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WACs > Title 174 > Chapter 174-116
Chapter 174-116 WAC
174-116-210 Purpose.
174-116-215 Regulations.
174-116-220 Authority.
174-116-225 Emergencies.
174-116-230 Liability of college.
174-116-235 Enforcement.
174-116-240 Parking permits—General information.
174-116-241 Parking permits—Special exceptions.
174-116-242 Parking permits—Issuance and display.
174-116-243 Parking permits—Validity and revocation.
174-116-250 Responsibility and presumption in reference to illegal parking.
174-116-255 Designated and assigned parking areas.
174-116-261 Parking—Prohibited places.
174-116-262 Impounding and immobilization of vehicles.
174-116-266 Disabled or inoperative vehicle.
174-116-270 Access.
174-116-275 Parking of motorcycles.
174-116-280 Citations, late fees and fines.
174-116-285 Severability.
DISPOSITION OF SECTIONS FORMERLY CODIFIED IN THIS TITLE
174-116-010 Purpose. [Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-010, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-010, filed 9/22/83; Order 72-7, § 174-116-010, filed 10/27/72.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-011 Regulations. [Statutory Authority: RCW 28B.40.120(11). WSR 84-13-056 (Order 84-2, Resolution No. 84-28), § 174-116-011, filed 6/19/84; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-011, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-020 Authority. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-020, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(12). WSR 88-19-097 (Order 88-3, Resolution No. 88-32), § 174-116-020, filed 9/20/88. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-020, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-020, filed 9/22/83; Order 72-7, § 174-116-020, filed 10/27/72.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-030 Enforcement. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-030, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-030, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-030, filed 9/22/83; Order 72-7, § 174-116-030, filed 10/27/72.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-040 Parking permits—General information. [Statutory Authority: RCW B40.120(12) [28B.40.120(12)]. WSR 99-24-014, § 174-116-040, filed 11/22/99, effective 12/23/99. Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-040, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(12). WSR 88-19-097 (Order 88-3, Resolution No. 88-32), § 174-116-040, filed 9/20/88. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-040, filed 6/24/87; WSR 85-03-048 (Order 85-1, Resolution No. 85-5), § 174-116-040, filed 1/14/85; WSR 84-13-056 (Order 84-2, Resolution No. 84-28), § 174-116-040, filed 6/19/84; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-040, filed 9/22/83; Order 72-7, § 174-116-040, filed 10/27/72.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-041 Parking permits—Special exceptions. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-041, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-041, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-041, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-042 Parking permits—Special permits. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-042, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-042, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-042, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-043 Parking permits—Issuance and display. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-043, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(12). WSR 88-19-097 (Order 88-3, Resolution No. 88-32), § 174-116-043, filed 9/20/88. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-043, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-043, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-044 Parking permits—Validity periods. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-044, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-044, filed 6/24/87; WSR 84-13-056 (Order 84-2, Resolution No. 84-28), § 174-116-044, filed 6/19/84; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-044, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-045 Parking permits—Housing residents. [Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-045, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-045, filed 9/22/83.] Repealed by WSR 88-19-097 (Order 88-3, Resolution No. 88-32), filed 9/20/88. Statutory Authority: RCW 28B.40.120(12).
174-116-046 Parking permits—Revocations. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-046, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-046, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-050 Responsibility and presumption in reference to illegal parking. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-050, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-050, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-050, filed 9/22/83; Order 72-7, § 174-116-050, filed 10/27/72.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-060 Designated and assigned parking areas. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-060, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-060, filed 9/22/83; Order 72-7, § 174-116-060, filed 10/27/72.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-070 Speed. [Statutory Authority: RCW 28B.40.120(11). WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-070, filed 9/22/83; Order 72-7, § 174-116-070, filed 10/27/72.] Repealed by WSR 87-14-020 (Order 87-2, Resolution No. 87-13), filed 6/24/87. Statutory Authority: RCW 28B.40.120(11).
174-116-071 Parking—Prohibited places and fines. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-071, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-071, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-071, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-072 Impounding of vehicles. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-072, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-072, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-072, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-080 Access. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-080, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-080, filed 9/22/83; Order 72-7, § 174-116-080, filed 10/27/72.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-090 Motor vehicle registration. [Order 72-7, § 174-116-090, filed 10/27/72.] Repealed by WSR 83-20-016 (Order 83-4, Resolution No. 83-42), filed 9/22/83. Statutory Authority: RCW 28B.40.120(11).
174-116-091 Special parking and traffic regulations and restrictions authorized. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-091, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-091, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-091, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-092 Parking of motorcycles. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-092, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-092, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-092, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-100 Parking permits required. [Order 72-7, § 174-116-100, filed 10/27/72.] Repealed by Order 73-4, filed 11/27/73. Later promulgation, see WAC 174-116-105.
174-116-105 Valid parking permits required. [Order 77-3, § 174-116-105, filed 12/16/77; Order 75-2, § 174-116-105, filed 8/12/75. Formerly WAC 174-116-100.] Repealed by WSR 83-20-016 (Order 83-4, Resolution No. 83-42), filed 9/22/83. Statutory Authority: RCW 28B.40.120(11).
174-116-110 Parking permits. [Order 72-7, § 174-116-110, filed 10/27/72.] Repealed by Order 73-4, filed 11/27/73. Later promulgation, see WAC 174-116-115.
174-116-115 Parking permit regulations. [Statutory Authority: RCW 28B.40.120(11). WSR 81-19-092 (Order 81-3, Motion No. 81-36), § 174-116-115, filed 9/18/81; WSR 80-06-034 (Order 80-2, Motion No. 80-13), § 174-116-115, filed 5/9/80, effective 9/1/80; Order 77-3, § 174-116-115, filed 12/16/77; Order 75-2, § 174-116-115, filed 8/12/75. Formerly WAC 174-116-110.] Repealed by WSR 83-20-016 (Order 83-4, Resolution No. 83-42), filed 9/22/83. Statutory Authority: RCW 28B.40.120(11).
174-116-119 Fines. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-119, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(12). WSR 88-19-097 (Order 88-3, Resolution No. 88-32), § 174-116-119, filed 9/20/88. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-119, filed 6/24/87; WSR 84-13-056 (Order 84-2, Resolution No. 84-28), § 174-116-119, filed 6/19/84; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-119, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-120 Parking permit valid. [Order 72-7, § 174-116-120, filed 10/27/72.] Repealed by Order 73-4, filed 11/27/73. Later promulgation, see WAC 174-116-125.
174-116-121 Election to pay or contest a notice of infraction. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-121, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-121, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-121, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-122 Appeal/hearing procedure. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-122, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-122, filed 6/24/87; WSR 84-13-056 (Order 84-2, Resolution No. 84-28), § 174-116-122, filed 6/19/84; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-122, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-123 Establishment of infraction review committee. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-123, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-123, filed 6/24/87; WSR 85-21-067 (Order 85-4, Resolution No. 85-32), § 174-116-123, filed 10/18/85, effective 1/1/86; WSR 84-13-056 (Order 84-2, Resolution No. 84-28), § 174-116-123, filed 6/19/84; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-123, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-124 Jurisdiction of the infraction review committee. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-124, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-124, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-125 Appeal/hearing procedure—Rules of evidence. [Statutory Authority: RCW 28B.40.120(11). WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-125, filed 9/22/83; Order 77-3, § 174-116-125, filed 12/16/77; Order 75-2, § 174-116-125, filed 8/12/75. Formerly WAC 174-116-120.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-126 Appeal/hearing—Procedure—Review decision. [Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-126, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-126, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-127 Appeal/hearing—Mitigation and suspension of fines. [Statutory Authority: RCW 28B.10.560 (1)(a). WSR 95-16-093, § 174-116-127, filed 7/31/95, effective 9/1/95. Statutory Authority: RCW 28B.40.120(11). WSR 87-14-020 (Order 87-2, Resolution No. 87-13), § 174-116-127, filed 6/24/87; WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-127, filed 9/22/83.] Repealed by WSR 17-02-076, filed 1/3/17, effective 2/3/17. Statutory Authority: RCW 28B.40.120.
174-116-130 Display of parking permits. [Order 72-7, § 174-116-130, filed 10/27/72.] Repealed by Order 73-4, filed 11/27/73. Later promulgation, see WAC 174-116-135.
174-116-135 Parking permits displayed. [Order 75-2, § 174-116-135, filed 8/12/75. Formerly WAC 174-116-130.] Repealed by WSR 83-20-016 (Order 83-4, Resolution No. 83-42), filed 9/22/83. Statutory Authority: RCW 28B.40.120(11).
174-116-140 Parking areas. [Order 75-2, § 174-116-140, filed 8/12/75; Order 73-4, § 174-116-140, filed 11/27/73; Order 72-7, § 174-116-140, filed 10/27/72.] Repealed by WSR 83-20-016 (Order 83-4, Resolution No. 83-42), filed 9/22/83. Statutory Authority: RCW 28B.40.120(11).
174-116-150 Violation, penalty, impounding. [Statutory Authority: RCW 28B.40.120(11). WSR 78-08-088 (Order 78-2, Motion 78-26), § 174-116-150, filed 7/31/78; Order 75-2, § 174-116-150, filed 8/12/75; Order 73-4, § 174-116-150, filed 11/27/73; Order 72-7, § 174-116-150, filed 10/27/72.] Repealed by WSR 83-20-016 (Order 83-4, Resolution No. 83-42), filed 9/22/83. Statutory Authority: RCW 28B.40.120(11).
174-116-160 Bicycle parking. [Order 72-7, § 174-116-160, filed 10/27/72.] Repealed by WSR 83-20-016 (Order 83-4, Resolution No. 83-42), filed 9/22/83. Statutory Authority: RCW 28B.40.120(11).
174-116-170 Bicycle travel. [Order 72-7, § 174-116-170, filed 10/27/72.] Repealed by WSR 83-20-016 (Order 83-4, Resolution No. 83-42), filed 9/22/83. Statutory Authority: RCW 28B.40.120(11).
174-116-180 Bicycle equipment, brakes, lights. [Order 72-7, § 174-116-180, filed 10/27/72.] Repealed by WSR 83-20-016 (Order 83-4, Resolution No. 83-42), filed 9/22/83. Statutory Authority: RCW 28B.40.120(11).
174-116-190 Pedestrian right of way. [Order 72-7, § 174-116-190, filed 10/27/72.] Repealed by WSR 87-14-020 (Order 87-2, Resolution No. 87-13), filed 6/24/87. Statutory Authority: RCW 28B.40.120(11).
174-116-260 Fines and penalties. [Statutory Authority: RCW 28B.40.120(11). WSR 83-20-016 (Order 83-4, Resolution No. 83-42), § 174-116-260, filed 9/22/83.] Repealed by WSR 87-14-020 (Order 87-2, Resolution No. 87-13), filed 6/24/87. Statutory Authority: RCW 28B.40.120(11).
These rules govern motorcycle and motor vehicle parking at The Evergreen State College (college). The purpose of these rules is to facilitate campus safety and access. Specifically, these rules are intended:
(1) To expedite college business, protect state property, provide maximum safety and convenience for all;
(2) To assure access at all times for emergency vehicles, equipment, and personnel;
(3) To provide funds to establish, maintain and manage suitable, self-sustaining campus parking facilities through a principled and fairly administered process;
(4) To protect and control vehicular traffic;
(5) To protect pedestrians; and
(6) To encourage and support sustainable transportation, including travel to and from the college by means other than single occupancy vehicles.
[Statutory Authority: RCW 28B.40.120. WSR 17-02-076, § 174-116-210, filed 1/3/17, effective 2/3/17.]
Drivers and owners of vehicles on the property of the college are responsible for safe and lawful operation of those vehicles. Individuals operating or parking vehicles on college-owned property must at all times comply with the campus regulations, ordinances of Thurston County and laws of the state of Washington.
(1) The college through its board of trustees is authorized to establish traffic and parking regulations as stated in RCW 28B.10.560.
(2) The college is authorized to issue permits to park on the campus. All outstanding campus parking violations must be satisfactorily settled before a permit will be issued or renewed.
The vice president for student affairs, or their designee, has the authority to suspend, modify or repeal any or all provisions in this chapter for an authorized college event or in the event of an emergency, disaster or other like contingency. Such action must be limited in duration and scope to meet the institutional needs of the college and/or address the dangers of the contingency.
Liability of college.
The college assumes no liability for motor vehicles or their contents when such motor vehicles are on campus. The college offers parking permits to those desiring to park on campus. A parking permit licenses the holder (licensee) to park one motor vehicle in the lots designated on the permit. The college is not responsible for fire, theft, damage, or loss of vehicle or any article left in such vehicle. A parking permit is a license to park and no bailment is created. A "motor vehicle" is defined as a vehicle that is self-propelled; for example cars, trucks, and motorcycles. Motor vehicles include a neighborhood electric vehicle as defined in RCW 46.04.357 and a medium-speed electric vehicle as defined in RCW 46.04.295. Electric personal assistive mobility devices and power wheelchairs are not considered motor vehicles.
Enforcement.
(1) Whenever an unattended vehicle is parked in violation of these regulations, the college may take the registration number and other identifiable information and may affix to such vehicle a parking citation in a conspicuously visible location.
(2) When an attended vehicle is parked in violation of these regulations, and upon request of a designated college official, the driver may be required to move the vehicle immediately to a designated parking area or off college property. Refusal to move the vehicle is a violation of these regulations and may warrant a parking citation.
Parking permits—General information.
(1) Parking permits are issued by the college following application and the payment of the appropriate fees. All privately owned motor vehicles parked or left unattended on college property are required to display a currently valid Evergreen parking permit during specified days and hours. These hours are posted in each parking area at the entrance to the parking areas, or along the roadways where parking is indicated. The college maintains the authority to sell and require the display of special event parking permits during times and days, including weekends, as established by the college. Vehicles parked on campus are required to display valid parking permits at all times and days of the week as established by these rules. A complete list of parking permits issued by the college is available in the parking services office and on the college web site.
(2) Fees for parking and the effective date thereof, will be approved by the president of the college. Prior to approval by the president, the college will, after notice, hold a hearing on the proposed fee schedule. The hearing will be open to the public, and will be presided over by a presiding officer designated by the president. The presiding officer will prepare a memorandum for consideration by the president, summarizing the contents of the presentations made at the hearing. Approved fee schedules will be available in the public area of the parking services office and on the college's web site.
Parking permits—Special exceptions.
All persons parking vehicles on campus will park in available space as established by the college parking regulations and will pay the established parking fee except as follows:
(1) Vehicles with government tax exempt licenses will be allowed to park without charge.
(2) Members of the press, television, radio and wire services, on official business, after obtaining a permit from the parking office, may park without charge.
(3) Taxis and commercial delivery vehicles may enter the campus without payment of the parking fee only for pick-up and delivery of passengers, supplies and equipment.
(4) Permanently and temporarily disabled persons may request a disability parking placard from the parking office. Vehicles parked in handicapped-accessible spaces must display a paid parking permit and a state of Washington or college-issued temporary disabled parking placard to be valid.
Parking permits—Issuance and display.
(1) All parking permits must be entirely visible and displayed on the vehicle in accordance with the instructions printed on the permit, with permit numbers and relevant dates visible. Vehicles that do not have visible and properly displayed permits may be cited for the violation of improperly displaying a permit.
(2) Ownership of permits is not transferable except when approved by parking services. If a registered vehicle is sold, the permit must be removed and returned to parking services for a replacement or any refund.
(3) Persons not residing on campus may apply for a duplicate permit for a second car either personally, family, or employer owned. Proof of ownership or appropriate authorization must be presented prior to issuance of a second permit. Two vehicles displaying the same numbered permit may not be parked on campus at the same time unless one also displays a valid daily permit.
(4) Vehicles displaying a valid permit may be parked in any designated campus parking lot authorized by the permit. Vehicle parking in the modular housing area is restricted to residents and other users authorized by parking services. F lot parking permits are valid in B, C, and F lots. Modular housing permits are valid in all of the campus parking lots.
(5) Permit holders may obtain a complimentary temporary daily permit for a vehicle being used as a temporary replacement.
(6) No vehicle may be parked on campus for the purpose of using such vehicle as a living unit. Any exception must be approved by the director of police services or their designee.
Parking permits—Validity and revocation.
(1) Parking permits will be valid from the date of purchase through the expiration date and/or time stated on the permit.
(2) Parking permits are licenses and remain the property of the college. Parking permits may be revoked for any of the following reasons:
(a) When the purpose for which the permit was issued changes or no longer exists.
(b) When a permit is used in an unauthorized manner.
(c) Falsification of a second car parking permit application.
(d) Counterfeiting or altering a permit.
Responsibility and presumption in reference to illegal parking.
(1) The registered owner or permit holder will be responsible for all parking violations involving the vehicle on which the permit is displayed.
(2) In any review, appeal or hearing alleging the violation of any parking regulation, proof of the following will create a presumption that the registered owner or permit holder was the person who parked or placed the vehicle in the location where the violation occurred:
(a) Proof that the vehicle described was stopped, standing or parked in violation of a regulation; and
(b) Proof that the person named in the citation was the registered owner or permit holder of the vehicle when the citation was issued.
Designated and assigned parking areas.
(1) The motor vehicle laws of the state of Washington and these rules will be applicable at all times in areas covered under the scope of this policy including all college-owned property.
(2) No vehicle may be parked on the campus except in those areas set aside and designated as parking areas.
(3) No vehicle may be parked in any parking area without a valid, current permit for that area issued by parking services.
(4) Vehicles may park only within marked spaces provided in each parking lot.
(5) Metered parking spaces require appropriate payment in the corresponding parking meter for valid parking, regardless of any passes or permits displayed on the vehicle.
(6) Vehicles parked in electric vehicle charging spaces are required to display a valid parking permit, and must be actively charging as indicated by the charging station.
Parking—Prohibited places.
(1) No vehicle may stop, stand or park so as to obstruct traffic along or upon any street or sidewalk or in any parking lot.
(2) No vehicle may park, stop or stand in a location likely to interfere with traffic flow except momentarily to pick up or discharge passengers.
(3) No vehicle may be parked on any lawn or grass areas except as required for maintenance or construction authorized by parking services.
(4) No vehicle will be parked so as to occupy any portion of more than one parking space or stall as designated within the parking area. The fact that other vehicles may have been so parked as to require the violator to occupy a portion of more than one space or stall will not constitute a rationale for a violation of this section.
Impounding and immobilization of vehicles.
(1) The expense of such impounding and storage will rest solely on the owner or permit holder of the vehicle. Neither the college nor its employees will be liable for loss or damage of any kind resulting from impounding and/or storage services provided by a private vendor.
(2) Any vehicle parked upon property of the college in violation of these regulations, including the motor vehicle and other traffic laws of the state of Washington, with at least three unpaid citations, with the oldest being at least thirty days old, may be either immobilized or impounded and removed for storage.
(3) Notice of intent to impound will be posted on the vehicle twenty-four hours prior to impound.
(4) If the vehicle is parked in a metered or timed space, notice of intent to impound will be posted on the vehicle for four hours prior to impound.
(5) Any vehicle that blocks, hinders, or obstructs a legally parked vehicle, crosswalk, sidewalk, disability access area, service drive, or loading zone may be cited, and after the college has made a reasonable attempt to contact the owner, the vehicle may be impounded immediately.
(6) Immobilization is defined as impounding the vehicle in place through the installation of a wheel boot, which is a device designed to prevent vehicles from being moved. It consists of a clamp that surrounds a vehicle wheel, and designed to prevent removal of both itself and the wheel. Release from in-place immobilization is contingent on payment of all outstanding fines and charges.
Disabled or inoperative vehicle.
No disabled or inoperative vehicle will be parked on the campus for a period in excess of seventy-two hours without approval of parking services. Vehicles which have been parked for periods in excess of seventy-two hours and which appear to be disabled or inoperative may be impounded and stored at the expense of the registered owner. It is the responsibility of the owner or permit holder of a disabled vehicle to notify police or parking services of the vehicle's location and estimated time of removal or repair. A valid parking permit or pass must be displayed on the disabled vehicle while it is parked on campus.
Privately owned motor vehicles will be driven only on those roadways designed and built for their use. Driveways and roads marked "service" may be used only by college employees in college-owned vehicles conducting official business, emergency vehicles, and authorized delivery vehicles. All other vehicles are prohibited from traveling or parking in these areas unless authorized by parking services. Brick-paved and other designated areas are for pedestrian and bicycle traffic only, except as needed for emergency vehicles or for maintenance of buildings or grounds. No person without authorization from the director of facilities or the director of police services will install, move, deface, or in any way change a sign, barricade, structure, marking or direction so placed, or previously placed, for the purpose of regulating traffic or parking.
Parking of motorcycles.
(1) Motorcycles are, for the purpose of these regulations, considered to be motor vehicles and are subject to all parking regulations.
(2) Motorcycles may be parked in designated motorcycle parking areas in addition to the stalls in the regular parking lots.
(3) Motorcycles are not permitted on paths, sidewalks, in buildings or in pedestrian areas at any time.
Citations, late fees and fines.
(1) Payment.
(a) Persons cited for violation of these regulations are required to pay a fine within ten days of the date of issuance of the citation. All parking fines and fees are due upon issuance. Thirty days after the issuance of the citation, a late fee will be added to the unpaid parking fine. For example, a parking citation issued on May 1st would be assessed a late fee on May 31st.
(b) All fines are payable at the cashier's office or other designated locations on campus. Fines may be paid in person or by telephone during normal business hours or by mail or online. The notice of citation, citation number or vehicle license plate number must accompany any fine payment.
(2) Types of citations:
(a) No valid permit;
(b) Overtime parking;
(c) Improper position;
(d) Parking in a restricted space;
(e) Disabled zone;
(f) Parked at painted curb;
(g) Prohibited zone;
(h) Obstructing traffic;
(i) Parking in bus zone;
(j) Fire lane;
(k) Parked on grass;
(l) Altered permit;
(m) Nondesignated parking space;
(n) Expired meter; and
(o) Wheel-lock.
(3) Fine amounts:
(a) When a citation is issued, fines are determined in accordance with a fine schedule. The fine schedule and the effective date thereof is approved by the vice president for student affairs or their designee, and available in the parking services office and on the college web site.
(b) Adjustments: When mitigating circumstances exist, the vice president for student affairs or their designee may reduce or dismiss fines.
(4) Unpaid fines. If any fine remains unpaid after sixty days from the date of the notice of citation, the account may be referred for collection and subject to the college policy for accounts receivable collection process.
(5) Appeals: Citations may be appealed by submitting a written appeal to parking services within ten calendar days of the date the citation was issued. Appeals must be submitted to parking services in person, mail, or using the college web site. If a timely appeal is not filed, the citation becomes final. Appeals will be reviewed by a board consisting of voting members from the following groups: Students, classified staff, faculty, and exempt staff. A parking services representative will act as a consultant to the board and will vote only to break a tie. The board may uphold or dismiss the citation. If the board upholds the citation, it may reduce the fine amount. In no event may the board impose a fine exceeding the amount set forth in the fine schedule. Within ten calendar days following the board's review, parking services will notify the appellant, by mail or by email, of the board's determination. Additional appeal rights are governed by RCW 28B.10.560.
Severability.
If any provision of this chapter or its application to any person or circumstances is determined to be invalid, the remainder of the chapter and its application to other persons or circumstances is unaffected.


These annual awards were created to bring recognition to the games and people in the RPG community who have introduced games to the market using their own resources and talents.
Once a year, players and creators of games that are listed on RPGfix may nominate the best games to receive an award.
If a game receives significant nominations in a particular category, it will be entered into the final community voting round.


Long before I saw the forecast of wind or woke to it howling around my house at midnight, I had decided to sit on the rollers this morning. I wanted to get a bit more than the normal spin until the legs free up. I decided on an hour, something I've not done on the rollers before (trainer is another story).
It started off not too badly, sure the wind had knocked out the telly aerial, but the internet connection and thus iView still worked. An eternity passed and I finally gave in and checked the clock. I'd only been going for 25 minutes. I got back to the task of smooth pedal strokes and tried to ignore the fidgeting in my brain.
With Vic CX champs this weekend, the Warny next weekend and the state of my legs, I'm going to go for a modified two week taper rather than my customary one. Stay tuned for updates on how stir crazy I'm going.


Zulu Bible is an offline Bible solution containing Zulu edition of the Bible. Download this app specially made for Zulu speakers in South Africa, Zimbabwe, Lesotho, Malawi, Mozambique and Swaziland. After downloading, read the Bible even when you're not connected to the internet. Browse the verses you want to learn and enjoy daily the Word of God on your Android phone or tablet.


text:
Migrants invited to church pulpits for Labor Day
Lindsay Wasserberger on September 7, 2009
Romana was seven months pregnant, she told the congregation at Lakeshore Avenue Baptist Church on Sunday, when she walked for six hours from Mexico to the United States -– mostly without water, sometimes without shoes.
She crossed through brush where sticks ripped at her skin, she said. Her brother and grandmother dragged her when she felt too tired to walk. When Romana and her family arrived at a tunnel where they thought they could rest, she said, US immigration workers arrested them.
"They threw us in the van like we were animals. They didn't care that I was pregnant," Romana said in Spanish while standing Sunday beside her interpreter in the Lakeshore pulpit. "I asked for water and they told me to be quiet, that there was no water for me."
The congregation members, a crowd of varied ages and ethnicities scattered throughout the pews, fixed their eyes on Romana while she spoke. When the interpreter, now a 30 year old woman, revealed that she was the child Romana had said she felt kick inside her during her journey, many of the parishioners were visibly moved .
"I am here to ask President Obama for the immigration reform he promised," Romana's daughter translated for her mother, "so no one has to suffer like I did."
Linda Crockett, an organizer for the East Bay Interfaith Committee for Workers Justice, the organization that arranged for Romana to speak during the special Labor Day weekend service, entered the pulpit afterward in tears.
"Immigrant workers are people of faith just like you," Crockett said. "I hope Romana's story inspired you to continue or join in the fight for justice."
Pastor H. James Hopkins invoked Leviticus 25, an Old Testament chapter that addresses work and the erasure of debt. He repeated the words "humanity" and "desperation," urging his congregation to help others rise out of debt rather than sink deeper.
"This has been a hard year for all people in the United States: citizens and those who are not–those who are here quote legally and those who are here quote not," he said. "Find a way to provide a rope, a ladder, a road for others to find their way out of desperation."
Romana spoke as part of Labor in the Pulpits, an event that brought Labor Day-related sermons and guest speakers this weekend to 16 churches throughout the East Bay. The organizers, the East Bay Interfaith Committee for Workers Justice, a project of the East Bay Alliance for a Sustainable Economy, produced the event to provide local religious communities with information about the issues facing low-wage workers.
With the help of the Black Alliance for Just Immigration and East Bay Housing Organizations, the committee focused this year on bringing the stories of immigrants into East Bay churches in the effort to ease the tension the committee sensed was escalating between local unemployed US citizens and immigrants – many undocumented – as a result of the recession, according to Diana Rashid, an organizer for East Bay Interfaith.
Other Oakland Labor in the Pulpits participants included Faith Presbyterian Church and Our Lady of Lourdes Catholic Church.
At Our Lady of Lourdes on Saturday, a woman who identified herself as Maria spoke of the challenges she has faced since moving to the United States 20 years ago from Mexico, where she said she often witnessed violence and felt unsafe. Without legal US residency, Maria said, she first worked as a housekeeper. When her employer learned she was undocumented, he gave her three days to fix her status – an inadequate amount of time, she said. When the three days passed, Maria was fired.
Maria applied for her visa ten years ago. She has been told she has to wait six more years, she said, but has not been told why.
Before passing the floor back to the pastor, Maria said, "I am a person of faith. I pray President Obama give us immigration reform. That is my dream."
Interfaith members said they hope the speakers, who were mostly from Spanish-speaking countries but also included immigrants from Africa and Asia, inspired the clergy as well as the congregants to support the committee's efforts, Rashid said.
The Rev. Hopkins said he participated in the event to encourage parishioners to see beyond the statistics and politics of immigration policy to the human beings seeking security and a way to keep their families together, he said. He hoped, he said, that his parish's congregants – mostly middle class workers, some in management positions – would take away a sense of empathy for Romana and other immigrants.
"Justice is a simple biblical mandate," Hopkins said during an interview, "and [churchgoers] are people who act on their beliefs."
Business | Culture | Economy | Immigration | Labor | Politics | Religion
Video: Shipping out to watch the Bay Bridge retrofit
Chabot opens as construction nears unveiling

redpajama_set_name: RedPajamaCommonCrawl


text:
7250 TTV Agrotron Tier 3: TOP GERMAN TECHNOLOGY. Success is achieved by enterprises that are able to tackle every task throughout the season using the lowest possible amount of reso
/RMI - Repair Maintenance Information
7250 TTV Agrotron Tier 3
/7 Series Stage 4 (Tier4 Final)
/Agrofarm TB
/5 Series
/5G TB Series
/6.4 Series (Stage IV)
TOP GERMAN TECHNOLOGY.
Success is achieved by enterprises that are able to tackle every task throughout the season using the lowest possible amount of resources and being effective with the time available. Equally, every penny counts when it comes to reducing production costs. DEUTZ-FAHR has developed the new 7 Series for such businesses. The new 7250 TTV Agrotron E3 delivers 246 HP and stand out for a range of trademark German characteristics: high quality, high precision and reliability combined with cutting-edge technology in terms of both cost-effectiveness and comfort.
The 7250 TTV Agrotron E3 represents the culmination of highly innovative and intelligent tractor technology manufactured at our new facility in Lauingen, Germany, offering the exceptional comfort of the MaxiVision 2 cab, intuitive controls, a newly designed armrest, state-of-the-art precision farming systems, highly efficient engine and transmission technology, innovative axles and brakes which ensure optimal driving comfort and maximum safety. Furthermore, the hydraulic systems and lift capacity both front and rear, compliment the overall 7 Series specification making this tractor the ideal partner for increasing the productivity of professional contractors and agricultural businesses – both in the field and on the road.
Download Browse online
DEUTZ 6.1 L06 SIX CYLINDER ENGINES: POWERED BY EFFICIENCY.
The new 7250 TTV Agrotron E3 is equipped with a completely redesigned engine in terms of performances and cost-effectiveness. The new Deutz 6.1 engine is more responsive, offers higher reserve torque and has a higher torque at lower rpms. At the same time, consumption of fuel has been reduced by 5 %, compared to the previous generation.
The efficiency of the engine is supported by Electronic Visco fan and the redesigned, compact and high-performance cooling system. The patented cooling system can be opened completely, which makes servicing much more straightforward. A pre-cleaning system on the air intake slots prevents dirt from entering the air filter, making a substantial difference to service costs. The Deutz 6.1 engine – modern technology made in Germany – is synonymous with the highest levels of know-how and efficiency, allowing you to achieve maximum productivity.
TTV TRANSMISSION. TURN POWER INTO TRACTION.
The new 7250 TTV Agrotron E3 is fitted with state-of-the-art electronically controlled continuously variable TTV transmission technology. It is a solid concept that has been tried and tested over the years, now with new software for even better performance. DEUTZ-FAHR is the only manufacturer that offers a 60 Km/h tractor in such a compact design. All final speeds of 40 Km/h, 50 Km/h or 60 Km/h can be reached at a reduced engine speed, which is extremely fuel efficient. Overall, the speed range extends from 0.2 Km/h to 60 Km/h. For field work, the TTV transmission system operates most efficiently at all speeds.
The highly comfortable TTV transmission is extremely efficient thanks to its four mechanical stages, which change automatically. Engine and transmission management are fully integrated, providing a comfortable automatic system for mechanical range shifting. Up to four cruise speeds can be stored in order to optimise work processes. A new trailer stretch mode increases driving safety on hilly terrain. Every single detail of the TTV transmission system has been designed to maximise the productivity of the new 7250.
WORKING WITH ALL IMPLEMENTS IN ANY SITUATION.
The productivity of a tractor depends directly on the equipment it can drive. The aim is to reduce the amount of distance travelled through increasingly large and heavy attachment combinations. This is where the new 7250 TTV Agrotron E3 clearly demonstrates its strength. With three PTO speeds (540ECO/1000/1000ECO) at the rear and 1000 or 1000ECO at the front, all work can be carried out very efficiently. Thanks to the reduced engine speed characteristics, coupled with the ECO power take-off sysytems, fuel consumption is significantly reduced. All hydraulic and PTO functions can be easily set and controlled from the armrest in the cab. An external control of hydraulics, rear lift and PTO from both sides of the tractor is also possible.
LIFT UP YOUR PRODUCTIVITY.
The new 7250 TTV Agrotron E3 provides attachment area technology that was previously available only in higher power classes. The rear attachment area features up to five hydraulic control valves, while the front attachment area has two. The absolute highlight is that all valves are proportionally controlled.
The new hydraulic system of the 7250 TTV Agrotron E3 allows a position-controlled front linkage to be equipped. Until now, such advanced technology was only available on the 9 Series. The lifting capacity is up to 5480 kg, an impressive figure. Additionally, equipment is positioned much closer to the tractor with the newly integrated front linkage concept, which substantially enhances lifting capacity. The front mounting area also features an integrated ISOBUS socket. The hydraulic pump delivers an output of 120 L/min as standard or 160 L/min on request. The load sensing system is included as standard. A separate pump supplies the steering so that the working hydraulic system is not compromised. The rear lifting capacity is 10,000 kg.
FOR UNIQUE SAFETY IN THIS TRACTOR CLASS.
Productivity needs maximum vehicle control - both in the field and on the road. At top speed as well as on challenging ground conditions and with the heaviest equipment. The 7250 TTV Agrotron E3 gives you a uniquely valuable sensation: safety in every situation.
The new heavy duty front axle with suspension and the braking system of the 7250 TTV Agrotron E3 are unique in this tractor class. The intelligent and adaptive suspension system give the new 7250 TTV Agrotron E3 maximum stability – both during heavy tillage operations and when driving loads. The innovative suspension concept stabilises the tractor during all transport operations and boosts driving safety. In order to ensure better handling on uneven ground, the dampers are mounted on the front side of the axle.
The 7250 TTV Agrotron E3 is the only standard tractor that is fitted with high performance dry disc brakes on the front axle. The additional Booster brake system ensures maximum braking performance with the lightest pedal pressure. No other standard tractor offers a heavy duty front axle, dry disc brakes combined with the booster brake system and improved safety.
ERGONOMIC OPERATION. MAXIMISING DRIVER'S COMFORT.
The unique features offered by MaxiVision 2 cab can be seen inside and also from outside. When it comes to ease of use and ergonomics, the new 7250 TTV Agrotron E3 takes care of work for the driver to the extent permitted by current technology. The new InfoCentre Pro on the dashboard features a 5" colour display, providing the driver with a stream of data about the tractor's operating conditions. It offers maximum convenience in terms of settings and information. In order to allow precise, stress-free work in all lighting conditions, the tractor can be fitted with an optional 40.000 lumen LED package with four working headlights. The interior is designed for the driver's comfort - reduced engine noise, vibrations and heat due to the cab and engine hood being separated. The new hood encapsulates the latest, highly contemporary look of the DEUTZ-FAHR tractor family, while its compact design also ensures outstanding forward visibility. The best possible view of rear attachments is guaranteed by the rearward position of the cab.
Other features that reduce stress are the bright, welcoming colours of the interior, the large windows and a sunroof, available in three designs: an easy-to-open green roof, a glass roof with 84% UV radiation absorption and a FOPS safety version for optimal visibility during front loader operation. The air conditioning system – with automatic functions – provides the largest number of air outlet openings in this tractor class guaranteeing uniform air distribution like no other model. Every detail of the MaxiVision 2 cab has been designed to help the driver work for long periods without stress, thus achieving maximum productivity.
The newly designed armrest makes the 7250 TTV Agrotron E3 even more relaxing and more intuitive to operate. All routine functions can be automated. Simply pre-select the desired basic settings and activate them at any time with a tap on the screen. The new, highly ergonomic joystick on the armrest controls everything from driving speed, direction of travel and cruise control selection to the ComfortTip system, lift controls and front lift controls. It is an intuitive system that requires no change of hand position and no more than a moments thought to operate. All of the controls are clearly marked with colours and are logically arranged according to their frequency of use. The driver has complete control over the new 7250 TTV Agrotron E3 at all times and can work for very long periods at a highly productive rate.
Deutz Common Rail. High pressure common rail injection system, with two lubricated injection pumps integrated in the engine block, and an innovative combustion chamber configuration for superlative power and torque over a wide range of engine speeds.
A system maximising the potential of the Sense Clutch shuttle for superlative manoeuvrability. Ideal for jobs involving frequent stops and starts.
ELECTRONIC ENGINE GOVERNOR
The system monitors a set of engine parameters, and optimises engine function by adjusting injection accordingly. Automated for total simplicity: lets the driver set, store and recall the most appropriate engine speed for the job in hand.
An electronic system which automatically engages and disengages drive to the front wheels and the differential lock in relation to steering angle and speed.
Hydraulic reverse shuttle with two multiplate wet clutches and an electronic control unit.
A system ensuring an effective and immediate response from the brake system in all conditions with minimal pressure on the pedal by maintaining a constant pressure in the relative hydraulic circuit.
COMFORT TIP
The Operating Sequence programming allows the tractor to perform a series of functions automatically. The pre-set program is automatically executed while successive operations are performed manually (by pressing a button) or automatically (following a defined period of time or distance) if the tractor is equipped with an iMonitor.
Optimised power curve usage, reduced consumption and improved driver comfort by maintaining lower engine speeds and using the engine at speeds close to maximum torque.
COMMANDER CONTROL
Joystick-controlled control functions. The operator can easily and conveniently control selected tractor functions.
This advanced technology tractor is equipped with several sensors monitoring the correct operation of all machine systems and functions. Sensor signals are collected and analysed by a control panel which adjusts tractor settings on the basis of this information in order to meet current needs.


text:
Cross-talk between SIM2s and NFκB regulates cyclooxygenase 2 expression in breast cancer
Garhett L. Wyatt1,
Lyndsey S. Crump2,3,
Chloe M. Young2,3,
Veronica M. Wessells2,3,
Cole M. McQueen1,
Steven W. Wall1,
Tanya L. Gustafson1,
Yang-Yi Fan4,
Robert S. Chapkin4,
Weston W. Porter1 &
Traci R. Lyons ORCID: orcid.org/0000-0002-8876-03912,3
Breast cancer is a leading cause of cancer-related death for women in the USA. Thus, there is an increasing need to investigate novel prognostic markers and therapeutic methods. Inflammation raises challenges in treating and preventing the spread of breast cancer. Specifically, the nuclear factor kappa b (NFκB) pathway contributes to cancer progression by stimulating proliferation and preventing apoptosis. One target gene of this pathway is PTGS2, which encodes for cyclooxygenase 2 (COX-2) and is upregulated in 40% of human breast carcinomas. COX-2 is an enzyme involved in the production of prostaglandins, which mediate inflammation. Here, we investigate the effect of Singleminded-2s (SIM2s), a transcriptional tumor suppressor that is implicated in inhibition of tumor growth and metastasis, in regulating NFκB signaling and COX-2.
For in vitro experiments, reporter luciferase assays were utilized in MCF7 cells to investigate promoter activity of NFκB and SIM2. Real-time PCR, immunoblotting, immunohistochemistry, and chromatin immunoprecipitation assays were performed in SUM159 and MCF7 cells. For in vivo experiments, MCF10DCIS.COM cells stably expressing SIM2s-FLAG or shPTGS2 were injected into SCID mice and subsequent tumors harvested for immunostaining and analysis.
Our results reveal that SIM2 attenuates the activation of NFκB as measured using NFκB-luciferase reporter assay. Furthermore, immunostaining of lysates from breast cancer cells overexpressing SIM2s showed reduction in various NFκB signaling proteins, as well as pAkt, whereas knockdown of SIM2 revealed increases in NFκB signaling proteins and pAkt. Additionally, we show that NFκB signaling can act in a reciprocal manner to decrease expression of SIM2s. Likewise, suppressing NFκB translocation in DCIS.COM cells increased SIM2s expression. We also found that NFκB/p65 represses SIM2 in a dose-dependent manner, and when NFκB is suppressed, the effect on the SIM2 is negated. Additionally, our ChIP analysis confirms that NFκB/p65 binds directly to SIM2 promoter site and that the NFκB sites in the SIM2 promoter are required for NFκB-mediated suppression of SIM2s. Finally, overexpression of SIM2s decreases PTGS2 in vitro, and COX-2 staining in vivo while decreasing PTGS2 and/or COX-2 activity results in re-expression of SIM2.
Our findings identify a novel role for SIM2s in NFκB signaling and COX-2 expression.
Ductal carcinoma in situ (DCIS) is a heterogeneous disease characterized by tumor cells that are confined to the ductal system of the breast [1]. DCIS progresses to invasive ductal carcinoma (IDC) through events such as epithelial mesenchymal transition (EMT), basement membrane degradation, inflammatory signaling, and other pathways associated with a wound-healing milieu [2,3,4]. It is estimated that ~ 20% of mammography-detected breast cancers are DCIS [5] and ~ 65,000 cases of DCIS are diagnosed per year [6]. Provided that DCIS is removed surgically, as is the standard of care, a woman diagnosed with DCIS without recurrence is more likely to die of other causes than of breast cancer [7]. However, it is estimated that ~ 15–20% of DCIS patients develop invasive disease within a decade [8, 9]. Recently identified risk factors for DCIS recurrence include age < 40 at diagnosis, African American ethnicity, hormone receptor negativity, and HER2 positivity [7]. However, these high-risk groups only account for 20% of the DCIS patient population [9]. Therefore, identifying additional risk factors for, or markers that will predict, DCIS aggressiveness is an extremely important goal for preventing invasive cancer in DCIS patients.
There is increasing evidence that inflammation plays a key role in breast cancer progression [10]. One such specific inflammatory pathway is nuclear factor kappa b (NFκB). The NFκB signaling pathway includes five members: NFκB1 (p105/p50), NFκB2 (p100/p52), RelA (p65), RelB, and c-Rel. Dimers of the aforementioned proteins are held in the cytoplasm by inhibitor kappaB kinase (IκB) proteins, primarily IκBα. The mechanism of activation of NFκB requires phosphorylation of IκBα by inhibitor of kappaB kinase (most commonly IKKα and IKKβ), which leads to degradation of IκBα. Upon degradation of the IκBα, NFκB heterodimers, specifically the canonical heterodimer p50/p65, translocate to the nucleus and bind to promoters of target genes, leading to activation of their transcription [11, 12]. Known transcriptional targets of NFκB include mediators of inflammation, such as cytokines/chemokines and immunoreceptors, as well as proteins involved in antigen presentation, cell adhesion, stress response, apoptosis, growth factor receptor signaling, and transcription [13]. Two NFκB consensus sites are located in the promoter region of the human PTGS2 gene, which encodes for pro-inflammatory enzyme cyclooxygenase 2 (COX-2) [14]. These NFκB consensus sites contribute not only to cancer progression by preventing apoptosis but also to the activation of COX-2-mediated signaling [15]. COX-2 is the inducible form of cyclooxygenase, which is the key enzyme involved in the biosynthesis of the pro-inflammatory prostaglandins [16,17,18,19,20,21]. Importantly, COX-2 has been implicated in DCIS progression through promotion of proliferation, migration, invasion, and metastatic spread in pre-clinical models [22,23,24]. Additionally, expression of COX-2 is frequently observed in patients with invasive disease and is associated with DCIS recurrence. Furthermore, the therapeutic benefit of inhibiting COX-2 has been observed in the colon, esophagus, lung, bladder, breast, and prostate cancers [18, 19, 25,26,27,28,29,30,31,32,33,34,35]. Thus, it is logical to expect that inhibition of COX-2 signaling in breast cancer patients could enhance overall prognosis.
Previously, we have shown that Singleminded-2s (SIM2s; expressed from SIM2), a member of the bHLH/PAS family of transcription factors, is a tumor suppressor that is expressed in breast epithelial cells and downregulated in the transition from DCIS to IDC [36,37,38,39]. Specifically, using the MCF10-DCIS.COM progression model, we demonstrated that re-expression of SIM2s inhibits growth, invasive phenotypes, and progression to metastasis. Furthermore, overexpression of SIM2s in breast cancer cells promotes a more luminal-like phenotype, whereas downregulation of SIM2s leads to increased invasive potential [39]. Consistent with the role for SIM2s in cancer progression, we have also shown that the NFκB signaling pathway is negatively regulated by SIM2s in normal mammary tissues during postpartum mammary involution [40], which has been identified as a driver of tumor progression and metastasis. In this study, we demonstrate a relationship between SIM2s, the NFκB signaling pathway, and COX-2 in breast cancer cells. We suggest that re-expression of SIM2s could be mediated by inhibition of COX-2 signaling, which may serve to reduce breast cancer progression.
MCF7 and SUM159 cells were purchased from American Type Culture Collection (ATCC) and were maintained in accordance to their guidelines. MCF10A-DCIS.COM (DCIS.COM) cells were generously donated by Dr. Dan Medina (Baylor College of Medicine, Houston, TX, USA). Cells were plated into 6-well plates for RNA isolation experiments according the guidelines from ThermoFisher Scientific. Celecoxib experiments were performed as follows: cells were first dosed with 10 μM celecoxib for 24 h, then media was changed and treatment was performed at 20 μM celecoxib for 24 h, and then cells were harvested for analysis. DHA and PGE2 experiments on cell lines were performed as follows: cells were dosed with 50 μM DHA or 100 μM for 24 h and then harvested for analysis.
Generation of cell lines
Point mutations in the SIM2 gene were generated via long cDNA synthesis (Invitrogen). Plasmids were amplified using Subcloning Efficiency DH5a competent cells (Life Technologies). Plasmid DNA was isolated using the HiPure Plasmid Maxiprep Kit (Life Technologies) or the ZymoPURE Plasmid DNA Isolation Kit (Zymo Research). Viral transduction was performed as previously described [38]. Puromycin selection (2 μg/mL) was started the following day and maintained for a week.
Two hundred thousand DCIS.COM cells stably expressing anti-COX-2 shRNAs (a generous gift from Kornelia Polyak and Andriy Marusyk) were orthotopically injected and tumors harvested as previously described [22, 23].
RNA isolation and real-time PCR
RNA isolation and real-time PCR were performed as previously described [39]. Primers can be found in Additional file 1: Table S2.
Immunoblotting
Cells were washed with cold PBS and lysed in high-salt lysis buffer (50 mM HEPES, 500 mM NaCl, 1.5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 1% Triton X-100, pH 7.5) supplemented with 1 mM Na3VO4 (Sigma) and 1 mM complete ULTRA tablets mini EDTA-free Easy pack (Roche). Protein concentration was determined using the DC Protein Assay (Bio-Rad) with bovine serum albumin as a standard. Immunoblotting and zymography were then conducted as previously described [38]. Antibodies can be found in Additional file 1: Table S1. Blots were imaged on a ChemiDoc MP (Bio-Rad) after incubating in ProSignal Pico ECL Spray (Genesee Scientific) for 3 min. Quantification was performed using ImageJ.
Immunohistochemistry (IHC) for COX-2 was performed as previously described [22]. Analysis for positive nuclei was performed as previously described [24]. Antibodies can be found in Additional file 1: Table S1.
Transient transfection
MCF7 or 293T cells were used for transfections for luciferase activity. One hundred nanograms (0.1 μg) of plasmid containing transcription factor was mixed with up to 1 μg (amount varies per construct) of plasmid containing promoter construct. Three microliters of Genejuice (Novagen) was used per microgram of DNA. DNA and Genejuice were mixed in Opti-MEM media (Invitrogen). Protein was harvested 2 days after transfection, using Reporter Lysis Buffer (Promega). Luciferase activity and total protein were measured as described previously [37]. Luciferase activities were normalized to total protein values and are represented as the means ± SE for three wells per condition.
For chromatin immunoprecipitation (ChIP) assays, 2 μg of plasmid containing repressor and 2 μg of plasmid containing the SIM2 promoter construct were transfected into 293T cells in a 10-cm plate. Chromatin was harvested 2 days after transfection.
Oncomine analysis
Analysis of SIM2 in primary breast cancer versus metastasis was performed using the Oncomine software (oncomine.org). The TCGA dataset was analyzed for SIM2 with the threshold p value set at 0.05 and the threshold fold change set at 2. Additionally, using Oncomine gene expression signatures, we evaluated the breast cancer metastasis concept, setting the odds ratio threshold at 2 and the p value at 0.01.
To address scientific rigor, all experiments in cell lines and xenografts were conducted at a minimum in biological triplicates and technical duplicates and were repeated three times. Normal distribution was confirmed before conducting unpaired t test. ANOVA analysis was performed using JMP Pro 14 to asses that means are in fact different, and then the post hoc Student's t test was performed. Correlation analysis was performed using Prism7; Pearson's r and a two-tailed test were performed to examine significance. Significance was considered at p < 0.05 unless otherwise noted.
SIM2s downregulates NFκB signaling
To test the hypothesis that SIM2 negatively regulates NFκB/p65-mediated transcription in breast cancer cells, we co-transfected a reporter plasmid encoding a NFκB binding site upstream of the luciferase gene (5x NFκB-luc) with the p65 subunit along with SIM2s in MCF7 cells and measured relative light units as a readout for NFκB activity. As expected, we observed that p65 strongly activated the reporter construct, but this response was blocked by co-transfection of SIM2s (Fig. 1a). Furthermore, we repeated the 5x NFκB-luc transfection in MCF7 cells with stable transduction of a SIM2 sh-RNA or control plasmid (Additional file 1: Figure S1). In the shSIM2 cells, the 5x NFκB-luc was significantly increased over the controls (Fig. 1b). To determine whether inhibition of the 5x NFκB-luc reporter by SIM2s was dependent on the transcriptional repressor activity of SIM2s, the transfection was repeated with a SIM2s expression construct missing the Pro/Ala transcriptional repression domain (SIM2sΔR). Interestingly, this construct also significantly attenuated the activation of the 5x NFκB-luc construct by NFκB/p65, demonstrating that the repression domain of SIM2s is not required for inhibition of NFκB signaling (Fig. 1c; Additional file 1: Figure S2A,B). As an alternative, we performed immunoblot analysis of key players in the NFκB signaling pathway to determine whether SIM2 modulates expression levels of key mediators of NFκB/p65 signaling in our breast cancer cell lines that could downregulate signaling in a posttranscriptional/posttranslational manner. We found that IKKα, IKKβ, phosphorylated-p65, and p65 protein expression, all of which mediate NFκB activation, are decreased in SIM2s overexpressing SUM159 cells (Fig. 1d). Similarly, we found that these same NFκB pathway protein levels are increased in SIM2s knockdown MCF7 cells (Fig. 1d). These results suggest that SIM2s may affect NFκB-mediated transcription via modulation of protein expression and/or phosphorylation of key mediators of NFκB signaling. Akt is a known activator of NFκB signaling through its ability to phosphorylate and activate IKKα/IKKβ, which leads to nuclear translocation [41, 42]. Thus, we analyzed whether activation/phosphorylation of Akt was modulated by SIM2 expression. Indeed, we observed that overexpression of SIM2s results in a modest decrease in pAkt, while SIM2s knockdown strongly restored pAkt. Together, our results suggest a SIM2s-mediated negative regulation of NFκB/p65 involves de-activation of Akt signaling.
a Luciferase activity in MCF7 cells co-transfected with 5x kB binding sites upstream of the luciferase gene (5x NFκB-luc) and NFkB p65 and/or SIM2s. (Diagram of promoter construct is shown above for reference.) b Luciferase activity in MCF7 control or MCF7 SIM2-shRNA cells with 5x NFκB-luc. c Luciferase activity in MCF7 cells co-transfected with 5x NFκB-luc and NFkB p65 and/or SIM2s with its repression domain deleted (SIM2sΔR). d SUM159 plpcx emp (control), SUM159 plpcx SIM2s-FLAG (overexpression), MCF7 psil SCR (control), and MCF7 psil SIM2-shRNA (knockdown) were analyzed by western blot for levels of IKKα, IKKβ, phospho-p65, p65, and beta actin as a loading control. e SUM159 plpcx emp (control), SUM159 plpcx SIM2s-FLAG (overexpression), MCF7 psil SCR (control), and MCF7 psil SIM2-shRNA (knockdown) were analyzed by western blot for levels of phospho-AKTs473, pan AKT, and GAPDH as loading control. ANOVA and Student's t test were performed to test significance. a, b, c All significant at p < 0.05, *p < 0.05. Analysis was performed via ImageJ for comparison of protein expression
NFκB signaling downregulates SIM2s expression
Unexpectedly, we also observed that stable overexpression of inhibitor kappa kinase beta (IKKβ), which normally functions to phosphorylate IκBα in the cytoplasm, allowing for activation of NFκB-mediated signaling, significantly decreases SIM2s gene expression in the DCIS.COM cells suggesting a reciprocal relationship between NFκB and SIM2s (Fig. 2a). Conversely, when we suppressed NFκB activation via stable transduction of the inhibitor kappaB alpha super repressor (IκB-SR), which maintains the NFκB heterodimer (p50/p65) in the cytosol, SIM2s expression was increased (Fig. 2b). To confirm that activation of NFκB downregulates SIM2 expression in breast cancer cells, we cloned a 2-kb portion of the SIM2 promoter upstream of the luciferase gene and co-transfected with increasing amounts of p65 in MCF7 cells. We observed dose-dependent repression of SIM2s promoter activity (Fig. 2c; Additional file 1: Figure S2C). Importantly, co-transfection with IκB-SR, as well as IκB-SR with NFκB p65, successfully reversed the downregulation of SIM2 promoter activity (Fig. 2d; Additional file 1: Figure S2D), suggesting that this response was not a dominant negative effect. Analysis of the SIM2 promoter identified two consensus NFκB binding sites near the transcriptional start site for SIM2. Utilizing ChIP analysis, we showed that p65 directly binds to the SIM2 promoter around the transcriptional start site (Fig. 2e). To determine whether binding of p65 to the NFκB binding sites is necessary for downregulation of SIM2s expression, we mutated the two NFκB sites in the SIM2 promoter reporter construct and performed additional co-transfection experiments with p65. The NFκB double mutant site promoter failed to inhibit SIM2 promoter activity when compared to the wild-type promoter (Fig. 2f; Additional file 1: Figure S2E), implicating a direct interaction of NFκB/p65 on the SIM2 promoter to repress SIM2s transcription. These results suggest that NFκB-mediated transcriptional repression of SIM2s may serve to reverse SIM2-mediated downregulation of NFκB signaling, allowing for its activation and promotion of transcription of known pro-inflammatory target genes such as PTGS2.
a SIM2s expression in DCIS.COM control cells and cells overexpressing IKKβ by qPCR as fold change. b SIM2s expression in DCIS.COM control cells and cells overexpressing IκB-SR by qPCR as fold change. c SIM2s promoter activity in MCF7 cells co-transfected with SIM2 promoter upstream of the luciferase gene and increasing amounts of NFκB p65 (50 ng, 100 ng, and 150 ng). d SIM2s promoter activity in MCF7 cells after co-transfection of promoter with control vector (pcDNA3), NFκB p65, and/or IκB-SR. e ChIP assay for NFκB p65 binding after transient transfection of SIM2 promoter with NFκB p65 in HEK293T cells. f SIM2s promoter activity in MCF7 cells co-transfected with SIM2s promoter upstream of the luciferase gene and 150 ng NFκB p65 compared with the SIM2s promoter activity in MCF7 cells co-transfected with NFκB double mutant SIM2s promoter upstream of the luciferase gene. ANOVA and Student's t test were performed to test significance. a, b All significant at p < 0.05, unpaired t test: *p < 0.05
SIM2s expression downregulates COX2
To explore the relationship between SIM2s and PTGS2 expression in breast cancer, we analyzed three different breast cancer cell lines including MCF7, DCIS.COM, and SUM159 cells. The non-invasive MCF7 cell line and the highly invasive triple-negative SUM159 cell line were utilized to examine the differential expression of SIM2s, and subsequent regulation of PTGS2, as it relates to invasion. DCIS.COM cells (also triple negative) were used for their unique ability to mimic basal-like DCIS in vivo and their ability to progress to invasive disease upon acquisition of COX-2 protein expression [22, 43]. We have previously shown that the invasive competent DCIS.COM cells have more SIM2s expression when compared with the non-invasive MCF7 [37, 38]. Confirming and extending this observation, qPCR analysis reveals the lowest PTGS2 expression in MCF7 cells, which was increased 130-fold in DCIS.COM cells and the highest in the SUM159 cells (Additional file 1: Figure S3). To determine whether reduction of SIM2s in the non-invasive cells could increase expression of PTGS2, we analyzed control and shRNA-SIM2s DCIS.COM and MCF7 cells by qPCR. Our results revealed that downregulation of SIM2s led to a significant increase in PTGS2 gene expression in both cell lines (Fig. 3a, b). Moreover, we found that overexpression of SIM2s in highly invasive SUM159 cells significantly inhibited PTGS2 expression (Fig. 3c). In previous studies, we showed that overexpression of SIM2s in DCIS.COM cells blocked invasion in vivo, whereas loss of SIM2s or overexpression of the protein product of PTGS2, COX-2, resulted in increased invasion and metastasis [22, 39]. To determine the relationship between SIM2s and COX-2 protein expression in vivo, we performed IHC analysis for COX-2 in tumors derived from control and SIM2s DCIS.COM xenografts to reveal that COX-2 levels were decreased with overexpression of SIM2s (Fig. 3d). Taken together, our results suggest that SIM2s may repress invasion in the DCIS.COM model by promoting downregulation of COX-2.
a PTGS2 expression in MCF7 control cells and cells overexpressing SIM2s by real-time qPCR as fold change. b PTGS2 expression in DCIS.COM control cells and cells with SIM2-shRNA by real-time qPCR as fold change. c PTGS2 expression in SUM159 control cells and cells overexpressing SIM2s by real-time qPCR as fold change. d Immunohistochemistry for COX-2 in DCIS.COM cells stable transduced with control vector or SIM2s-FLAG (overexpression). Unpaired t test: *p < 0.05
COX-2 downregulation restores SIM2s
Since the invasive potential in DCIS.COM positively correlates with, and depends upon, expression and activity of COX-2 [22, 44], we tested the hypothesis that the loss of invasive phenotype observed with blocking of COX-2 expression was due, in part, to re-expression of SIM2s. Thus, we measured SIM2 protein levels by IHC analysis of tumors generated from control and shPTGS2 DCIS.COM cells, which are less invasive [22]. Our results reveal an increase in positive nuclear staining for SIM2 with PTGS2 knockdown (Fig. 4a, b; Additional file 1: Figure S4A). We also observed a significant negative correlation between expression of SIM2 and COX-2 and confirmed increased SIM2 in shPTGS2 DCIS.COM and control cells via immunoblot (Fig. 4c, d; Additional file 1: Figure S4B). Additionally, in this study, 87.5% of tumors in the control group had progressed to fully invasive disease compared with 25% in the shPTGS2 group (Fig. 4e). To determine whether COX-2 activity drives the inverse relationship between SIM2 and COX-2 and cell invasion, we treated the highly invasive SUM159 cells with a dose of the selective COX-2 inhibitor celecoxib that had previously been shown to decrease invasion of COX-2-expressing cells [22]. We observed a significant increase in SIM2s expression (Fig. 4f). Additionally, celecoxib also inhibited activation of the 5x NFκB-luc reporter MCF7 cells, suggesting that COX-2 activity can also feedback to promote NFκB-mediated suppression of SIM2s (Fig. 4g). Indeed, treatment of MCF7 cells with PGE2, the product of COX-2 activity, inhibited SIM2s gene expression (Fig. 4g). Furthermore, we extend our findings to show that docosahexaenoic (DHA), a n-3 polyunsaturated fatty acid (PUFA) that can also result in a shift to a more anti-inflammatory gene expression profile [45] and can reduce COX-2 expression [46,47,48,49], significantly increases SIM2s expression (Fig. 4i). Thus, our driving hypothesis is that reduction of inflammatory pathways via inhibition of activity and/or decreased COX-2 expression results in re-expression of SIM2s and may be one mechanism for preventing progression of DCIS to invasive or metastatic breast cancer [23]. Consistent with this hypothesis, Oncomine analysis reveals that SIM2s is in the top 5–10% of under-expressed genes in a breast cancer metastasis concept signature and in the top 10% of copy number loss genes (Table 1). Further, overall expression is significantly lower in a small number of metastasis in this dataset when compared to the expression in the primaries (Additional file 1: Figure S5), supporting our previous studies showing loss of SIM2s with DCIS progression to IDC [39, 50]. Thus, our results may be relevant for preventing metastasis in women with breast cancer.
a IHC analysis for COX-2 positive nuclei in tumors generated from control (Ctl) and shPTGS2 (KD2) DCIS.COM cells. Prism7 was utilized for statistical significance. Unpaired t test, *p value < 0.02. b IHC analysis for SIM2s positive nuclei in tumors generated from control (Ctl) and shPTGS2 (KD2) DCIS.com cells. Prism7 was utilized for statistical significance. Unpaired t test, *p value < 0.0001. c Correlation data for SIM2s and COX-2 positive nuclei in tumors generated from control and shPTGS2 DCIS.com cells. Prism7 was utilized for statistical significance. Unpaired t test, **p value < 0.01. d Images of IHC analysis for SIM2s in tumors generated from control and shPTGS2 DCIS.COM cells (left); DCIS.COM control, shPTGS2 (KD1), and shPTGS2 (KD2) were analyzed by western blot for SIM2s and GAPDH as loading control (right). e Pie Chart to show percent tumor progression DCIS+IDC or IDC only in the control group (n = 8) and shPTGS2 (n = 4). f SIM2s expression in SUM159 control cells and cells dosed with 20 μM celecoxib by qPCR as fold change. g SIM2s expression in DCIS.COM control cells and cells dosed with 50 μM DHA by qPCR as fold change. h SIM2s expression in MCF7 cells dosed with vehicle or 100 μM PGE2 for 24 h by qPCR, unpaired t test: p < 0.08. i SIM2s expression in DCIS.COM cells treated with vehicle (control) or 50 μM DHA by qPCR. Unpaired t test: *p < 0.05
Table 1 Oncomine analysis
Through transgenic mouse models and in vitro studies, SIM2s has been identified as a novel player in several key aspects of mammary gland development. Specifically, genetic ablation of SIM2s in mammary epithelial cells revealed that SIM2s is required for ductal morphogenesis and differentiation of luminal cells for milk production during lactation. Furthermore, mammary-specific overexpression of SIM2s resulted in a delay in post-lactational mammary gland involution through suppression of Stat3 and NFκB signaling, as well as maintenance of markers of epithelial cell differentiation normally observed only during lactation. These results suggest that SIM2s has tumor-suppressive activities in the mammary gland through maintenance of epithelial cell differentiation. Consistent with this, loss of SIM2s expression in the mammary epithelium results in EMT events, such as loss of E-cadherin and increases in matrix metalloprotease activity, and similar results are also observed in breast cancer cell lines. SIM2s is also downregulated in breast cancer patient samples, further validating its potential role in tumor suppression [39]. In this study, we demonstrate a novel role for SIM2s as a negative regulator of tumorigenesis via downregulation of the NFκB pathway, which normally results in transcriptional activation and expression of the pro-inflammatory/pro-tumorigenic enzyme COX-2, which in turn promotes DCIS invasion. Interestingly, we also identify a novel link between SIM2s and preventing signaling of the pro-tumor/pro-survival kinase Akt, which has been shown to promote tumorigenesis in part through NFκB-mediated COX-2 expression [51]. Additionally, we also show that SIM2s is directly targeted for suppression by NFκB signaling, suggesting a regulatory pro-tumorigenic feedback loop. Consistent with a role for SIM2s preventing this pro-tumorigenic cycle, loss of SIM2s also drastically increases COX-2 expression, while loss of COX-2 activity and expression results in re-expression of SIM2s and downregulation of tumor cell invasion. Thus, we have identified a reciprocal relationship between a molecule with known tumor-suppressive activities, SIM2s, and a well-established tumor promotional pathway that involves pro-survival and pro-invasive signaling mediated by Akt, NFκB, and COX-2 (Fig. 5).
A model depicting SIM2s and NFκB cross-talk regulated to COX-2
Based on our previous results reporting a role for COX-2 in promotion of DCIS invasion [22], and results showing that SIM2 is lost in IDC compared with DCIS in patient samples [39, 50], we predict that loss of SIM2s may be important for progression of in situ lesions to invasive disease via upregulation of COX-2 expression and activity. Consistent with this hypothesis, in the DCIS.COM model, loss of SIM2s is associated with increased invasiveness and enhanced tumor aggressiveness and progression, all of which are also observed with gain of COX-2 [22, 23, 37,38,39,40, 50]. Specifically, upon loss of SIM2s in tumors, increased co-localization of keratin 5 and vimentin has been observed [39], which is indicative of mesenchymal and invasive phenotypes; furthermore, gain of COX-2 results in increased collagen deposition in the tumor microenvironment, which tumor cells utilize to invade the surrounding tissue and access the vasculature to form metastasis [22, 52, 53]. Matrix metalloproteinases (MMPs), which are associated with basement membrane degradation during mammary gland development and cancer, are also significantly increased with loss of SIM2s [54,55,56]. These changes likely promote an increased potential for progression of DCIS to IDC and furthermore for tumor cell escape from the primary site. Interestingly, COX-2 has been shown to promote angiogenesis [57] and to inhibit anoikis via activation of Akt [58], suggesting that this pathway can also promote dissemination and survival in circulation. Furthermore, increased COX-2 and increased proliferation are associated with subsequent recurrence of DCIS in patients [21]. Here, we show that cells with low invasive potential exhibit increased expression of COX-2 upon knockdown of SIM2s and endogenously express moderate levels of SIM2s compared with the low level of SIM2s observed in the more invasive cells [37]. Likewise, overexpression of SIM2s in invasive cells decreases COX-2 expression. Coincidently, SIM2s overexpression also significantly decreased COX-2 staining in tumor sections and all point toward a role for SIM2 in preventing metastasis. This is consistent with data from the TCGA showing downregulation of SIM2s in a metastasis gene signature. Since it is well known in the literature that COX-2 inhibition is associated with better prognosis for breast cancer patients [59, 60], further studies on strategies for re-expression of SIM2s may be beneficial in improving prognosis of breast cancer patients. Furthermore, an additional implication is that SIM2s could be utilized as a marker to identify DCIS patients that are of low risk for acquisition of COX-2 expression and progression to IDC and/or metastatic disease. However, relevance for this mechanism beyond local invasion, such as in metastatic spread, remains an unanswered question that we will address with future studies.
These findings support a role for SIM2s in the prevention of breast cancer progression through its ability to repress PTGS2 expression via modulating the NFκB signaling pathway. It has long been established that NFκB regulates genes involved in cell proliferation and cell survival. Specifically, blocking NFκB in tumor cells can lead to susceptibility to anti-cancer agents. However, due to the complexity of the tumor microenvironment, NFκB signaling also has been found to have anti-cancer effects in various cancer cells. Thus, it is important to investigate a mechanism, specifically in mammary tissue, in which the targeted pathways are highly involved with cell proliferation, survival, migration, and invasion. Due to elevated COX-2 expression correlating with poor prognosis, it is imperative to investigate reducing COX-2/PTGS2 expression. In the data provided here, we have demonstrated an integral role for SIM2s involvement in mediating NFκB signaling to decrease expression of COX-2/PTGS2, which could lead to an improved prognosis for breast cancer patients.
COX-2:
Cyclooxygenase 2
DCIS:
Ductal carcinoma in situ
DHA:
Docosahexaenoic acid
EMT:
IDC:
Invasive ductal carcinoma
IKKα:
Inhibitor kappa b kinase alpha
IKKβ:
Inhibitor kappa b kinase beta
IκB:
Inhibitor kappa b
IκBα:
Inhibitor kappa b alpha
IκB-SR:
Inhibitor kappa b alpha super repressor
NFκB:
Nuclear factor kappa b
PUFA:
SIM2s:
Singleminded-2s
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We gratefully acknowledge Kornelia Polyak and Andriy Marusyk for providing the shCOX-2 DCIS cell lines and Pepper Schedin for providing the shCOX2 tumors and controls from the in vivo studies. Sarah Tarullo is acknowledged for independent/blinded confirmation of the invasion analysis.
This research is supported by an NRSA F31CA236140 (LSC/TL), the University of Colorado Department of Medicine Outstanding Early Career Scholars Program (TL), and the National Cancer Institute R21CA197896 (WP), R01HD083952 (CO-PI WP, MR), R21CA185226 (TL), and R01CA211696 (TL).
Department of Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA
Garhett L. Wyatt, Cole M. McQueen, Steven W. Wall, Tanya L. Gustafson & Weston W. Porter
Department of Medicine, Division of Medical Oncology, University of Colorado Anschutz Medical Campus, Aurora, USA
Lyndsey S. Crump, Chloe M. Young, Veronica M. Wessells & Traci R. Lyons
The University of Colorado Cancer Center Young Women's Breast Cancer Translational Program, Aurora, CO, USA
Department of Nutrition, Texas A&M University, College Station, TX, USA
Yang-Yi Fan & Robert S. Chapkin
Garhett L. Wyatt
Lyndsey S. Crump
Chloe M. Young
Veronica M. Wessells
Cole M. McQueen
Steven W. Wall
Tanya L. Gustafson
Yang-Yi Fan
Robert S. Chapkin
Weston W. Porter
Traci R. Lyons
GW performed the in vitro experiments, formal data analysis, interpretation of the data, production of figures, writing of the original draft, and reviewing and editing the final draft of the manuscript. CY and LSC were involved in animal husbandry, tumor immunostaining, and immunoblot analysis. VW was involved in designing and performing immunostaining of the xenograft studies. CMM and SWW performed treated cells, harvested proteins, harvested RNA, and assisted in the experimental design and western blots. TG was involved in animal husbandry, in vivo experiments, and ChIP analysis. YF and RC provided the polyunsaturated fatty acid resources. WP and TL oversaw all the experiments, experimental design, and interpretation of the data; provided the resources; and contributed to the writing as well as reviewing and editing of the manuscript. All authors read and approved the final manuscript.
Correspondence to Weston W. Porter or Traci R. Lyons.
Animal studies were approved by the Institution's laboratory care committee.
Additional file 1: Figure S1:
Verification of effective transduction knockdown of SIM2 via sh-RNA. Infection of MCF7 cells with the SIM2-shRNA construct results in decreased SIM2 protein levels in comparison to a nonspecific scrambled shRNA construct. Figure S2: Verification of NFkB promoter luciferase and SIM2 promoter luciferase assays in the HEK293 cell line. A. Luciferase activity in HEK293T cells co-transfected with 5x kB binding sites upstream of the luciferase gene (5x NFkB-luc) and NFkB p65 and/or SIM2s. (Diagram of promoter construct is shown above for reference.) B. Luciferase activity in HEK293T cells co-transfected with 5x NFkB-luc and NFkB p65 and/or SIM2s with its repression domain deleted (SIM2sΔR). C. SIM2 promoter activity in HEK293T cells co-transfected with SIM2 promoter upstream of the luciferase gene and increasing amounts of NFκB p65 (50ng,100ng, and 150ng). D. SIM2 promoter activity in HEK293T cells after co-transfection of promoter with control vector (pcDNA3), NFκB p65, and/or IκB-SR. E. SIM2 promoter activity in HEK293T cells co-transfected with SIM2 promoter upstream of the luciferase gene and 150ng NFκB p65 compared with the SIM2 promoter activity in HEK293T cells co-transfected with NFκB double mutant SIM2 promoter upstream of the luciferase gene. ANOVA and Student's t-test was performed to test significance. A, B, C all significant at p<0.05, *p<0.05. Figure S3: Verification of PTGS2 in various breast cancer cell lines. PTGS2 expression in MCF7, DCIS.COM, and SUM159 parental breast cancer cell lines. ANOVA and Student's t-test was performed to test significance. A, B, C all significant at p<0.05. Figure S4: No correlation between Tumor size and SIM2s or COX-2 gene expression. A. %SIM2s positive nuclei compared to tumor size (cm2). B. %COX-2 (M+S) compared to tumor size (cm2). Prism7 was utilized for statistical significance analysis. Two-tailed t-test was performed to test significance. Figure S5: SIM2 expression in TCGA Breast primary site and metastasis. Oncomine analysis of SIM2 expression in TCGA Breast primary site (n=529) compared to metastasis (n=3). t-Test performed to test significance, p-value=0.006. The Oncomine™ Platform (Thermo Fisher, Ann Arbor, MI) was used for analysis and visualization. For further information, refer to the terms of use. Table S1: Antibodies used in study. Table S2: Primer sequences used in study.
Wyatt, G.L., Crump, L.S., Young, C.M. et al. Cross-talk between SIM2s and NFκB regulates cyclooxygenase 2 expression in breast cancer. Breast Cancer Res 21, 131 (2019). https://doi.org/10.1186/s13058-019-1224-y
COX-2
NFκB
Prostaglandin E2


Offering a terrace and sun terrace, Casa a Mare is set in the Sorrento City Centre district in Sorrento, 1.1 km from Corso Italia.
The rooms include a private bathroom. For your comfort, you will find bath robes and free toiletries. Casa a Mare features free WiFi throughout the property. Private parking is available at the bed and breakfast.
You can engage in various activities, such as cycling and fishing. Museo Correale is 1.1 km from Casa a Mare, while Marina Grande is 100 metres away. The nearest airport is Capodichino Airport, 29 km from the property.