of PIWI/AGO gene family, is expressed in the germline stem cells (GSCs) of testis for gametogenesis but not in adult somatic and stem cells. It has been implicated to play an important role in tumor development. We have previously reported that precancerous stem cells (pCSCs) constitutively express Piwil2 transcripts to promote their proliferation. Here we show that these transcripts de facto represent Piwil2-like (PL2L) proteins. We have identified several PL2L proteins including PL2L80, PL2L60, PL2L50 and PL2L40, using combined methods of Gene-Exon-Mapping Reverse Transcription Polymerase Chain Reaction (GEM RT-PCR), bioinformatics and a group of novel monoclonal antibodies. Among them, PL2L60 rather than Piwil2 and other PL2L proteins is predominantly expressed in various types of human and mouse tumor cells. It promotes tumor cell survival and proliferation in vitro through up-regulation of Stat3 and Bcl2 gene expressions, the cell cycle entry from G(0/1) into S-phase, and the nuclear expression of NF-κB, which contribute to the tumorigenicity of tumor cells in vivo. Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers. Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB. These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction. The identification of PL2L proteins provides a novel insight into the mechanisms of cancer development as well as a novel bridge linking cancer diagnostics and anticancer drug development.

Show MeSH
Related in: MedlinePlus